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DSC Data Analysis in Origin®

1. 10 20 1g at a0 BO ro au au 100 Temperature OC Cp tkealmole C TI DSC Data Analysis Tutorial Guide e Select File New Graph and click OK A new graph will become the active graph in Origin e Double click on the Layer 1 icon located in the upper left hand corner of the new graph e Click the bindproteinon cp dataset from the Available Data list to highlight it then click the right pointing arrow gt to place this dataset in the Layer Contents list box and click OK Note The apostrophe at the end of the data set name indicates that this data has been corrected for the Layer 1 Available Data Delete bindligandlds cp bindingbasel cp bindproteimon cp bindligandlds cp dm bindprateinon ep Layer Contents l mE Sarl Layer Properties Plot Associations TITIUS Edit Range response time of the instrument Show Range Sot v Rescale on DK Show current folder only To determine ACp e Select the menu item Peak Start Baseline Session Origin calculates and plots in red the left and right linear line segments from which to determine the baseline A new menu bar appears containing the baseline session menus e Select the menu item Adjust Move Segments by Cursor adjust the low temperature and high temperature segments then double click an endpoint or press enter to set the baseline segments See Lesson 4 Baseline Determinations for g
2. pc20904012 DSC 1 pc20904013 DSC 1 od NEN n3 L3 L2 65 DSC Data Analysis Tutorial Guide 66 To apply a Y Offset Many times when reading in multiple files the curves will overlap each other making it difficult to view the individual curves You may use the Y Offset button to offset each curve so that at the peak the closest point of the next curve is offset by the amount entered e Ifyou are not continuing from the previous example read in a series of scans pc20904001 DSC as described above e Click the Y Offset button and the following dialog box will pop up Offset all curves Cancel Value 2750 e You may select the default value or enter a different value and click OK Each curve will be offset so that the closest point of each adjacent curve will be equal to the amount entered as shown below Cp kcal mole C 40 60 Temperature C Find Two Tm Lesson 6 Autosampler Data Optional Accessory To Find Two Tm e Select File New Project to open a new Origin project e Click on the Read Data button The Import Multiple ASCII dialog box opens with the Auto 0 00015 DSC Data dsc selected as the Files of type Only DSC A files that have an 001 before the ey 4 AER 0 00020 extension dot will be listed S ome S 0 00035 p mp e Navigate to the 0 00040 Lf AS2peais0D3DSC cp C VOrigin70 Samples folder m ASA
3. 0 66 95 76 57 O O O AS2Peaks D9 DEC O 1 Aiz 1 44 0 56 95 76 57 AS2Peaks lD DEC 1 A19 1 44 0 6565 95 76 67 O 67 DSC Data Analysis Tutorial Guide Ligand Protein Binding Constants from Autosampler DSC Data Data obtained from DSC scans may be used to calculate binding constants for protein ligand or protein protein interactions for further information refer to page 101 of the Appendix The addition of a ligand into a protein solution acts to shift the transition midpoint to higher temperatures if that ligand binds to the low temperature native form of the protein The more strongly the ligand is bound the greater the increase in Tm Accurate estimates of binding constant Kg may be obtained from DSC data in the presence and absence of ligand The parameters required to calculate Kg are the protein concentration ligand concentration the heat AH and the heat capacity ACp change in the absence of ligand and the temperature midpoint of the transition in the presence Tm and absence T of Ligand The project BindingConstants OPJ is available in the C VOrigin70 Samples directory for the following example to determine binding constants from DSC data For the following example we will first determine the ACp then AH for the protein with no ligand The Tm s for both the bound and unbound protein can be read from the previously determined values refer to the table on page 65 e Launch a
4. When extrapolating the binding constant to a different temperature you must determine AHg and ACpg from an ITC experiment or other independent information e Click the Calculate Kg at a different temperature Tr check box to insert a checkmark then click OK e Enter the following values in the parameter text boxes T 25 C AH at Tp 15000 cal mole Input For Different Temperature Fa xj ACp z 1000 cal mole deg 22 e Click OK The binding constant at Tg will be calculated and the following dialog box will open listing the entered parameters and the extrapolated Kg You may print the results or click OK or Close to close the dialog box Binding Results 73 DSC Data Analysis Tutorial Guide Ligand Protein Binding Constants from Multiple Samples e Launch an new instance of Origin e Select File Open navigate to the C Origin Samples directory select the project BindingConstants OPJ and click Open Your project should look similar to what is shown below This data has had a baseline subtracted has been corrected for instrument response time has been normalized on concentration and the Tm has been determined Subtract Reference M x Absolute Cp m Q o n 2 o z pa o ae Sa et Q Od S MR om BM so BH OD Temperature C e Click the Auto Binding Constants button from the Auto DSC button group The DSC Ligand Protein Binding Constants dialog box will open e En
5. 63 64 DSC Data Analysis Tutorial Guide To apply the Response Time correction Due to the very fast scan rates available up 250 deg hr for the Capillary DSC and the finite response time of the instrument there may be some Tm shifting and or peak broadening A Response Time button is available that when activated will apply a response time correction for all scans plotted in the active layer This functions similar to the Response Time button available for the standard DSC scans see pages 88 91 After you apply the time constant the traces will change color from black to blue The original data will be removed from memory Click the Response Time button Note make sure to click the Response Time button located in the Auto DSC group of buttons and not the button associated with the standard DSC The traces will change in color from black to blue indicating the Response time correction has been applied and the original data has been removed from Origin Note This action may take a minute or two if there are many datasets plotted Hormalize Concentration To Normalize Concentration When you click the Normalize Concentration button the data is divided by the number of moles of the sample substance in the cell to convert from cal degree to cal mole degree Concentration normalization is always carried out after subtracting the reference data The concentrations mM of the proteins are usually entered in VP Viewer wh
6. LabTalk script language 34 Layer Control dialog box 34 35 Line Types for fit curves 91 92 M Math dialog box 23 Menu levels 5 6 N Nonlinear least squares fitter 20 22 O Offsetting multiple DSC files 10 Opening and Analyzing Previous Versions of Origin ORG Documents 8 Opening DSC data files 11 12 Opening Worksheet window 14 15 P Page View mode 7 23 Pick Peaks tool 80 Plot window maximizing 38 Print View mode 7 23 R Reference data subtracting from sample data 13 14 Registering with MicroCal Software 5 Response time autosampler data 63 FeedBack modes 89 relaxation time constant 89 Response Time 68 91 Response time button 89 106 S Scan rate normalization 10 and instrument calibration 24 Scripting 34 Sequential model equations 97 99 Set Default Directory 12 simplex minimization nonlinear least squares fitter 21 Starting Origin 5 9 Subtracting reference data 13 14 System requirements for Origin 3 T Text formatting 23 32 33 Tolerance 50 Two state model 20 22 Two state with dCp 22 23 V View mode and screen refresh rates 6 available types 6 W Window View mode 7 Worksheet viewing data 14 15
7. a O 50 Temperature C Upon setting the third Tm Origin calculates all three sets of fitting parameters six parameters in all two for each transition and displays the initial fit curve 5 As before select 100 Iter from the menu bar to fit the curve to the data After several fitting cycles you should get a pretty good fit implying that the three transition model describes this data better than the two transition model Fitting Example 2 For multisubunit proteins it often happens that the subunits dissociate simultaneously with the unfolding of the protein 1 e A nA where n is the number of subunits When this happens the shape of the DSC peak is broadened relative to unfolding of the same protein where no dissociation occurs e g covalently linked subunits The mathematics for curve fitting to systems which undergo simultaneous unfolding and dissociation are included in the Appendix You may wish to try fitting the data set in the Origin project Dissoc OPJ contained in the Origin70 Samples folder The data was obtained on a helical polypeptide thought to contain two identical chains linked together in a coiled coil state These data have already been normalized on concentration and a progress baseline has been created so curve fitting may begin immediately Note that whenever using the dissociation model the concentration used for data normalization should be that for the associated low temperature state not
8. entered parameters and the calculated Kg You may print the results and or calculate the Kg at a different temperature Binding Results X gl x Results for Ligand Protein Binding Constants K dg SIGHS I ett 16 nl Input Parameters To 51 C Tm 55B5 C i 1 AH 110000 cal mole ACD 1100 cal mole dedg Corot 9E 5 moles l lig 0 01 males 8l DSC Data Analysis Tutorial Guide To extrapolate the binding constant to a different temperature e Click the Calculate Kg at a different i Input For Different Temperature zIbd x temperature Ty check box to insert a npa s or A p ra We Fo checkmark then click OK A new dialog box Ops e will open up allowing you to enter the f parameter information to extrapolate the Kz 15000 TE value to another temperature Choose the File AHgat Te Edi pc20904007 DSC to use for extrapolating the AC DOO cal mole deg binding constant to a different temperature Repeat the method described on the previous pages to determine the AH and the ACp at the Cancel different temperature Tp Enter the following values in the parameter text boxes Tr 25 C Hg at T 15000 cal mole deg ACpg 1000 cal mole e Click OK The binding constant at Tf will be calculated and the following dialog box will open listing the entered parameters and the extrapolated Kg You may print the results Binding Results E Fa Results for Ligand Frotein Binding Constants KIS 1o6ES M at 6
9. t E et 40 s BU TO Bl Jr 100 X Axis Title To set the markers press enter or double click on one of the markers Select Integrate from Baseline from the Peak menu The area of the peak between the markers will be determined and printed in the Script Window For this example the area was found to be equal to approximately 110 000 cal mole Your value may be different depending on where you placed the lower and higher temperature baseline segments during the baseline session Note The script window may be hidden near the bottom edge of the workspace if the Script Window is not open you may open it by clicking on the Script Window button located on the standard tool bar Ea Lesson 6 Autosampler Data Optional Accessory To calculate a single DSC ligand protein binding constant from autosampler data In the following example we will calculate the binding constant for a ligand to a protein then extrapolate the binding constant to a different temperature From the previous example we determined T 61 C AH at T 110 000 cal mole and ACp 1 100 cal mole deg for the protein only For the following example we will use the values from the pc20904006 DSC row of the summary table on page 65 From the table we can see that Tm 66 57 C the Protein Concentration 00009 moles liter and the Ligand Concentration 010 moles liter From previous information we know the stoichiometry n l Knowing the heat of binding AHg and AC
10. 0 folder and open the Setup exe application file by double clicking on the filename File Edit Go Tools 3 Cut Favorites 8 Up View z Y E Eormsard Select the AddOn Setup Folder cae Origin Setup Disks EHE D riginbU Origin OT P m datal cab 1KB Wi le 02 18 2002 3 00 PM P aa datal hdr AKE Beh File 02 18 2002 3 00 PM S lang dat 23 DAT File 02 18 2002 3 00 PM Cg NAG PDFs e layout biri IKB BIH File 2 18 2002 3 00 PM mfg Origin os dat 1KB DAT File 02 18 2002 3 00 PM 8 Palettes isi Setup bmg 113KB BMP File 02 18 2002 3 00 PM m 3l Setup eee f2KB Application 02 18 2002 3 00 Ph s Setup tril 1KB Configuration 02 18 2002 3 00 PM ae setup ins B4KB Intemet Com 02 19 2002 3 00 PM mm setup lid 1KB LID File 02 18 2002 3 00 PM Hun Note you may also launch the setup up program by selecting Run from the Start Menus then entering c Origin70 AddOn Open Setup Setup exe into the text box and clicking OK e Follow the instructions on screen Help B Copy Paste X Delete Properties A Undo edUKB EX File 02 16 2002 3 00 PM AKE Application 02 15 2002 3 00 Po S466 Application E 02 18 2002 3 00 PM Double click on Setup exe 838KB Disk free space 14 1 E34 My Computer Type the name af a program folder document ar Internet resource and Windows will open it For you C NOngin O 44dd0n Setup setup ex Cancel Brows
11. 5 Simultaneously Running DSC and ITC Configurations sse eene eene eene 6 Rar Aree T E 7 Note About Data IMPO eera R E A O A Loe io A 7 Analyzing Previous Versions of Origin ORG Documents and Projects OPJ 8 Lesson I Basic DSC Data Analysis siccsscscisceciavscscsccciziasssvaesetassaacesadesssnacusouuavacsereeciaaccbtesacesuicouseebacsevaaseseaseonce 9 Sanno ATO 1AM tate T ga tee T o a a gn ne see abe ata tic RENE 9 NS DSO BJ mc c E A 9 Surac ine Relerence Data ase rest teorie iP reset ndis o MM Da a Rus Ies ree ee eee 13 VIS WA WV On SIG Gl Aaa t speed pedi ate uertit s i sedges pn pP Nd erbe DsE pera eee ie pud eugene ocean Se eee peu EUR 14 INormializing The Ata os co sig oat EE aig ttn terea ier als ae ona Iu e EE 15 Creatine 3 ASC IN eienn th fet et e th ef iet eta ofa iC tft bam 17 GD A tHe DUI RET 19 Ther NNE SE Oa eee cise cee NP ne Coc Med DELI Ui E D DI ACC a 21 ANI ALON Faro a etate matis A un noauhaledele maa eau ME ORE EU ete idR ED O T Rd Po C a 24 Lesson 2 Using the Data Selector TooL eiue een re eee eoo licens Yap ea eno auno sa s ced oeo eoe E ep ode aepo Suena Rada as 25 Lesson 3 TGCS E ATION crisser aiaee E ys i depo pe dea e Ule e iaa vede dos PUE EVE aaa Oe DR UU odo aR O e ena Uns OU EE 29 Setuine Range and Intesratine the Daft s iuc ena RE HR EEG EE EN EE 29 Displaying the Intcbratlon B eSUliso oio olet detenta ECU OR ate det iras d raris EL Ee 32 Plotin the
12. E mm E pan I Q A r 0 0008 E3 0 0006 0 0004 lt a E 9 0 0002 Z E oO 0 0000 o Oo D 0 0002 Q M 0 0004 V 0 0006 0 20 40 B 80 Temperature C B n ii A v S 7 L Ix y x UNTITLEC Name Type View Size Mstat SIAR Ka A Eporing G C Printers 64 MAU120010 GY Exploring G FF Origin 7 EIRA 12 33PM The RawDSC window which contains buttons that are used to import DSC data Two DSC data files SAMPLEI DSC and REF1 DSC are included for your use with this lesson SAMPLE contains sample DSC data REF1 contains the associated reference data Both are located in the Samples sub folder of the Origin70 folder These files were obtained on the MC 2 DSC instrument using the Windows data collection software Files from MCS or VP DSC data collection software packages are similar but have different header information To open the SAMPLEI DSC sample data file and REF1 DSC reference data file Click on the Read Data button in the RawDSC plot window The Import Multiple ASCII dialog box appears 11 DSC Data Analysis Tutorial Guide Import Multiple ASCII Ei x Look in C3 Samples Tl ce 33 Sample dec Vchtl 5bs dsc _ Data samples dec 3 Vchtbs dsc Graphing E Sample5 dec L Programming Vchtl 2bs dec Refl dec Vchtl3bs dsc 3 Rers dsc 3 vchtl 4bs dsc File name Fef dec OF Files of type psc Data dsc Re
13. Files of type psc Data dsc Remove Cancel File Marne Vehtl tbs dec Vchtl2bs dsc Vehtl abs dac Vchtl5bs dsc MVchtBbs dsc 54 Lesson 5 Curve Fitting Your Origin graph will look like below El RawDSC Vebildbadac_cp Head Data Vebil thadac_ecp Vebil bide rp Subtract Reference VebulSbidse_ep chik adac rp Normalize Concentration x x Demon or o Temperature C Notice the square around the line type of the last file opened in this case data set Vcht9bs dsc in the legend of the graph This indicates it is the active data set When you click on the Absolute Cp button the routine will convert this data set Step 2 Converting raw data to absolute heat capacity values 1 Click on the Absolute Cp button Absolute Cp The Absolute Cp dialog box will open with the following default parameters Cancel Specific volume ml gm LUTAEI Thermal expans 1 deg 7 E 4 Concentration mgm ml 0 258 Cell Volume mljj0 512 File name VchtSbsdsc 2 For this example all the default parameters are correct Click OK l Literature value for the partial specific volume of chymotrypsinogen is 717 ml gm and the coefficient of volume expansion is 7x10 deg 55 DSC Data Analysis Tutorial Guide 56 A new window named AbsCp will be created With the converted data plotted as shown belt mmmn E AbsCp 2 B x Ee Read Data Ta TT WenS
14. K T RT AH T X TE K T exp aT gt 11 where again the right hand term in eq 10 must be repeated for each transition and where eq 11 must be evaluated for each transition In this case the fitting parameters are T 4 AH A Tmp AH mp but otherwise the curve fitting procedure is the same as described above Model for Independent Non two state Transitions Because of the extra fitting parameters required for the non two state vs two state model this model is only applied to transitions with no AC Before curve fitting with this model a progress baseline must be subtracted from the experimental data to remove the AC effects if they are present this also sets Con to zero at all temperatures To treat non two state transitions the appropriate place to begin 1s eq 6 which still includes both calorimetric and van t Hoff heat changes Indicating the temperature dependent parameters this can be rewritten as K T AH AH VS a 1 K T RT In this case the equilibrium constants will be calculated as in eq 11 with the important exception that the van t Hoff heat rather than the calorimetric must be used since now the two will be different 1 e 12 AH T K T exp 4 1 13 A T exp m gt 13 Eq s 12 13 are then used for curve fitting in the usual way and the parameter set will be Tors AH aAa lee A A a mB II Sequential Transitions Sequential transitions may occur in protei
15. K T K T AH AH AH AH D e rr EN 19 where the temperature dependent quantities have been indicated in the usual way The equilibrium constants can be evaluated in terms of the temperature independent AH parameters from 98 Appendix Equations Used in Deconvolution l l K T El R cl 20 Using eq s 19 20 and fitting parameters K AH K5 AEL the fitting procedure is carried out as described above for the models involving independent transitions III Single Two State Transition with Subunit Dissociation It sometimes happens with biological macromolecules having subunits that subunit dissociation occurs simultaneously with thermal unfolding 1 e A n K PE 21 4 where n is the number of dissociable subunits and where the brackets signify molar concentrations of species If fis the fraction of macromolecule in the A state and 1 f the fraction in the A state then e m where C is the total bulk molar concentration expressed as n mer equivalents The equilibrium constant using eq s 21 22 then becomes K FN unc 23 l f Using eq 23 and the expressions OlnK T AH T T AH T AH AC T T 4 24 and integrating from Tma where f 0 5 to T then gives AH T AC T K T 05 n C exp o T B vm c T rm2 25 mA mA Once K T is known then f T may be solved from eq 23 using numerical methods and the excess heat capacity calculated from
16. Will determine two Tm s for all scans plotted in the active layer Find Delta Tm This button will allow you to select a scan to use as a reference Tm The program will then calculate the temperature difference between the reference Tm and the Tm s of all other scans Y Offset When this option is selected you will be prompted to enter a value to offset each scan that is plotted in the active layer This may be useful when scans are overlapping and you want to view individual curves Normalize Normalizing the data divides the data by the number of moles of the Concentration sample substance in the cell to convert the data from cal degree to cal mole degree When you click this button all scans will be normalized by the concentration mmoles l for each sample Response Time This is used for removing small amounts of peak broadening due to using a feedback mode which has a response time too slow to resolve a peak adequately When you click on this button all scans plotted in the active layer will be corrected for their response time The original data will be deleted For more information about response time refer to the Response Time VP DSC section starting on page 88 Auto Binding Constants This button provides an automated method to calculate ligand protein binding constants for the multiple scans plotted in the active layer Kb Threshold Value When this button is selected you will be prompted to enter a Threshold value for Kb Al
17. an integral equation gives T T T AG l l fd and f AH T AC T T d T I T I T Integrating the above equation from Tm where AG 0 to another temperature T then gives AG Can Ti ag T AH T AC In gt T Cw TD a M Once the temperature T is assigned all other parameters in the above equation are known except AG which can then be calculated 103 Index A Absolute heat capacity equations 100 fitting example 54 57 Active data set 13 Autosampler 59 73 binding constants 68 73 buttons 60 Conc 1 amp Conc 2 64 find delta Tm 65 find one Tm 64 find two Tm 66 normalize concentration 63 response time 63 subtract baseline 62 67 Y offset 65 B Baseline 17 18 37 44 adjust linear segments 39 40 adjust the default baseline 43 baseline menu commands 38 baseline session 38 42 cubic connect baseline 41 42 default name 44 linear connect baseline 41 move baseline by cursor 42 progress baseline 40 41 restoring 22 23 select baseline type 40 step baseline 42 subtract from DSC data 44 viewing worksheet data for 44 Binding constants 68 82 68 82 from autosampler data 68 73 from standard DSC data 77 84 Buttons 10 autosampler 60 Calibration 24 Chi Sqr button 21 Chi Sqr minimization 88 Concentration 15 17 Curve fitting 19 23 choosing a model 45 46 Dissociation w dCp 51 53 fitting iterations 50 fitting mode
18. button in the RawDSC1 plot window The Import Multiple ASCII dialog box opens e Double click on Samplel dsc located in the Samples sub folder of the Origin70 folder Click OK to plot the files from the lower list box SAMPLE1 DSC opens into the RawDSC plot window Sample1dsc_cp O o o Q O 60 Temperature C Before integrating you must plot a baseline for the data You will then choose to either integrate from the baseline or subtract the baseline and integrate from zero see Lesson 4 for more about baseline determinations 29 DSC Data Analysis Tutorial Guide Plotting a baseline e Select Start Baseline Session from the Peak menu Origin starts the baseline session and plots two red linear baseline segments e Select Progress Baseline from the Baseline menu Origin connects the linear segments with a progress baseline e Select OK from the menu bar Origin asks if you want to subtract the baseline Click No Origin exits the baseline session Sample1dsc_cp SAMPLE1DSC CP BASE O qm 9 a O Temperature C To set the integration range Click on the Data Selector tool in the toolbox Two data markers appear on the SAMPLE curve note that these appear on SAMPLE1 and not on the baseline curve because SAMPLE1 is currently set as the active data plot in the Data List under the Data menu Using the mouse or the arrow keys position the dat
19. e Click OK Data sampleldsc_ cp Model dl pM2sT Chi 2 4135600 BLO 56 9 111 BL1 22 1 2 66 T 60 65 0 0446 m AH 9 69E4 255 AC 1 41E3 109 O a i oO m e m M eL Q o0 60 T Temperature C 23 DSC Data Analysis Tutorial Guide 24 Calibration We will close this lesson with a brief note about instrument calibration Every two or three months you will want to calibrate the DSC instrument against the Y axis scale in Origin To do this you will fill both cells with water then key in heat pulses to produce a calibration file as described in your DSC instrument manual You then open the calibration file into Origin and check that the heat pulses match Origin s Y axis scale readings 1 Note that the measured peak heights of 9 008 6 007 and 3 006 indicated on the figure below agree very closely with experimentally entered pulses of 9 6 and 3 mcal minute Pulse heights can be readily measured using the data reader tool from the Toolbox One way to do this is to take a reading immediately before the pulse and immediately after the pulse average these two readings and then subtract the average from a third reading taken in the middle of the pulse 9 008 mcal min 6 007 mcal min 3 006 mcal min X axis title When you open a calibration file you must remember not to normalize on scan rate This means you should remove the checkmark from the Scan Rate Normalization checkbox
20. e Click the Find Delta Tm button File Name p520904004 dsc The Select Method dialog box opens ee lee e Click the File Name option and enter the name pc20904004 dsc this is one of the protein only scans and click Ok Note you must enter the dsc extension The summary table will open listing the filename Tray Well Concentration 1 Concentration 2 the Tm the Delta Tm and any comments that might have been included in the data file 0c20904001 DSC 1 1d 9D 1 L72 4328 Baseline pc20904002DSC 1 3 1d o 2308 37 92 Baseline pc20904003DSC 1 5 1d 0 X Baseline pc20904004 DSC 1 7 0 09 10 81 0 Rnaseonly no ligand 9 0 09 10 827 amp 17 Rnase Phosphate 009 10 865 57 5 57 RNase 3 CMP 2c20904007 DS 009 10 703 3 133 HN se 2 CMP 2c20904008 DS 0 09 10 80 97 0 03 Hnaseonly no ligand 5c20904009 DS 0 09 10 862 63 1 63 Rnase Phosphate 0 09 10 665 A 55 RHNase 3 CMP 009 10 7036 9 30 RNAse Z CMP 0 09 10 60 93 0 07 Rnase only no ligand 0 09 10 82 75 1 75 Rnase Phosphate 27 10 09 10 86 554 5 54 RNase 3 CMP 29 0 09 10 70 5 9 35 RNAse 2 CMP 2c20904011 DS 2c20904012 DS 2c20904013 DS od od pc20904007 DSC 1 pc20904008 DSC 1 pc20904009 DSC 1 pc20904010 DSC 1 pc20904D11 DSC 1
21. first we need to retrieve the baseline we subtracted previously To do so e Select Simple Math from the Math menu The Math dialog box opens Lesson Basic Data Analysis e Select Sampleldsc cp then click on the upper gt button Sampleldsc cp copies to the Y1 text box select SAMPLEIDSC CP BASE this is the original baseline data set then click on the lower gt button SAMPLEIDSC CP BASE copies to the Y2 text box Click in the Operator text box and type a in the operator text box Click OK The original Samplel data is restored since the baseline which was subtracted earlier has now been added back Now that you have retrieved the baseline data go ahead and fit a curve using model 3 Select 2 State w dCp Cursor Init from the DSC menu Then follow the same steps as outlined for the previous two models Click on Done to exit the fitting session To format the fitting parameters text Right click on the text in the plot window and select Properties form the drop down menu The Text Control dialog box opens Change the text style for example change the font to Times New Roman and point size to 16 Note that to ensure a true What Y ou See Is What Y ou Get text display on screen you should select the Print View mode from the View menu View mode does not affect output so for the sake of screen refresh speed it is usually adequate to use Page View Window View or Draft View all of which are faster
22. in the RawDSC plot window before you read in the data file If you neglect to do this and leave the scan rate normalization on your calibration results will be wrong Also note that when unnormalized DSC data are read into Origin the Y axis units are mcal min rather than cal deg which is obtained when data are normalized You now know how to perform basic data analysis of DSC files using Origin The following lessons in this tutorial each look at a specific aspect of DSC data analysis in detail You may work through these lessons in any order Lesson 2 Using the Data Selector Tool Lesson 2 Using the Data Selector Tool The Data Selector is a toolbar tool that is used to select a segment of plotted data Subsequent math editing or fitting operations will be carried out on the selected segment only In this lesson you will learn how to select a plot segment delete the segment set the data plot display range and reset the display range A sample DSC data file SAMPLE2 DSC has been included for your use with this lesson To open the SAMPLE2 DAT data file Select File New Project to open a new project Click on the Read Data button in the RawDSC window Shortcut Click the New Project The Import Multiple ASCII dialog box opens butt the Standard i mon quus Double click on Sample2 dsc in the Files list box Sample2 dsc is located in the Samples sub folder of the Origin70 folder The file name will be added t
23. new instance of Origin or select File New Project to open a new Origin project e Select File Open navigate to the C Origin Samples directory and select BindingConstants OPJ and click Open Your project should look similar to what is shown below This data has had a baseline subtracted has been corrected for instrument response time has been normalized on concentration and the Tm s have been determined Subtract Reference Ll Ll mec Absolute Cp Cp kealfmole PC Temperature C Kb Threshold Value 68 Lesson 6 Autosampler Data Optional Accessory New x Cancel e Select File New Graph and click OK A new graph will be created e Double click on the Layer 1 icon located in the upper left hand corner of the new graph LabT alk Script Template e Click the pc20904004dsc cp dataset from the aco AME Path Ein Available Data list to highlight it then click the right An E reah pointing arrow gt to place this dataset in the Layer Name ORIGIN Set Default Contents list box and click OK To determine ACp e Select the menu item Peak Start Baseline Session Origin calculates and plots in red the left and right linear line segments from which to determine the baseline A new menu bar appears containing the baseline session menus e Select the menu item Adjust Move Segments by Cursor The cursor will change to a cross hair icon e Adjust
24. next with a straight line The XY coordinates of the point appear in the Data Display Tool You can place any number of points you wish You can also use the arrow keys to place points Click once with the mouse to place a cross hair on the screen Use the UP DOWN LEFT and RIGHT arrow Keys to move the cross hair Press RETURN to set the position of a point When you are satisfied click on the Pointer tool in the toolbox to set the baseline Once you have created a baseline you can use the Adjust Baseline option to fine tune the default baseline To adjust the baseline e Select Adjust Baseline from the Peak menu The pointer becomes a cross hair To adjust a baseline point click and drag with the mouse Or use the arrow keys select a point with LEFT or RIGHT arrows move up and down with the UP or DOWN arrows move left and right with CONTROL LEFT or CONTROL RIGHT arrows When you are satisfied double click on a data point or press Return to set the baseline The baseline points in Cursor Draw Baseline and Move Segments by Cursor connect with a straight line by default Connect type and other display options can be changed by double clicking on a point in the baseline and selecting new options in the Plot Details dialog box 43 DSC Data Analysis Tutorial Guide To change connect type for a baseline Double click on the baseline The Plot Details dialog box opens e Select a connection option e g spl
25. of Origin or to upgrade an existing copy insert the Origin 7 CD into your CD ROM A window opens with a number of options including installing Origin Click the link to install Origin If the CD does not start automatically browse the CD and run ORIGINCD EXE directly The Setup program prompts you to type in your Origin serial number and license key These numbers are located inside your registration card in the Origin product package Please refer to the Origin Getting Started Booklet for further information When choosing a Destination directory or folder name to place Origin make sure this name or any other name in the path does not include a space otherwise Origin will not operate properly After installation is complete you will see the Origin70 program folder with the program icons If you wish to create a shortcut desktop icon you may do the following Right click the MicroCal LLC DSC icon and select copy from the drop down menu then right click anywhere on the desktop and select paste to install a desktop icon for Origin DSC Installing Origin for DSC Custom Disk After installing Origin you will need to install the Custom disk for the DSC routines if you have purchased other options the relevant Autosampler PPC and ITC routines will be included on this disk e Insert the Custom Disk CD into your CD ROM DSC Data Analysis Tutorial Guide Using Windows Explorer navigate to the AddOn Setup sub folder of the Origin 7
26. proteins are performed to measure the Ty values Protein Protein Binding Constants from Standard DSC Data T lt Tm lt To e Launch a new instance of Origin for DSC Note Normally you would calculate the parameters ACp AH T for each protein and the Ty for the complex Since the method of these calculations has been described on pages 78 80 we will simply provide the parameters below e Click the Binding Constant button and the Binding Constants from DSC Wa E3 Binding Constants from DSC dialog box will open up C Ligand protein interactions e Click the Protein protein interactions To lt Tm lt To radio button then click OK The Protein protein interactions dialog box will open up as shown below C Protein protein interactions Tm T o T o Cancel e Enter the following values Protein Protein Interaction To lt Tm lt To FAE F3 Identification Protein Pretein Binding Constante Parameters To To 50 C Tm 58 3 C AH at T 150000 cal mole ACp at T 2000 cal mole deg Protein Conc P 00004 moles l Protein Conc P 00004 moles l E Tm SE AH atTo 150000 cal mole ACpatTo 2000 cal male deg Prot Cone P 400004 males Prot Cone P 400004 males TU Cancel i 83 DSC Data Analysis Tutorial Guide e Click OK The binding constant at Tm will be calculated and the Binding Results dialog box will open list
27. results are pasted to the Script Window To clear the integration shading from the plot window select Refresh from the Window menu The procedure that copies the integration results to the Script Window is a macro called EndInteg EndInteg is defined in the DSC CNF file and can be redefined to customize the output to suit your needs For more information see the LabTalk Reference Manual available separately from MicroCal 3l DSC Data Analysis Tutorial Guide Displaying the Integration Results The Script Window is a full featured text editor Use it to edit the integration text save the text to a file or copy and paste the text with the clipboard To copy the integration results to the plot window Click and drag with the mouse to highlight the integration results in the Script Window Choose Copy from the Script Window Edit menu The text is copied to the clipboard ocript Window 77 Alernatively to copy and paste you File Text BRM Hide O may right click on the highlighted Msg e Undo MUEPAMILEi1DSC CP BRSE f text select copy from the menu then Ctrl eX right click where you want to i Ctrl C position the text label and select Ctri Y paste Del e Choose Edit Paste from the menu bar along the top of the Origin project window The integration text is pasted to the RawDSC1 plot window the active window Sampleidsc_cp SAMPLE1DSC_CP BASE Integ of Sampleidsc from SAMPLE1DSC CP B
28. standard way of defining the best fit 1s to choose the parameters so that the sum of the squares of the deviations of the theoretical curve s from the experimental points for a range of independent variables is at a minimum For the ITC models where there is no weighting the theoretical models can be represented by y IO PeP where Di the fitting parameters the expression for y simplifies to l t 2b ex email where n the total number of experimental points used in the fitting p total number of adjustable parameters y experimental data points fGpupsps fitting function Note the difference d n p is usually referred to as the number of degrees of freedom The above equation states that the Chi squared value of the fit is equal to the sum of the squares of the deviations of the theoretical curve s from the experimental points divided by the number of degrees of freedom Since there is no weighting it can be seen that the calculated values are dependent on the magnitude of the scale and the number of data points After fitting this value is reported as Chi42 DoF Response Time VP DSC The VP DSC is the first calorimeter to have operator selectable response time It is made possible by passage of the amplified AT signal temperature difference signal between the two cells into the computer for digital processing before the feedback voltage 1s returned to provide feedback power to the sample cell Th
29. structure ABA 1 e two identical and non interacting A domains and a single B domain Head to head covalently linked dimers can readily form ABA type of structures By comparison of results from different fitting models structures of this type can be recognized from DSC data If you are continuing from the previous lesson first select File New Project or click on the New Project button to open a new project Step 1 Prepare the data The first step in any fitting procedure is to open the DSC data file and reference data file then prepare the data by subtracting reference data and normalizing the result on concentration We have dealt with these data preparation procedures in Lesson 1 of this tutorial Here we will briefly review the steps involved 1 Click on the Read Data button in the RawDSC window and open the SAMPLES DSC and REF5 DSC data files located in the Origin70 Samples folder 3 Click on the Subtract Reference button The Subtract Reference Data dialog box opens 4 Select SampleSdsc_cp for the Data drop down list box entry 5 Select Ref5dsc cp for the Reference drop down list box entry 6 Click OK to subtract RefSdsc_cp from Sample5dsc cp 7 Click on the Concentration Normalization button Lesson 5 Curve Fitting 8 Enter 0 074 mM and 1 22 ml in the dialog box and click OK After completing these eight steps your screen will show the normalized SAMPLES data plotted in the NormDATA plot window Cp k
30. that for the dissociated high temperature state 1 To open Dissoc OPJ click Done to exit the fitting session then select File Open navigate to the Samples sub folder of the Origin70 folder then double click on the file name in the Files list Dissoc OPJ opens to show the Iv2p0 cp data set with its associated baseline plotted in the NormDATA window 5 DSC Data Analysis Tutorial Guide 52 E NormDATA P3 Tem nz raturz c 2 Select fitting model 4 Dissociation w dCp Cursor init from the DSC menu 3 Step 2 may have caused an error message to appear warning you that no baseline is selected for this data If this happened you need to let Origin know that you want to use the baseline supplied with this data which is named lv2pO cp base To do this select the Peak Cursor Pick Baseline command then double click on the plotted baseline data When you have finished again select Dissociation w dCp Cursor init from the DSC menu 4 Select 2 as the Number of Subunits Note that the number of subunits n and the concentration Ct appear in the Fitting Function dialog box as non floating parameters 5 Assign value of 0 to the BLO and BL1 parameters and remove the checks from each Vary Box so the low temperature baseline is not allowed to float Click on 100 Iter a couple of times You will note that the overall fit curve is fairly good C a zB p e m Le D C Lesson 5 Curve Fitting NonLi
31. the Available Data list then click on the gt button My test appears in the Layer Contents list All data sets in the Layer Contents list will plot in the active layer Lesson 3 Integration Layer 1 Available Data Delete Layer Contents tl l OF sampleldsc cp j my best Rance sampleldsc cp base nteg area Layer Properties my test lt Plot Associations aroun Edit Hange Show Range Sort v Rescale on OF Show current folder only Click OK The Layer Control dialog box closes and my test plots as a line graph in the Graph1 plot window i 2 X t gt 48 50 52 54 56 58 60 62 64 66 68 70 72 74 X Axis Title 35 DSC Data Analysis Tutorial Guide This page was intentionally left blank 36 Lesson 4 Baseline Determinations Lesson 4 Baseline Determinations Origin creates a baseline for the reaction heat data by establishing a left linear segment and a right linear segment that are considered to be the baseline region Several methods can be used to connect these two segments to form the baseline In this lesson you will learn how to create format subtract and open the related worksheet for a baseline You will also learn about the various baseline creation options that are available Baseline commands are located under the Peak menu Starting the Baseline Session When you are ready to create a baseline for your data you must start the baseline session In t
32. to enter the tray number the second will allow you to enter the well designation The sample that was in that tray and well will be then used to subtract from all other traces that are plotted in the active layer Note Tray designations must be numbers and not start with zero 0 the Well designation was designated when the experiment was set up in VPViewer and stored in the data file header and may be the number of the well e g 1 2 3 etc or the matrix number e g Al A2 etc Sample Tray 3 4 5 b 7 8 939 10 11 12 2 Xx Ing x 1 2 DOGOOO0OG009e Qe 00090 c 6 G9 G G0 G9 GG G2 9 Go G9 Ge mm C Manually Select C File Mame 6 Tray Well Label Trap Well 5 Uk Cancel 6 GG 9 6 9 6 9 6 9 9 9 t 8 69 6 6 G G 69 69 6 Go G9 6 FOQOMSSOOOMDO LAMDADPODOOODOSOE 1 OOMOOHOOS Select Manually Select and click OK The cursor will change to a cross hair Double click on one of the baselines 1 e PC20904001 PC20904002 or PC20904003 The selected baseline will be subtracted from all scans plotted in the active layer your results should look similar to what is shown below Note There may be a slight Tm shift when using different baselines for subtractions for this example the PC20904003 dsc dataset was selected e m D e SS LLL _ Cp cal C y IAW IE WS 60 Temperature C
33. 0904015 DSC 1 29 9009 10 7035 11200 RNAse 2 CMP Eb Threshold Value You may easily find binding constants that exceed a designated value by entering a threshold value for Kg All values in the summary table that exceed the designated value will be highlighted To find Kp values that exceed a Kg Threshold Value e Return to the ARawDSC window by selecting Window ARawDSC e Click the Kb Threshold Value button located in the Auto DSC button group Enter Threshold Value The following dialog box opens Cancel it Threshold Value 1500 e Enter a threshold value e g 1500 and click OK The Summary Table will become the active window and cells in the table of all Kg values that exceed the entered value will be highlighted 75 DSC Data Analysis Tutorial Guide This page was intentionally left blank 76 Lesson 7 Other Useful Details Lesson 7 Other Useful Details Ligand Protein Binding Constants from Standard DSC Data Data obtained from standard DSC scans may be used to calculate binding constants for protein ligand or protein protein interactions as discussed on page 101 of the Appendix The addition of a ligand into a protein solution acts to shift the transition midpoint to higher temperatures if that ligand binds to the low temperature native form of the protein The more strongly the ligand is bound the greater the increase in Tm Accurate estimates of binding constant Kg
34. 10 FUNCTION Reads a data file into the project Use this button if the data were generated by MicroCal s data collection software After data files have been imported into Origin use this button to subtract reference data from the sample data Before you proceed to fit the data you will need to normalize the result by concentration Normalizing the data divides by the number of moles of the sample substance in the cell to convert from cal degree to cal mole degree Concentration normalization is always carried out after subtracting the reference data If this option is selected when you open a data file Origin converts from the experimental data units of mcal minute to units of cal degree You should always leave this box checked except when you are checking calibration of the instrument Click on this checkbox to select or de select this option If the box is not checked then unnormalized raw data will read in as mcal minute In raw data files separate columns are included for Y axis temperature and time for each data point When data are being Scan Rate Normalized the time and temperature data are used to determine scan rate whereupon the time data is discarded and only the normalized Y axis readings and temperature data are imported into the project The operator has the option of importing or not importing the time data fime extension into the project as active data though the time data is not plotted automatically This i
35. 6 6 C K Is 2 1266 M at 25 C Input Parameters To a eu Tm ib Bi Tf ERE n 1 AH 110000 cal mole ACD 1100 cal mole deg Cprot S9E 5 moles Clit 1 01 moles AHTF 15000 cal mole ACp 1000 moles pin oce 82 Lesson 7 Other Useful Details Protein Protein Binding Constants from Standard DSC Data When estimating the binding constant for protein protein interactions the approach is a little different than for ligand protein interactions since both of the proteins P and P will undergo their own thermal unfolding transition with transition temperatures T and To When P and P are mixed together in the same solution then a separate transition will be observed for the complex PP with transition midpoint Tw If Ty occurs at a temperature between T and T then the regular model for ligand protein is used to calculate the binding constant If Ty occurs at a temperature higher than both T and T then a different model is used which takes into account the temperature shift in both transitions Please refer to the Section VI of the Appendix for more information about protein protein binding constants The procedure for measuring binding constants of protein protein interactions is similar to that of ligand protein interactions as described in the previous pages Separate DSC experiments of each protein alone must be performed to determine the respective values of T AH and ACp and experiments on the complex of the two
36. ASE i 86 gt 212 T Range 47 80103 Area 0 00789 Tm 61 00183 T1 228 00049 Cp cal C 60 Temperature C The text as pasted is too large and placed poorly Let s reformat and reposition the text and add a text border To change the text style Right click on the integration results text in the plot window and select properties from the drop down menu The Text Control dialog box opens From here you can edit the text set font style color and point size rotate the text and add a border 32 Lesson 3 Integration Note that the Text Control dialog box is divided into two display boxes all text entries and formatting is done in the upper text box the lower text box displays the text as it will actually appear WYSIWYG in the graph window Type 12 into the center points text box e Select Black Line from the Background drop down list box Note that if you select none for the Background the box may not be erased till you perform a screen refresh select Window Refresh Click OK The Text Control dialog box closes and the integration results text updates to show your changes Click and drag on the integration results text to place the text in the upper right corner of the plot window To see the graph exactly as it will appear when printed switch to the Print View view mode View mode is chosen from the View menu To change the view mode Click once on the View menu The View menu command
37. B AH p gt AC B are given To begin curve fitting with Origin the operator indicates the number of transitions needed to fit the experimental DSC heat capacity curve C Texp and then initializes all of the Tm values so that Origin can provide guesses for each of the other fitting parameters Knowing these it then calculates CT from eq s 7 9 and compares these values with C T exp Using Marquardt methods based on non linear least squares the guesses for each parameter are then improved the calculations carried out again and this iterative process continued until there is no further improvement in the fit of Hs calculated CT to the experimental C T exp as indicated by a minimum value of chi Model for Independent Two State Transitions excluding DCp Effects When all heat capacity changes AC a gt AC B are assumed to be zero certain simplifications can be made in eq s 7 9 Before curve fitting begins the operator subtracts a baseline from the data which effectively sets Con equal to zero at all Appendix Equations Used in Deconvolution temperatures so that Bo and B1 are no longer used as fitting parameters Incorporating this setting all heat capacity changes to zero and recognizing that all AH T for each transition may be replaced by the temperature independent heat AH i e heat changes must be temperature independent if there is no change in heat capacity then eq s 7 9 simplify to K DAH TY T AAA 10 1
38. D then select AS2Peaks001 DSC m to add the data file series to the UOS Seis lower list box and click OK 0 00055 20 30 40 50 60 70 80 The AS2Peaks family of data files will open and your graph should look as shown Temperature C To Subtract a Baseline Tray Well Label method e Click the Subtract Baseline button The Select Method dialog box will open e Select the Tray Well Label option and enter the number 1 for the tray and A11 for the well matrix number The baseline AS2Peaks006 dsc will be subtracted from all scans that are currently plotted 40 60 As described in the previous example you may select the Response Time and Normalize Concentration buttons Note The Find Delta Tm does not operate for two Tm s Temperature C e Click the Find Two Tm button The following table will open listing the filename Tray Well Concentration 1 Concentration 2 Tm1 Tm2 and any comments that might have been included in the data file AS2Peaks D1 DEC 1 o ai 0 72 ol 66 88 766e O O O AS2Peaks 0U2 DSC O 1 a3 0 72 0 6 84 75 51 I AS2Peaks 0U3 DSC 1 AS 0 72 0 B6 84 77 45 AS2Peaks D4 DSC 1 A7 0 2 0 86 95 76 67 es AS2Peaks D5 DSC 1 ago 1 0 98 44 Baseline AS2Peaks DE DEC 1 All 1 0 J Baseline AS2Peaks D7 DEC O 1 A13 1 44 O 0 56 84 75 48 AS Peaks008 DSC 1 Al5 1 44
39. Details Calculate AG at temperature T From analysis of a DSC scan on a protein three experimental parameters for the unfolding transition are obtained These include midpoint Ty Enthalpy change at Ty AH Ty and Heat capacity change at Ty AC Using these three experimental parameters the Gibbs free energy change AG may then be obtained at any temperature T The routine assumes a reversible two state transition and also assumes AC is independent of temperature To calculate AG at temperature T e Click the Calculate Delta G T button Calculate Delta G T v ng The calculate Delta G T dialog box opens md Kal E m 5 t e Enter the following values for Tm AH Ty and AC AH Tm 110000 acral Ta 60 C Atp 2500 cal male deg AH Ty 110000 cal mole aw AC 2500 cal mole deg e Click OK A new window opens plotting AG from 0 to 90 C at 1degree intervals A dashed line is plotted at y 0 and crosses the AG curve at the temperature Ty Ed Use the Data Reader tool to read the value of AG at any integer temperature from 0 to 90 C 40 60 Temperature C e You may use the Data Reader tool to read the value of AG at any integer temperature from 0 to 90 C For example at 25 C AG is ca 6840 87 DSC Data Analysis Tutorial Guide 88 Chi square chi 2 Minimization The aim of the fitting procedure is to find those values of the parameters which best describe the data The
40. EEEEEEEEEUEUCUNT i im Script Window AStart Dsc ogs WordPad 88 Microcal Origin C 5 BY Microsoft Word DSC Tut HONE 402PM 89 DSC Data Analysis Tutorial Guide You may see from the that the data collected in the passive mode dashed line has a slightly higher Tm and 1s slightly broadened from the data collected at the High Gain FeedBack mode fastest response time Make sure the data set Vcht20b2ds cp is the active data set box in legend is around the dashed line type of the Ezg venz nzd cn Vcht20b2ds cp data set Vcniz nddz cn Click on the Response Time button The Response Time dialog box will open e Enter 0 for the FeedBack Mode No Gain and PEE 40 9 for the Relaxation Response Time The scanrate will be automatically entered AE Please Note Normally the FeedBack Mode and Relaxation Response Time will be read YP FEEDBACK MODE o in from your experimental data file Response Time sec 40 9 SCANRATE deg hr 60 02849 e Click OK A new curve will be generated and plotted as a blue line shown below as a wider line This is the modified curve for the passive mode that removed the small amount of response time broadening The smaller peak of the repeated scan using High Gain FeedBack is due to some non reversibility of the sample 90 Lesson 7 Other Useful Details VCHT20B2DS CP Passive with Response X Lec VERO dece __ a H A Time broadening
41. Integration Area Data eorr repas be oaa obo ipee tuo to repas bre aid aee A bdo eu eau ERR BOR M 34 Lesson 4 Baseline Determinations sivsccccsccescesscsssccsvecestecstecsscsvecenteretesssevtecentavsascsssdsndesesesosessssseseueavasusntacaveente 37 Sarine the Biscle Sess ON ete ditas ti eli a bo ibas cei beue aaa ui ue ee eee 37 ser AdiusunentobLrncat cbe BE 259552 eet besito e id cs be ter eesbatun cese estes betta aaa 39 Choosino a Baseline ODLOBR 3 oett tnde t DE PER v oos eauohin E AE i Sot Se 40 Cursor DC DETERIUS OE docesoenuees 43 Lesson 5 Curve PINN eem 45 Genera lC DII lb ads eec inu eom sedes da acted ch lena dea onda SR a ates 45 FEE cial di em T Rr ee 46 EE Oke ean IND operc RD E NE 5 Pattie Example 3 25 Hp Tc HIR 54 Lesson 6 Autosampler Data Optional ACCeSSOLY cccccccccccscsssssssssssssssssssssccccccccsssscesessssssssssscccccssccseees 59 Launching the DSC Autosampler Data Session en ccccccccssccecccecceceeceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeseessesseseseeeeess 59 AUO PIED UI telnet rem er nee ane ere eee tea er Oe ey tee 59 Ligand Protein Binding Constants from Autosampler DSC Data cccccccssscccccececeeeeeeeeeseeeseeeeeeeeees 68 Ligand Protein Binding Constants from Multiple Samples ssesesesesessssssseeeeeeenn nnns 74 DSC Tutorial Guide Lesson 7 Other Useful Detalls sco stescescccssscetesesszececessiveuceesstasacatawabadasesaseieestasiucss
42. MicroCal LLC 22 Industrial Drive East Northampton MA 01060 USA Phone 413 586 7720 Fax 413 586 0149 Stabilit DSC Data Analysis in Origin Tutorial Guide Version 7 0 January 2004 a ey ee Using Origin scientific plotting software to analyze calorimetric data from all MicroCal Differential Scanning Calorimeters Fit Parameters 4 RAW DATA AFTER Tmi 58 29 C BASELINE SUBTRACTION AHi 148 kcal ACp3 5 41 kcal deg Tm2 68 16 C AH2 201 kcal ACp2 6 13 kcal deg BEST FIT CURVE FROM NON LINEAR LEAST SQUARES l ACp kcal mole deg F TRANSITION 1 owe amp __TRANSITION 2 50 60 70 Temperature C NC MicroCal MAU120010 REV C Table of Contents Introduction to DSC Data Analysis sisssesssccsceshsvsasesascestsveccesveveacevedvasseseusesievestcevevensansesedencseseusasievbasaseaaneseastanss 1 Getin SUALLE Pee R 3 S VSICDTISCQUIECTHOLDLDS ctio iedc ite DE EedU Lipa a a ieee Ae De pere DIS E 3 Ins talline Orione Sinele User Licens ento tio E ER ent nte e pb A ed mae en om EE 3 Installins Orniem tor DSC Custom DISE eese eran Een evt erc Rcs te pd eae Ro n t ERR RC Un dS 3 Recisterme with OP DID D iocos costo ettet rp pro TOREM ccbdes due Set pA EREN o there edoctus on NARAN 5 SART DIE ESI LE eee ee N E Re eRe ERTS ALOE RT LOOT HUND RI E nN E E eee 5 Iie min E S e esse eerie mtn daas aps e antenna N A E E EE E A ese set cpu ebat dese e DATA I CILE CES uU DIE
43. PC20904003 etc and plotted in the graph Use the magnifying glass tool to rescale the graph around the peaks After rescaling your graph should look similar to what is shown 6l DSC Data Analysis Tutorial Guide 62 Temperature C Subtract Baseline To Subtract a Baseline You may select a baseline to subtract from all scans plotted in the active layer by one of three methods Manually Select File Name or Tray and Well label Each method is described below e Click the Subtract Baseline button select Method Ing xi The Select Method dialog box will open Manually Select File Name When you select Manually Select and click OK the Teme EIS cursor will change into a cross hair You may then double click or single click and press enter on any trace this trace will then be subtracted from all other traces that are plotted in the active layer E File Name Select Method EIE When you select File Name a text box will pop up Meis E ie Es and you may enter the filename of a file the file must have been read into Origin to be used to subtract from all other traces that are plotted in the active layer Note When entering the filename you must File Name 2n s include the dsc file extension C Tray Well Label Using C Manually Select Cancel Lesson 6 Autosampler Data Optional Accessory Tray Well Label When you select Tray Well Label two text boxes will appear one will allow you
44. Rescale to Show All All math editing and fitting operations will be done on the display range only The data outside the display range are not deleted they are merely hidden Use the Reset to Full Range command to bring the hidden data back into view To reset display to full range Choose Reset to Full Range from the Data menu Origin resets the data markers so as to display the entire curve 28 Lesson 3 Integration Lesson 3 Integration If you have created a baseline for a DSC data plot you can select a range of data to integrate Integration yields the following information about the data integration range temperature range area thermal midpoint and width of curve in degrees C at half height In this lesson you will use the Data Selector tool to set the integration range integrate the data copy the integration results to the plot window and plot the integration data Setting Range and Integrating the Data As you saw in Lesson 2 all math editing and fitting operations are performed on the currently selected plot segment or on the current display range This 1s true for integration as well The first step in integration is to use the Data Selector to set the integration range If you do not set the integration range the entire display range will be integrated Before beginning this lesson open a new project e Select the File New Project or click on the New Project button menu item e Click on the Read Data
45. Vcht20b3ds cp 0 00004 i removed High FB 0 00002 Passive 0 00000 O o O o O 0 00002 50 Temperature C Line Types for Fit Curves You may select a line type to plot your data or fit curves from the Plot Details dialog box The Plot Details dialog box is available by double clicking on the data plot right clicking on the data plot and selecting Plot Details from the shortcut menu or selecting the desired data plot from the Data menu data list and selecting Format Plot The Line Group Select the desired line connection from the associated drop down list The line connection type affects interpolation results The default line type for fit curves is Straight line The most common methods of connecting the fit curve data points are straight spline or B spline Straight A straight line is displayed between data points This type of line connection will not give a smooth representation of the fit curve if there are few data points Spline This option generates a cubic spline connection To use the connection the X values must be discrete and increasing Furthermore the number of data points cannot exceed 900 if the data set exceeds this number the operation will fail Since curvature information is held in memory the spline resolution remains the same regardless of page magnification The SplineStep variable in the ORIGIN INI file controls the spline calculation increment It is expres
46. a markers approximately as shown below If you are not sure how to position the markers refer to Lesson 2 in this tutorial 0 0014 Sampleidsc cp SAMPLE1DSC CP BASE 0001 00010 Cpi cal C Temperature C 30 Lesson 3 Integration Double click or press Return to set the markers The area between the markers is the range that will be integrated To integrate the selected range e Since you have created a baseline but have not subtracted it from the data integrate by choosing Integrate from Baseline from the Peak Menu If you had subtracted the baseline from the data you would integrate by choosing Integrate from 0 from the Peak menu The area between the two data markers is integrated The integration results copy to the Script Window Note The Script Window may hard to find It may be located at the bottom edge of the work area with just the title bar showing or be hidden You may show hide the Script Window by either holding down the Alt key and pressing the number 3 key not on the key pad or by clicking the Script Window button located on the standard toolbar H ampleidse_cp S leid SAMPLE1DSC CP BASE N M ax LETT ax Window Fle Tex Edit Hide Integ of Sampleidsc_cp from SAMPLEIDSC_CP BASE i 8b T Range 47 881H3 5 73 6060256 Area 4 60789 Tm 61 H8183 T1 2 8 885949 Temperature C If you wish you can reset the markers and try again Each time you integrate the
47. ansition might also be seen at either T or T depending on whether P or P is in excess 1 e in autosampler routine the program will have to determine the largest peak If Tm occurs at a temperature between T and T then the regular model for ligand protein may be used to calculate the binding constant If Ty occurs at a temperature higher than both T and T then a different model must be used which takes into account the temperature shift in both transitions In this latter case the binding constant at Ty Kp Ty can be obtained from the following equation AHS 1 1 a Ty n La SH expl p un x dnm tz lx dm nd AC Tw To lin pb e i o Tw Ku 32 eq where Por K oq d ae when Ec 2 33 K P _ Fo when P x P eq tot 2 tot tot 102 Appendix Equations Used in Deconvolution VII Using DSC data to calculate AG at temperature T by extrapolating from the midpoint Tm From analysis of a DSC scan on a protein three experimental parameters for the unfolding transition are obtained These include midpoint Ty Enthalpy change at Ty AH Ty Heat capacity change at Ty AC Using these three experimental parameters the Gibbs free energy change AG may then be obtained at any temperature T as indicated below The derivation assumes a reversible two state transition and also assumes AC is independent of temperature Derivation Starting from well known thermodynamic relation and transforming to
48. asdsc Abst p Subtract Reference Normalize Concentration Absolute Cp cal gnvdeg Temperature C 3 Now hold down the Ctrl key and press the tab key once to switch back to the RawDSC window 4 Click on the next data set in the legend to make it the active data set Vehtldbsdse cp VehtlZbsdse cp Vehtl3bsdse cp Vehtl5bsdse cp V cht2bsdsce cp 5 Click on the Absolute Cp button again Repeat steps 1 4 for each data set till you have completed the data conversion for all 5 data sets Your graph should now look like below Mcht15bsds AbsCp Mcht14bsds AbsCp Mcht13bsds AbsCp Mcht12bsds AbsCp Mcht9bsdsc AbsCp Absolute Cp cal gm deg 40 50 60 70 80 90 Temperature C Lesson 5 Curve Fitting You may wish to expand the view around 25 deg and add some grid lines Vchti5bsds AbsCp Mcht14bsds AbsCp Vcht13bsds_AbsCp Vcht12bsds_AbsCp Veht9bsdsc_AbsCp O o O O O Q O o 2 O ip Q lt 24 0 24 5 25 0 25 5 26 0 26 5 Temperature C 57 DSC Data Analysis Tutorial Guide This page was intentionally left blank 58 Lesson 6 Autosampler Data Optional Accessory Lesson 6 Autosampler Data Optional Accessory The Autosampler is an optional accessory for use with the Capillary DSC to allow multiple unattended scans There a
49. cal mole C 50 60 fall Temperature C It is important to remember that data must always be normalized on concentration before doing curve fitting to any of the fitting models Step 2 Plot a baseline As we have discussed elsewhere in this tutorial the next step 1s to plot a baseline for the data Note that the SAMPLES plot shows flat baseline segments on either side of the transition with a AC in between The transition itself actually starts and ends at about the positions shown by the arrows below Cp kcal mole C 5 B 70 Temperature CC To create a good progress baseline for this data you may wish to manually adjust the linear baseline segments so that they are parallel to each other and coincident with the baseline data Origin does not use the position of the endpoints of the linear segments to calculate the progress baseline only the slope and offset You are thus free to create 47 DSC Data Analysis Tutorial Guide linear segments of any length which makes it much easier to align the segments parallel to each other To create the baseline 1 Select Peak Start Baseline Session Origin calculates and draws the two linear baseline segments faintly visible in the figure below mn _ e z o O w o Q O 50 60 70 Temperature C 2 Select Adjust Move Segments by Cursor 3 Beginning with the left linear segment click and drag on first the left and then the
50. ckly removes small amounts of response time broadening for any of the four response times This post run capability sometimes avoids the necessity of introducing extraneous noise associated with faster response times as well as allowing the operator to quickly determine if peak resolution is satisfactory for any data set Whenever peak broadening is severe however this post run correction will not be completely accurate To illustrate the functionality of the Response Time button we have included the Origin project ResponseT opj in the C Origin70 Samples directory This Origin project contains the results of two successive experiments run on a sample at 60 deg hr The first experiment was scanned using the Passive FeedBack mode the scan was repeated using the High Gain Mode The data was scanrate normalized and a baseline has been subtracted from each trace Select File Open e Navigate to the Origin70 Samples directory double click the project name ResponseT opj The project will open as shown below iid Microcal Origin C OriginData ResponseTime Responsel RawDSC File Edi View Graph Data Analysis Tools Format Window Help 8 x cc ca al Ha BS A pen Aea t me ESI Eng Ven2on2ds_cp Vcni20b23ds cp NNN Subtract Reference T zi pe 0 00002 rom Passive 0 00000 Cp cal C Absolute Cp 0 00002 40 45 50 55 B Temperature C a s r 7 s n o EREEREEEEEEEEEEE
51. e Getting Started Choose Folder X Please choose location of next disk Path Je customdisk Cancel Directories CG fitfunc 3 origine C samples Drives zm e nain Network Registering with OriginLab OriginLab Corporation a separate company from MicroCal LLC produces and supports the Origin software package MicroCal LLC produces and supports the calorimetric fitting routines imbedded in the Origin for DSC and Origin for ITC packages MicroCal LLC will provide technical support for all aspects of the software without registration OriginLab will not provide technical support for the calorimetric fitting routines but if the copy is registered will provide standard technical support for the general purpose routines of the program Upon receipt of Origin please fill out and return the registration form included with your package to OriginLab You may also register at any time by contacting the Customer Support Department at OriginLab Starting Origin To start Origin double click on the Origin 7 0 program icon on the Desk Top Alternatively click Start then point to Programs Point to the MicroCal Origin70 folder then click on the MicroCal LLC DSC program icon from the submenu Menu Levels This DSC version of Origin comes with a minimum of three distinct menu configuration options or menu levels there are other levels available if you purchased the optional PPC so
52. e Test Edit Hide AStart EVABRE RKO Chi Sqr 1 Iter 100 Iter 100 Simplex Iter l4 cS Eudora In Gy Exploring zi Origin 7 f untitled Paint Mau 2001 38 A 9 16AM Click the Chi Sqr button to display the reduced chi 2 value for the current parameter values The reduced chi 2 value is displayed in the view box at the bottom of the dialog box This feature allows the user to change the values of the parameters 1 e Tm AH to see an update of the fit using the new parameters This option can also be used for a quick simulation of curves Click this button to perform one iteration of the Levenberg Marquardt LM routine At the end of the iteration the fit curve fitting parameters and Chi 2 are updated same as above but performs up to 100 LM iterations The number of Iterations can be changed in the Advanced Mode fitting sessions window If you are in the Basic Mode click on the More button and selecting Options Control Then change the Max Number of Iterations from the drop down list box Click this button to perform up to 100 Simplex iterations Normally you will not need to do this because the Simplex method is much less robust and reliable than the LM method However if the LM method for a set of data behaves poorly try the Simplex method 2l 22 DSC Data Analysis Tutorial Guide Basic Click this button to switch from the Advanced Mode of the curve fitting dialog box
53. e active layer as can be seen in the first and second scans of the table below Lesson 6 Autosampler Data Optional Accessory Filename Tray Well Conc 1 Conc 2 Tmi Comments 5c20904014 DS c20904015 DS C302 03 EE EN oo T J 1 7 909 RHnaseonly no ligand 1 9 0009 X 10 827 Rnase Phosphate 1 n 9009 10 86657 RHNase 3 CMP 1 13 8909 10 703 RNAse CMP 1 15 1009 410 8097 nase only no ligand EE ae od i aes C3 C3 c3 c3 17 009 10 582 63 M Hnase Phosphate 19 009 10 8665 BRHNase 3 CMP 1 009 00 7038 RHN se 2 CMP 23 0 09 10 680 93 Rnaseonly no ligandi TEESE C C3 62 75 Hnase Phosphate og 10 2a 009 10 29 oog 10 l 0 35 HMAse 2 CMP Conc I amp Conc 2 Conc 1 is the concentration mM of the protein in the cell Conc 2 is the concentration mM of the ligand in the cell These values are usually entered into the data files when setting up the experiments but may be edited in the table e Select the menu item Windows ARawDSC to return to the ARawDSC window At this point you may wish to save your project for use later in calculating binding constants Find Delta Tm To Find Delta Tm Y ou may select a scan to represent as a reference Tm to calculate the shift of Tm s for all other scans select MEN a i that are plotted in the active layer Using C Manually Select
54. e equation relating the shift in midpoint to the binding constant Kg has been derived earlier Brandts J F amp Lin L N 1990 Biochemistry 29 6927 6940 and is exp mcd PEN AC piu 40 4 nR VT d nR o us K a e 29 T M where K Ty is the binding constant at Tm AH and AC are the enthalpy and heat capacity change for the transition at T p M is the concentration of unbound ligand at Ty and n is the number of binding sites on the protein Transition temperatures are expressed in K The above equation is most useful when Ty and To are well separated in temperature A very large Kg insures there will be large separation in midpoint no matter what the ligand concentration while measuring Ty in the presence of high ligand concentration will produce large separation even for weak or moderate binding In the former case involving large binding constants addition of less than saturating ligand concentration results in separate transition peaks for the liganded and unliganded proteins so that T and Ty may be obtained in the same experiment The value of free ligand concentration at the midpoint Ty can be calculated from the equations P IL l x Es vx Lor a nP 30 El ait Us S nP 2 where Piot and L are the total concentrations of protein and ligand respectively in the solution While ligand release will occur at Ty and contribute to the total experimental heat of unfolding it is generally not p
55. e for the cooperative unit which actually participates in the reaction Substituting from eq s 3 5 into eq 2 then gives 95 DSC Data Analysis Tutorial Guide 96 K AC K AH AH Cet E pA a 6 I K 1 K RT Eq 6 is perfectly general at this point and can be applied to either two state or non two state transitions so long as all parameters are evaluated at the same temperature T Model for Independent Two State Transitions including ACp Effects If we assume each transition is two state then all of the above van t Hoff AH values become equal to the calorimetric AH values If we further assume that C can be expressed as a linear function of temperature Con Bo Bi T then eq 6 becomes K T AC K T AR TY CADE Pot BPH GT RP p 7 where the temperature dependent parameters CT K T and AH T have been indicated We can express AH T in terms of its temperature independent value at the midpoint T a and the heat capacity change for the transition AC Abe AH T AH AC T T 8 and then integrate eq 5 from T where K T is unity to an unspecified temperature T AH T NT KAT een a eem an l 9 1 exp eae e rn e mA Now eq 7 using substitutions from eq s 8 and 9 for each transition A B may be used to calculate the value of the system heat capacity C T at any temperature T once values of the temperature independent fitting parameters Bo B1 T na AH ya AC As Ta
56. eak while the van t Hoff heat H is determined only by the shape of the transition peak The sharper the transition the larger is H and vice versa The relationship between H and H can sometimes provide insights not accessible from H alone For example if a protein is composed of two identical domains which unfold independently with the same Tm and H then the ratio of H H will be 2 0 while it would be 1 0 if the protein had only a single domain If on the other hand the protein dimerized and the dimer underwent only a single coupled transition then the H H ratio would be 0 5 It is clear from this that the calorimetric heat H refers to heat change per mole while H 1s heat change per unfolding unit called the cooperative unit Thus the ratio H H can in simple cases be thought of as the number of cooperative units per mole Note It is important to realize that the ratio of calorimetric to van t Hoff heat depends on concentration normalization since calorimetric heat is always expressed on a per mole 45 DSC Data Analysis Tutorial Guide 46 basis Thus for the protein dimer example considered above the ratio will be 0 5 if concentration normalization is carried out on a per mole of monomer basis while it will be 1 0 if concentration is entered in terms of moles of dimer present In model 1 it is possible to prescribe that overlapping transitions are either independent or sequential in nature For example if two structural do
57. ed as rescans in VP Viewer when the experiment was run had a sequential alphabet character 1 e a b c etc appended after the scan number When you select the Files of type to be Auto Rescans dsc then select the first file of the series 001 DSC for file import all files of the series will be read into Origin and plotted To open serial Auto DSC 001 DSC files e Click on the Read Data button The Import Multiple ASCII dialog box opens Select Auto DSC Data dsc as the Files of type Only DSC files that have an 001 before the extension dot will be listed e Navigate to the C Origin70 Samples folder then select PC20904001 DSC from the files list Please Note Raw data file names should not begin with a number nor should they contain any hyphens periods or spaces Note You may select a default folder for Origin to Look in for a data file by selecting File Set Default Folder and entering the default path e g for this tutorial you may wish to choose the path to be C Origin70 Samples Import Multiple ASCII EIEI Look in E Samples ES je e Programming 2 AS2PeaksO01 dec File name pc20904001 dec Add File s OF Files af type Auto DSC Data dsc Remove Files Cancel File Mame pe20904061 dec e Select the PC20904001 DSC file and click Add File s e Click OK The PC20904001 family of files is then read in sequentially 1 e PC20904001 PC20904002
58. en setting up the experiment and stored in the data file header You may verify the concentration values in the summary table Conc 1 and correct them in the table if necessary To view the summary table click the Window menu item and select MicroCal VP DSC Autosampler Scans Summary Table from the Windows list The summary table will be displayed similar to what is shown below i e without the Tm1 entries you may edit any of the entries for Conc 1 Select Window ARawDSC menu item then click the Normalize Concentration button from the Auto DSC group of buttons All data scans will be normalize on the protein concentration conc 1 the y axis scale will change to kcal mole C and the data will be replotted Note Normalization on Concentration is applied to all data files not just the data plotted in the active layer For baselines and other scans where the value for Concentration 1 may have been entered as zero Origin will insert a one 1 for the value to prevent division by zero 0 Find One Tm To Find One Tm Click the Find One Tm button The program will find one Tm for all scans plotted in the active layer then display a summary as shown in the table below Note Ifa scan was not plotted in the active layer in this case the baseline pc20904003 DSC was subtracted and automatically removed from the active layer then a Tm will not be listed The program will try to find a Tm for baselines if they are plotted in th
59. ent For example you could delete the segment To delete the selected plot segment Choose Cut or Clear from the Edit menu or press the Delete key The selected plot segment is deleted from the plot window The selected values are also deleted from the SAMPLE2 worksheet The data markers are removed You may select Edit Undo to replace the data 1 Sample2dsc_cp O o wm Oo Sa O 40 60 80 Temperature C Data markers are only meant to be displayed temporarily They will disappear as soon as another data set or plot window becomes active To select a segment of data for a longer period of time use the Set Display Range command Say that you would like to display or manipulate a central range of data in the SAMPLE2 data plot but you do not want to delete any of the data This is an ideal use for the Set Display Range command To set data plot display range Click on the Data Selector tool click on the right data marker and position it as shown below Then click on the left data marker and position it as shown 24 DSC Data Analysis Tutorial Guide L Sample2dsc cp 40 60 80 Temperature C Double click or press Return to set the markers Choose Set Display Range from the Data menu The data outside the selected range are hidden from view L Sample2dsc cp Cp cal C 40 60 80 Temperature C You may expand your data to fill the graph by selecting Graph
60. er macromolecule is composed of a number of structural domains A B C each of which is involved independently in a transition between the folded and unfolded forms A A B B Equilibrium constants will be expressed as fractions KA fA f A Kg fp fg with the usual designation for the actual 1 e calorimetric molar enthalpy changes AH AH as so The total molar enthalpy of the system H will be H H def AHU E Sf UNT Ta 1 The first term for the totally native form where all domains are in their folded state Hy must be included since all AH values are measured relative to the folded form The total molar heat capacity of the system Cp is the temperature derivative of the enthalpy in eq 1 SO C Cp FedCy art Ss b 2 where C yn is the molar heat capacity of the totally folded state AC is the change in heat capacity for unfolding the A domain and where the term in brackets will be repeated for each domain involved in unfolding Remembering that f 1 fa then the fractional concentration of the primed species can be readily expressed in terms of the corresponding equilibrium constant K 1 K 3 fa which can then be differentiated to give a K 2 7 oT 1 K J OF The derivative on the right of eq 4 is known from elementary thermodynamics un AH i5 OT RT where AH i is the so called van t Hoff heat change for the reaction which corresponds to the heat chang
61. ever memory is running low To access the worksheet of integ area data or use it for any other purpose you should first copy integ area to a permanent data set To copy the integration area data to a new data set Click on the Edit menu in the Script Window menu bar Note that the Script Execution command is checkmarked which means you can currently execute LabTalk scripts directly from the Script Window Onanew line type the following script exactly as it appears Be sure to include the single space before integ area and also before my test Please note that the data set name must consist of two parts separated by the underscore character The first part will be the worksheet name in this case the worksheet will be named my the second part will be the column name of the data set copy integ area my test Press Return Origin should append a semicolon to the text you just entered This indicates that the script was executed For more information about the Lab Talk scripting language please refer to the LabTalk manual or select the online Help Programming LabTalk Reference menu option My test is now an Origin data set that can be plotted and manipulated like any other data set To plot the my test integration area data e Select File New Graph to open a new plot window The Graph1 plot window opens Double click on the layer icon in the Graph1 plot window The Layer Control dialog box opens Click on my test in
62. f each of the baseline data points To use the Move Baseline by Cursor option first create a baseline by selecting one of the baseline creation commands described above Next select Move Baseline by Cursor from the Baseline menu Origin places twelve data points along the baseline you just created To move the data points either click and drag with the mouse or use the left and right arrow keys to select a point and the up and down arrow keys to move the selected point points can only move vertically When you are satisfied with the baseline double click or press Return A spline connection option is automatically applied to the new baseline You can remove the spline connection by changing the baseline s plot display options see page 42 below Lesson 4 Baseline Determinations Cursor Draw Baseline Cursor Draw Baseline is another method for creating a baseline The Cursor Draw Baseline method does not require you to enter the baseline session set linear segments or select a baseline option In this method you simply draw the baseline directly in the plot window To use the Cursor Draw Baseline option If you have not yet exited the baseline session do so now by clicking OK or Cancel in the baseline session menu bar Select Cursor Draw Baseline from the Peak menu in the standard menu display bar The cursor changes to a small cross hair Double click with the mouse to place each baseline point Each point connects to the
63. f you are unfamiliar with the basic operation of Origin you may find it helpful to read through OriginLab s Origin Getting Started Manual particularly the introductory chapter Note that this DSC tutorial contains information about Origin only in so far as it applies to DSC data analysis If you have questions or comments we would like to hear from you Technical support and customer service can be reached at the following numbers Toll Free in North America 800 633 3115 Telephone 413 586 7720 Fax 413 586 0149 Email info microcalorimetry com Web Page www microcalorimetry com Getting Started Getting Started In this chapter we describe how to install Origin on your hard drive how to configure Origin to include the DSC add on routines and how to start Origin System Requirements Origin version 7 requires the following minimum system configuration e Microsoft Windows 95 or later or Windows NT version 4 0 or later e 133 MHz or higher Pentium compatible CPU e 64 megabytes MB of RAM e CD ROM drive e 50 MB of free hard disk space e Internet Explorer version 4 0 or later we recommend version 5 0 or later Internet Explorer need not be your default browser but it must be installed for viewing Origin s compiled HTML Help Installing Origin Single User License Note We recommend that when installing Origin you do not accept the default directory but choose the folder C Origin70 To install a new copy
64. from your hard copy device Exact font placement and size is guaranteed at some sacrifice to screen appearance since the printer driver fonts must be scaled to fit their positions on the page this will not harm the appearance of true vector fonts This 1s a slow process and screen refresh speed suffers as a result Thus reserve the Print View mode for previewing your work prior to printing Origin automatically changes to Print View mode when graphics are exported to another application and when printing The view mode automatically returns to the selected view mode after the operation is complete Page View provides faster screen updating than Print View but does not guarantee exact text placement on the screen unless you are using typeface scaling software such as Adobe Type Manager Use Page View mode until your application is ready for printing or copying to another application Change to Print View mode to check object placement before exporting copying or printing Window View expands the page to fill up the entire graph window Labels buttons or other objects in a graph window that reside in the gray area of the page are not visible in Window View mode Draft View has the fastest screen update of the four view modes In Draft View the page automatically sizes to fill the graph window This is a convenient mode to use when you are primarily interested in looking at on screen data Note that view mode will not affect your prin
65. ftware the Autosampler DSC software or the ITC software Each menu level has its own distinct menu commands After Origin has opened you may change a menus level option under the Format Menu command option The seven menu levels are DSC Data Analysis Tutorial Guide General Full Menus Select this option to run Origin in the generic non instrument mode This menu level contains no instrument specific routines but does contain many general data analysis and graphics routines that you may find useful for other applications DSC Data Analysis Select this option to run Origin in a configuration that includes the instrument specific DSC data analysis routines Auto DSC Data Analysis Select this option to run Origin in a configuration that includes the instrument specific autosampler with DSC data analysis routines for handling multiple data files PPC Data Analysis Select this option to run Origin in a configuration that includes the instrument specific PPC data analysis routines Note that this menu level is available only if you purchased the optional PPC attachment for the DSC ITC Data Analysis Select this option to run Origin in a configuration that includes the instrument specific ITC data analysis routines Note that this menu level is available only if you purchased the optional ITC software module Auto ITC Data Analysis Select this option to run Origin in a configuration that includes the instrument specific autosample
66. he progress baseline The actual end points of the linear segments are not used During the calculation of a progress baseline Origin integrates over the entire display range of the active data set If you prefer you can restrict the region of integration to just the peak area After adjusting the low and high temperature linear baseline segments use the Data Selector tool described in Lesson 2 to set the region where integration should be carried out If you now select the Baseline Progress Baseline command integration will be restricted to the region that lies between the two data markers Linear Connect This baseline is formed by connecting the two linear segments with a straight line as shown below Whereas the progress baseline uses only the linear segment slope and intercept to form the baseline linear connect uses only the position of the two end points 41 DSC Data Analysis Tutorial Guide 42 Cubic Connect similar to Linear Connect except the connection is a cubic polynomial The end points are used to create the baseline as with Linear Connect but with Cubic Connect the slope is used also Step Baseline There are two step baseline options Step at Peak is based on a linear connect baseline and places the step directly under the peak Step at Half Area places the step at the position of half the integrated area Move Baseline by Cursor This option lets you manually adjust the baseline by specifying the position o
67. his lesson we will create a baseline for the SAMPLE1 DSC data file To open the SAMPLEI data file Shortcut You can program Origin to always Look in the Samples folder or any other folder by selecting File Set Default Folder and entering a new path in the text box e If you are continuing from a previous lesson first select File New Project or click on the New Project icon to open a new Origin project Click on the Read Data button in the RawDSC plot window The Import Multiple ASCII dialog box opens Double click on Samplel1 dsc in the Files list SAMPLE1 DSC is located in the Samples sub folder of the Origin70 folder The path and file name will be added to the lower list box Click OK SAMPLE1 opens into the RawDSC plot window Sample1dsc cp 60 Temperature C 37 DSC Data Analysis Tutorial Guide Before creating the baseline you would normally subtract reference data and then normalize the data on concentration For the sake of brevity we will bypass those steps here They are discussed in detail in Lesson 1 Basic DSC Data Analysis To start the Baseline Session Choose Start Baseline Session from the Peak menu Origin enters the baseline session and automatically analyzes the data to determine the linear segments to be drawn on either side of the peak fay Microcal Origin UNTITLED NormDATA OK Adjust Baseline Cancel Window x j ce es E S amp Cp kca
68. independently we will extrapolate the Kg value to another temperature Tp e Return to the ARawDSC window z e Click the Binding Constant button from the standard DSC group of buttons Del The Binding Constants from DSC dialog box will pop up Binding Constants from DSC X Dg X C Protein protein interations Tox Tm T o C Protein protein interactions T mo T a T o Cancel Select the Ligand protein interactions radio button and click OK The Ligand Protein Interaction dialog box will open up e Enter the following values in to the Ligand Ligand Protein Interaction Protein Interaction dialog box as determined in E WE the preceding pages Identification Protein Ligand Binding Constant Parameters T 61 C To L Tm 66 57 C p n 1 AH at Ts 110000 cal mole n sites per male ACp 1100 cal mole deg Protein Conc 00009 moles l AH at To 110000 cal mole Ligand Conc 01 moles l ACp 17100 Reece Protein Conc 4 000045 moles l Ligand Cone Dl moles e Click OK The binding constant at Tm will be calculated and Cancel the Binding Results dialog box will open listing the entered parameters and the calculated Kg 71 DSC Data Analysis Tutorial Guide Binding Results You may print the results and or extrapolate the Kz to a different temperature 72 Lesson 6 Autosampler Data Optional Accessory To extrapolate the binding constant to a different temperature
69. ine straight step from the Connect drop down list box Click OK Each time you create a baseline Origin names the baseline data set and appends a column to the active dataset worksheet for the baseline data The baseline data set 1s given the name NAME BASE NAME DRBASE in the case of Cursor Draw Baseline where NAME is the name of the data set for which you created the baseline To see the baseline data open the associated worksheet To open the worksheet containing baseline data S AS ho Double click on the baseline to open the Plot Details dialog box or select the baseline Worksheet Right name from the Data menu then select Plot from the Format menu to open the Plot click anywhere on the type dialog box data trace and select Open Worksheet from Click on the Worksheet button the drop down menu The baseline worksheet opens with the baseline data set named CP BASE Y To subtract the baseline from the DSC data e Select Subtract Baseline from the Peak menu The baseline is subtracted 44 Lesson 5 Curve Fitting Lesson 5 Curve Fitting In Lesson 1 we discussed Origin s basic curve fitting procedures Here we will look at the curve fitting process in greater detail In the first section below we will make some general observations about the curve fitting models used in Origin Following that we will work through two fitting examples to illustrate some of the flexibility of the curve fitting process General C
70. ing the entered parameters and the calculated Kg You may print the results and or calculate the Kg at a different temperature Binding Results x X Results for Protein Protein Binding Constants fe 2 Sele I gL Pe OC Input Parameters o sm im siens s AH 150000 cal male ACp 2000 cal mole dedg prot 4E 5moles l Cliq 46 5 moles Calculate Kg at a different temperature Te E Print Close Protein Protein Binding Constants Ty gt T To If Ty occurs at a temperature higher than both T and T then a different model is used which takes into account the temperature shift in both transitions To calculate the binding constant at Ty Kg Ty when Ty gt To To e Launch a new instance of Origin for DSC Note Normally you would calculate the parameters ACp AH T for each protein and the Tm for the complex Since the method of these calculations has been described on pages 78 80 we will simply provide the parameters below Binding Constants from DSC fa w x e Click the Binding Constant button and the DSC Binding Constants from DSC dialog box will open up C Ligand pratein interactions C Protein protein interations Ta Tmz To e Click the Protein protein interactions Tm gt To To radio button then click OK The Protein protein interactions dialog box will open up as shown below e Enter the following values 84 Lesson 7 Other Useful Details Protein Protei
71. is Note that NormDATA contains two buttons which are used in the subtract baseline procedure Creating a Baseline The final step before deconvolution is to start the baseline session and plot a baseline for your data You will then decide whether or not to subtract the baseline and based on that decision choose from one of three curve fitting models To start the baseline session Choose the Start Baseline Session command from the Peak menu Based on the normalized data Origin calculates and plots in red the left and right linear line segments from which to determine the baseline A new menu bar appears containing the baseline session command menus fay Origin 7 UNTITLED NormDATA E OK Adjust Baseline Cancel Window lj x Sample1dsc cp Cp kcal mole C 50 60 TO Temperature C x UNTITLE Name Type View EX 2d 1 TUM Tm Script W cript x pleidsc cp 1 283 INomDATA NUM A Start e 57 wm RS Ko Il 6 icin 7 3 Eseloing f MAUT200 Me untitled Mictocal Fes Origin 7 ya 9 42AM Origin lets you adjust the left and right linear segments and provides several options for connecting the segments to create the baseline For this example we will simply accept the 17 DSC Data Analysis Tutorial Guide default segments as plotted and connect them using a progress baseline For greater detail see Lesson 4 Baseline Determi
72. is allows software control of the gain the ratio of power applied to the sample cell relative to the magnitude of the AT signal which in turn controls the response time of the instrument to heat effects occurring in the cells The passive mode with zero gain is best to use for studying systems having very slow transients broad transition peaks slow scan rates or isothermal studies since the baseline is quieter and more stable due to the absence of feedback noise For systems with fast transients sharp thermal transitions and or fast scan rates shorter response times must be used to avoid distorting the shape of the Lesson 7 Other Useful Details thermogram even though this will result in lower sensitivity In the current version of VPViewer there are four response time selections in the experiment set up window No Gain Passive Low Gain Mid Gain and High Gain with respective relaxation time constants of 40 9 18 0 10 4 and 5 6 seconds The respective designations in the header of the data files are 0 Passive 5 Low Gain 75 Mid Gain and 1 High Gain Some protein transitions as well as those of polynucleotides are fairly sharp on the temperature axis so that detectable peak broadening may result when using the passive mode at usual scan rates In such a case faster response times or slower scan rates may be used to improve peak resolution in real time However Origin contains a Response Time button which when activated qui
73. l to convert from cal degree to cal mole degree Concentration normalization is always carried out after subtracting the reference data To normalize Sampleldsc by concentration Click on the RawDSC window to make it active or select RawDSC from the Window menu Click on the Normalize Concentration button A dialog box opens Enter the sample concentration in mM and the cell volume in ml For this example enter 0 08 into the Concentration mM text box Samplel dse was at a concentration of 80 uM and 1 12 in the Cell Vol ml text box The concentration will automatically appear if you originally entered it into the header of the raw data file For the VP DSC Cell volume is set in the VPViewer ini CELL1 CON for the MCS and CALDSC dat for the MC 2 file in the folder containing the data acquisition program and is stored in the header information of each data file calfdeg gt cal deg M for OK sampleldsc cp Cancel Concentration mM 0 08 Cell Vol ml 1 12 Click OK The Sampleldsc cp data plot is removed from the RawDSC plot window RawDSC closes and a new plot window NormDATA opens The normalized data are plotted as Sampleldsc cp in the NormDATA plot window EF Ongin 7 UNTITLED HormDrAT A Sampleldsc_cp Un Cp kcal mole C EET m EN F AMAA lpas oa NM M sue EHAS AS FED pner Gyr Eua junii vices Fon 7 EEEE Lesson Basic Data Analys
74. l binding constants that exceed this value will be highlighted in the summary table To input the data Raw data files from the autosampler are typically a Files of type Auta DSC Data dsc series of data files of the form Auto DSL Data dec FamilyName001 DSC FamilyName002 DSC DSCData dsc FamilyName003 DSC etc The family name may Auto Hescans have up 9 alphanumerical characters VPViewer adds the sequential 3 digit number therefore there may be a maximum of 12 characters for the filename To open the file series click on the Read Data button and the Import Multiple ASCII dialog box will open Select the Auto DSC Data dsc as the Files of type the upper File Name list box will list only the first file of the series 001 DSC but when selected for import all files with the same family name except for rescans see below will be read in until the end of the series 1s reached 60 Lesson 6 Autosampler Data Optional Accessory Alternatively you may open individual files or a selection of files similar to the normal DSC method When you select the Files of type to be DSC Data dsc then all files with the DSC extension will be listed in the upper list box You may then manually select the files to add to the lower list box and read into Origin A third option will allow you to read in all data files with the same family name including rescans Note Scans that were designat
75. l mole C 15 ic C Sam pleidsc_cp E o Temperature c Malte riz Lol alel For Help press F1 Sampleldsc cp NomDATA 2 Note that a new command menu bar now appears at the top of the project These command menus provide complete control over the baseline determination process as follows OK Accepts the current baseline provides an opportunity to subtract this baseline from the data curve and then exits the baseline session Adjust Allows the user to adjust the two linear segments Baseline Choose one of five options for creating a baseline or draw a baseline manually Cancel Exits the baseline session unconditionally The current baseline if any is deleted If you are having difficulty seeing the linear segments you can maximize the plot window for a better view To maximize the RawDSC plot window e Click on the Maximize button at the upper right corner of the window The window zooms to full size 38 Lesson 4 Baseline Determinations User Adjustment of Linear Segments If you are not satisfied with the two automatically determined linear segments use the Adjust menu to change the segments as you see fit The Adjust menu provides two options refit the segments or move the segments by cursor To refit the linear segments e Choose Refit Right Segment from the Adjust menu Baseline Cancel Mind Refit Left Segment Refit Right Segment Move Segments by Cursor Click the cr
76. ld be to two transitions so enter a 2 and click OK 3 Double click on or near each peak to set a Tn thermal midpoint for each transition As soon as you set the Tm s Origin initializes the fitting parameters in this case T4 Hi Ty H5 displays the parameters in the Fitting Session dialog box and draws an initial fit curve An Attention dialog box notifies you when initialization is complete Click OK to proceed with the fitting NonLinear Curve Fitting Fitting Session Category Function Action Options Scripts Ess 4 The 1 Iter and 100 Iter commands let you control the iteration of the fitting cycles Click on the 100 Iter button a couple times Origin iterates the fitting cycle until the change in Chi 2 between two successive iterations is less than a pre set value this value is determined by the value set in the Tolerance Text box of the Control Parameters dialog box MicroCal has set the default value to be 10 Select 100 Iter once or twice more At the end of each fitting cycle Origin updates the Chi 2 and fitting parameters values and draws a new fit curve By now you should have a pretty good fit to the 70 degree peak but the fit to the 60 degree peak is not so satisfying At this point you can begin to think about improving the fit by adding complexity to the model Step 4 Add complexity You did not get a good fit using model 1 with two 2 state transitions While the high peak fit p
77. le subunits rather than four 53 DSC Data Analysis Tutorial Guide Fitting Example 3 The Absolute Cp button will facilitate calculation of Absolute heat capacities for proteins this is discussed in more detail in Appedix IV starting on page 100 To illustrate the function of this button there are 5 raw data files of chymotrypsinogen solutions at five different concentrations covering a 10 fold range from 0 258 to 2 58 mg ml Step 1 Input the data The 5 raw data files used for this example are Vcht9bs dsc Vcht15bs dsc Vcht12bs dsc Vcht13bs dsc and Vcht14 dsc The concentration of the sample in each scan is 258 516 1 03 1 55 and 2 58 mg ml respectively The samples were run on a VP DSC at 60 deg hr using the passive response mode for each scan A buffer buffer reference trace has already been subtracted from each data scan 1 Click on the Read data button in the RawDSC window navigate to the C Origin70 Samples folder and open the Vcht9bs dsc Vcht12bs dsc Vcht13bs dsc Vcht14bs dsc and Vcht15bs dsc data files Import Multiple ASCII Fa Look in E Samples E El ce 3 pc20904008 dec pc 0S0401 4 dec Sample5 dec E pc20904009 dse BS pe20904015 dec B Vchtl2bs dsc E pc20904010 dsc B Ref dec I3 vchtl 3bs dec E pc2Z0904011 dsc Z RefS dsc 2 vchtl 4bs dsc 3 pc20904012 dec Samplel dsc Vchtl5bs dsc 3 pc20904013 dsc Sample2 dsc VchtSbs dsc File name Vchtl5bs dsc Vchtl2bs dsc f
78. ll models except model 4 offer three menu selection options model 4 offers only a Cursor Init option 1 Cursor Init allows cursor initialization of each transition T m s 2 No Init accepts the previously defined parameters if any 3 Simulate allows you to create a transition by entering user defined parameters Tools Format Window Help Set Indep Sequ Model Indep State Cursor Init State Mo Init State Simulate Man 2 State Cursor Init Non 2 State Mo Init Man 2 State fram State Maon 2 5tate Simulate z State we d Cursor Irit z State w dCp No Init Shate w dLp Simulate Dissociation wid p Cursor Init We will be using only the Cursor Init options in this lesson In the previous section you subtracted a baseline from your normalized data Models 1 and 2 require you to subtract the baseline before fitting while model 3 requires that you NOT subtract the baseline Model 1 is the simplest model so let s try fitting to this model first 19 20 DSC Data Analysis Tutorial Guide To fit a curve with the 2 state model model 1 Select 2 State Cursor Init from the DSC menu Origin integrates the Samplel data then opens a dialog box asking you to enter the number of transitions You will fit this data with a single Tm so enter a 1 and click OK EN QE Number of peaks I Cancel e Double click to set the Tn for this peak Or click once use the arrow keys to
79. ls described 19 45 46 fitting session 20 22 formatting parameters text 23 Line types 91 92 sequential model equations 97 99 Two state model 20 22 Two state w dCp model 22 23 D Data files automatic scan rate normalization 10 keeping time data 10 offsetting data 10 opening 11 13 Data Selector tool 25 28 hiding data markers 34 setting integration range 29 31 70 79 Data set create baseline for 17 18 create new data set 34 deleting selected segment 27 fitting a curve to 19 23 importing 7 integrating 29 35 normalizing 15 17 opening worksheet for 14 15 44 plotting 34 35 set as active 13 subtracting reference 13 14 Deconvolution See Curve fitting Delta G 84 87 Dissociation w dCp 51 53 E EndInteg macro 31 F Fitting See Curve fitting Fitting parameters defined 45 46 for four models 19 format parameters text 23 Fitting Session 20 22 H H parameter heat change 45 46 Hv parameter van t Hoff heat change 45 46 I Import Multiple ASCII dialog box 11 12 105 DSC Data Analysis Tutorial Guide Importing data 7 Installing Origin 3 Integration 29 35 copy integration area data 34 displaying integration results 32 EndInteg macro 31 formatting integration results 32 33 integrating selected range 31 integration results 29 31 plot integration area data 34 35 setting integration range 29 31 Isothermal scan 7 L
80. m The area of the peak between the markers will be determined and listed in the Script Window For this example the area was found to be equal to approximately 110 000 cal mole Your value may be different depending on where you placed the lower and higher temperature baseline segments during the baseline session Note The script window may be hidden near the bottom edge of the workspace if the Script Window is not open you may open it by clicking on the Script Window button located on the standard tool bar Ey 79 DSC Data Analysis Tutorial Guide To find the peak temperature Shortcut You may make a data set the active data set by clicking on the plot type icon in the legend 80 e Return to the NormDATA window select Window NormDATA Select the Tools Pick Peaks menu item The Pick Peaks dialog box will open Enter the options that are shown The Pick Peaks tool operates on the active data plot in the graph window Refer to Origin s on line help for more information about the Pick Peaks tool Make the bindproteinon cp data set the active data set by selecting Data and clicking on the BINDPROTEINON temp x cp y menu item to place a check mark next to the name Click the Find Peaks button on the Pick Peaks dialog box Origin will place a cross at the peak and print the temperature The peak temperature T should be ca 61 0 C for the protein only scan Make the data set bindligandld cp the acti
81. mains are interacting strongly it is possible that their transitions will be coupled in a sequential manner whereas the independent model might better describe two transitions which are completely uncoupled from one another In practice this choice is often not critical since the sequential and independent models lead to virtually identical results whenever the Tm s of two transitions are separated by a couple of degrees or more To switch between select the DSC Set Indep Sequ Model command and answer the prompt that appears Origin provides the option to float all parameters during the fitting procedure or to assign some parameters as fixed and float others In fixing a parameter it may be given an invariant numerical value The mathematical derivations for each model are included in the appendix In general the objective of most investigators is to use the simplest model e g fewest floating parameters which provides a good fit of the data Thus if data are satisfactorily described by the two state model using two transitions this 1s to be preferred over a two state model with three transitions or a non two state model with two transitions It is usually only when a given model is unable to fit the data that complexity should be increased by adding more fitting parameters Fitting Example 1 To better understand the flexibility of Origin s curve fitting process work through the following example The example uses data from a protein of
82. may be obtained from DSC data in the presence and absence of ligand The parameters required to calculate Kz are the protein concentration ligand concentration the heat AH and the heat capacity ACp change in the absence of ligand and the temperature midpoint of the transition in the presence Tm and absence T of Ligand The equations and parameters required to calculate binding constants for the standard DSC are the same as used with autosampler DSC data The methods used are a little different ACp and AH are determined by the same method see below and pages 69 70 for both autosampler and standard DSC data The peak temperatures T and Tm are determined automatically by autosampler routines but require some user interaction see page 80 for standard DSC data e Launch a new instance of Origin or select File New Project to open a new Origin project e Select File Open navigate to the C VOrigin70 Samples directory and select Binding Data from standard DSC OPJ and click Open This project contains 3 data files one with the protein in the absence of ligand one with the protein in the presence of ligand 1 and another with the protein in the presence of ligand2 The data has had a baseline subtracted corrected for the response time of the instrument and has been normalized on concentration EE NormDATA al 1 Bu Mra Baline z0 Unda Su biract 15 BINDPROTEINON CP BINDLIGAND1DS CP
83. move File Cancel File Name Ret dec Sample dsc Navigate to the Samples folder from the Look in drop down list box Note You may configure Origin to always look in the Samples folder or your own folder by selecting Files Set Default Folder and entering the path in the text box e To add the file Samplel dsc to the lower list box double click on the file Samplel1 dsc in the File Name list box alternatively you may click on the file and click on the Add File s button Note When assigning filenames to raw data no hyphens dashes or spaces may be used in the filename the names should not begin with a number nor should the letter e be followed by a number e Repeat the above procedure for the file Refl dsc Click OK to plot all files in the lower list box into the RAWDSC plot window Refldsc cp L Sample1dsc cp Cp cal C 60 Temperature C 12 Lesson 1 Basic Data Analysis You can read any number of data files into the same RawDSC window Note that when multiple data plots appear in the same window you can set the active data plot by clicking on the plot type icons next to the filename in the legend A box around the line symbol type indicates the currently active data plot Editing fitting and other operations can only be carried out on the active plot Subtracting Reference Data In most experiments you will have obtained both sample reaction heat data and reference data and will need
84. n Interaction Tm gt To To a B Es T 50 C Protein Protein Interaction Tm gt To To AH at T 150000 cal mole ACp at T 2000 cal mole deg Protein Conc P 0001 moles l To 65 C AH at T 150000 cal mole ACp at T 2000 cal mole deg Protein Conc P 0001 moles l TM 68 4 C e Click OK The binding constant at Tm will be calculated and the Binding Results dialog box will open listing the entered parameters and the calculated Kg You may print the results and or calculate the Kj at a different temperature Binding Results 85 DSC Data Analysis Tutorial Guide To extrapolate the binding constant to a different temperature When extrapolating the binding constant to a different temperature you must determine AHg and ACpg from an ITC experiment or other independent information Click the Calculate Kg at a different temperature T check box to insert a checkmark then click OK A new dialog box will open up allowing you to enter the parameter information to extrapolate the Kg value to another temperature Enter the following values in the parameter text boxes Input For Different Temperature AE A T 25 C Hg at T 15000 cal mole ACpg 1000 cal mole deg Click OK The binding constant at Tf will be calculated and the following dialog box will open listing the entered parameters and the extrapolated Kg You may print the results Binding Results 86 Lesson 7 Other Useful
85. nations To plot the baseline Select Progress Baseline from the Baseline menu Origin calculates and plots the progress baseline ENS PLN QO o nn 2 o c U o AX M o OQ 50 60 TO Temperature C The progress baseline is created such that each point of the baseline in the peak region reflects the extent of progress of the reaction For a single transition such as this the area between the progress baseline and the data approximates the total heat change due to the transition Click OK in the menu bar to exit the baseline session Origin asks if you want to subtract the baseline data Click YES Origin subtracts the baseline data from the normalized data then exits the baseline session Cp kcal mole C 50 60 T Temperature C 18 Lesson 1 Basic Data Analysis Fitting the Data Origin provides four models with which to fit a curve to your data All four models use the Levenberg Marquardt LM non linear least square method In general you will want to get the best possible fit using the simplest model that fits the data Curve fitting is discussed in greater detail in Lesson 5 This section will serve to acquaint you with the basic procedure The four curve fitting models are located in the DSC menu They are Model Parameters Model 1 2 State Tin H Model 2 Non 2 State Tas HE Ey Model 3 2 State w dCp Tn H Cp BLO BL1 Model 4 Dissoc w dCp Tm H Cp BLO BL1 n A
86. near Curve Fitting Fitting Session fel X Category Function Action Options Scripts speso Parameter Value Vary Error Dependency BLO o i O BLI D T m 3 px r 9 Tm 044 v foo o5878 Successfully progressed 4 rounds Reduced Chi sqr 34140 33225 Total 4 rounds in this session Successfully progressed 1 rounds Reduced Chi sqr 33838 60167 Total 5 rounds in this session 3 7 7 Levenberg M arquardt Chi sqr is not reduced Reduced Chi sqr 33838 60167 Total 5 rounds in this session ChiSq 1 Iter terif 100 Simplex Iter Done Perform a specified number of Levenberg Marquardt Basic Mod iterations Press ESC to stop asic Mode TU a an Temperature Cc 6 Now change the value of n from 2 0 to 1 0 Click on 10 Iter several times Note that the new fit curve is much worse than the original fit curve Chi 2 1s ca Five times larger and that the experimental data curve is now substantially broader than the fit curve This means the data are most consistent with the idea that dissociation into subunits occurs simultaneously with unfolding for this polypeptide 7 Change the value for n from 1 0 to 4 0 and fit the data again It is again obvious that the fit is not as good as the original fit using an n value of 2 0 which suggests that the polypeptide is morre likely to contain two dissociab
87. ns or other macromolecules when the stability of each domain A B C is dependent on whether other domains are folded or unfolded The sequential model imposes a precise order in which domains must unfold for every molecule in solution 1 e 97 DSC Data Analysis Tutorial Guide K K K K popncpcp qe AH AH AH AH 14 where each step in the reaction scheme involves the unfolding of one of the domains in the appropriate sequence 1 e step 1 is always the A domain step 2 always the B domain etc The total molar heat content of the system relative to a value of zero being assigned to the Po state will be H fy 0 f AH f AH AH f AH AH AH 15 where the f s indicate the fractional concentration of each state and the AH s are the molar enthalpy changes for each step Using the K values all of the f values can be related to f so that lec a Oe oe Oe eee l f 1 K K K K K K nT ny Ae ee 16 Q Q Q where Q I K K K K K K 17 Eq s 16 may be substituted into eq 15 which may then be differentiated with respect to temperature to obtain the system heat capacity Since AH values are treated as temperature independent in Origin s sequential model most of the derivatives involve using the relation AKK KK pue T K K K OT RT The final expression for the heat capacity of the system then becomes E s Ai ary tn 9 987 1 e ra
88. o the lower list box Click OK The Sample2 dsc file opens into the RawDSC plot window Sample2dsc cp Temperature C 25 DSC Data Analysis Tutorial Guide To select a segment of plotted data Click on the Data Selector tool in the toolbox The Data Selector becomes highlighted The pointer turns into a cross hair Two data markers appear one at either end of the Sample2dsc_cp data plot in the RawDSC 1 plot window Es amplezdsc cp C T L E i T G z IH EG ad 16 ic Temperature c Position the cross hair on the left data marker hold down the mouse button and drag the marker to the right approximately to the position shown below Or use the arrow keys to move the marker use the Left or Right arrow keys to select a marker use Control Left or Right to move the selected marker hold down the Control Shift key to increase the speed 5 ampie24ss cep C p cal c Temperature Cy 26 Lesson 2 Using the Data Selector Tool To set the markers double click in the plot window press Return pressing the escape key with your left index finger is equivalent to pressing Return and this allows you to keep your right hand on the mouse or click on any toolbar tool The markers are set and the Pointer tool is re selected unless you selected a different tool Any math editing or fitting operation now performed will affect only the selected plot segm
89. omment Origin provides four models with which to fit a curve to your data All four models use the Levenberg Marquardt non linear least square method but differ in the number of parameters involved as shown below Model Parameters Model 1 2 State with zero DCp m Tm H Model 2 Non 2 State with zero DCp THE IS Model 3 2 State with non zero DCp Tn H Cp BLO BL1 Model 4 Dissoc with non zero DCp Tn H Cj BLO BLI n Tm is the thermal midpoint of a transition H is the calorimetric heat change AH Hy is the van t Hoff heat change AH Cp is the AC for each transition BLO and BL1 define the slope and intercept of the low temperature baseline segment and n is the number of sub units All of the models except model 4 can be used to fit to one or more transitions In the case of multiple transitions each transition has its own complete parameter set e g if model is used to fit two overlapping transitions there will be two independent parameter sets Tin1 H and Tm2 H5 These specify the thermal midpoint Tm and the heat change AH at the T for each transition The BLO and BL1 parameters are an exception to this rule These parameters appear only once in the model they are not repetitive for each transition While all four models use a calorimetric heat change AH only the non 2 state model model 2 has a van t Hoff heat change AH The calorimetric heat H is determined only by the area under a transition p
90. on you should have Origin up and running If you are in Windows Mieracal LLC e From the Desk Top double click on the MicrCal LLC DSC icon Or alternatively DSC select Start Programs OriginLab MicroCal LLC DSC When you start Origin the program automatically opens the RawDSC plot window Note that this window contains several buttons These buttons let you execute certain DSC routines Reading DSC Data Before we proceed you should understand the difference in Origin between a data file and a project or document for versions of Origin previous to 4 0 A data file contains your experimental data In order to plot the data you must read the data file into an Origin project A project or document will contain both the experimental data and any plots you have made from the data To save a project select the File Save Project or File SaveProject As command The project saves as a OPJ file in Origin 4 0 or higher To open a project use the File Open command To import a data file you will use the buttons that are attached to the RawDSC plot window The RawDSC plot window contains nine buttons four of which are used for data import The buttons are described below DSC Data Analysis Tutorial Guide BUTTON NAME Read Data Subtract Reference Normalize Concentration Scan Rate Normalization Delete Time Data Apply Y Offset Response Time VP DSC Absolute Cp VP DSC Binding Constant Calculate Delta G T
91. oss hair on or near the data you want as the first point of the linear segment A red cross appears on that point Use the left and right arrow keys to move the red cross until you have selected the point you want Press Return This sets the selected point as the first point of the linear segment Repeat the previous three steps to select the last point of the linear segment Origin now generates a new linear segment by fitting the data between the two points to a straight line Now for the left linear segment Instead of using the refit command lets adjust the left segment by cursor To move linear segments by cursor e Select Move Segments by Cursor from the Adjust menu e Select and move one of the left segment end points by either Clicking on one of the terminal black squares and dragging the mouse or e Selecting an end point with the LEFT or RIGHT arrow keys then using the UP and DOWN arrow keys to move the point up or down Use the CONTROL LEFT or CONTROL RIGHT arrow keys to move the point left or right Repeat the previous step for the other segment end point When you are satisfied with the placement of the left linear segment double click in the plot window or press the Return key 39 DSC Data Analysis Tutorial Guide 40 LJ c aq ce e ty CMS cL a0 Bu fa Temperature C Choosing a Baseline Option Once you have set the linear segments you are ready to select one of
92. ossible to obtain a reliable estimate of this since the heat from ligand release usually constitutes such a small fraction of the total heat of unfolding However If one knows the heat of binding independently e g from ITC experiments it then becomes possible to extrapolate the Kg value obtained from equations 29 and 30 at Ty to another temperature Tp using the equation Brandts J F amp Lin L N 1990 Biochemistry 29 6927 6940 AH A T T K T K T exp LN Peel rere ee 31 R Ch d R F Ty where AHg and AC are the heat and heat capacity of ligand binding respectively at Tp 101 DSC Data Analysis Tutorial Guide VI Calculating Protein Protein Binding Constant from DSC Data 0 00040 0 00035 0 00030 O ud 0 00025 Z Q O 0 00020 0 00015 0 00010 0 00005 20 40 60 80 100 Temperature C Wher RCM MN tr ee CONCRA n m RR RO trie peto ee ge CET ee ee eee RM PP P P PP Kees P P the approach is a little different than for ligand protein interactions since both of the proteins P and P will undergo their own thermal unfolding transition This is illustrated in the above Figure where To AH and AC refer to transition parameters for P alone and Tv AH and AC are the corresponding parameters for P alone When P and P are mixed together in the same solution then a separate transition will be observed for the complex PP with transition midpoint Ty A second tr
93. pleldsc with the Y data in a column named cp Thus the Sampleldsc data set is called Sampleldsc cp To open the Sampleldsc worksheet e Select Sampleldsc from the data list in the Data menu BEES Math Peak DSC Tool Fo Seb Weoley Hange Reset to Full Range Data Markers Move Data Paints Remove Bad Data Points vw Sampleldsc temp cph Lesson Basic Data Analysis The Plot Details dialog box opens Note that you can also open this dialog box by double clicking on the data plotted in the RawDSC plot window Plot Details E E RawDSC Line a Layer Connect Straight Style Solid Width 0 5 v Color EI Black r Sam ple d E C i tem pt 2 Fill rea Under Curve Mormal zi Plot Type Line T gt Worksheet Cancel Apply Click on the Worksheet button The Sampleldsc worksheet opens ms Microcal Origin UNTITLED 5a Ez File Edt View Plot Column Ma 30 79999 a1 2 891 45E 5 31 20001 6 99336E 5 31 40002 3 04161E 5 31 60004 4 9134E 5 31 80005 5 97 786E 5 32 00006 f 35419E 5 Normalizing the Data Before you proceed to fit deconvolute the data you will need to normalize the result by concentration Normalizing the data divides by the number of moles of the sample 15 DSC Data Analysis Tutorial Guide To quickly switch between Origin windows press and hold down the Ctrl key while pressing the Tab key 16 substance in the cel
94. position the cursor then press RETURN Set Tm equal to about X 61 Origin initializes the fitting parameters displays the parameters and Chi 2 in the Fitting Session dialog box and draws an initial fit curve When initialization 1s complete the Attention dialog box appears with the message Done init with fixed Tm s Click OK Attention i Done mit with fed Tm s The Fitting Session Lesson 1 Basic Data Analysis You are now in the Advanced Mode of the Fitting Session dialog box Note that a new window displays the fitting button commands Please refer to the Origin User s Manual or press the F1 key for Online help for more information about the fitting procedures z ha 7 UNTITLED Hable Ea T Peak DS L8 x C Tools Format Window Help 8 x m O S E o o gt M O oO 30 40 Et ase aia al aie Amal ASe e E TFE gr fe fel t Fd Subtract Baseline Undo Subtract NonLinear Curve Fitting Fitting Session Category Function Action Options Scripts amam Parameter Value Vary Error Dependency Tm 60 77 Ww 0 02452 0 00914 H 3 503E4 M 2721 0 00914 Press Esc key to stop fitting iterations 50 60 70 a0 90 Temperature C Chi Sqr 1 Iter TO0 Iter 100 Simplex Iter AeL OAEOAENERM 62152112 y osy E See Enter fitting session and perform nonlinear curve fitting 4 ma ils rm I Script Window Fil
95. r with ITC data analysis routines for handling multiple data files Note that this menu level is available only if you purchased the optional AutoITC software module Short Menus Select this option to run Origin with menus that are an abbreviated version of the General Full Menus configuration Note that you cannot switch to a new menu level if there is a maximized plot window or worksheet in the current project A warning prompt will appear if you try to switch levels while a window is maximized If this happens simply click on the window Restore button B You will then be able to switch levels Simultaneously Running DSC and ITC Configurations If you purchased the DSC ITC Autosampler and PPC software modules the installation program will have automatically created icons in the MicroCal OriginLab program group for the relevant software This allows you to run the configurations simultaneously The most likely reason to do this would be if you have the MicroCal DSC with the PPC or Autosampler attachment and the MicroCal ITC microcalorimeters and you intend to run them on the same computer Double click on any icon to run that configuration Getting Started View Mode Each Origin plot window can be viewed in any of four different view modes Print View Page View Window View and Draft View Print View is a true WYSIWYG What You See is What You Get view mode This view mode displays a page that corresponds exactly to the page
96. raph to be located e Select Edit Paste Special e Select Origin Graph Object from the As list box e Select the Paste radio button e Click OK Linking your graph into Word e You must first create your graph in Origin and then save it as part of an Origin project OPJ e Open the saved Origin project if it is not already opened that includes the desired graph window e Make the desired graph window active select Edit Copy Page e Open your Word document and and click at the location where you want to insert the graph Lesson 7 Other Useful Details e Select Edit Paste Special e Select Origin Graph Object from the As list box e Select the Paste Link radio button e Click OK After your Origin graph is linked to Word you may return to the original Origin graph and make changes to the graph These changes can be reflected in the Word document by e Select Edit Update Client from the Origin menu to make immediate changes to the Word document graph Shortcut You may quickly start Origin and load the linked graph by simply double clicking on the graph while in Word Origin will be started with the original document loaded the changes can be made and by selecting Edit Update Client the changes will be transferred to the Word document 93 Appendix Equations Used in Deconvolution Appendix Equations Used to Deconvolute DSC Data I Independent Transitions It will be assumed here that a protein or oth
97. re two pharmaceutical applications where multiple scans may be required The first is optimizing solution conditions for protein formulation stability where a number of solution variables pH salts antibacterial agents sugars etc are examined to find the appropriate blend of additive which maximize protein stability by increasing its melting temperature Tmn Remmele R L Nightlinger N S Srinivasan S amp Gombotz W R 1998 Pharm Res 15 200 208 The second application is determining binding constants of protein target molecules to small ligands or other proteins where Tm shift resulting from addition a of a potential drug candidate allows calculation of binding constant to target protein Brandts J F amp Lin L N 1990 Biochemistry 29 6927 6940 The DSC can determine the Tms for different drug target complexes at varying concentrations thereby allowing determination of individual binding constants Launching the DSC Autosampler Data Session Auto DSC files are located in a separate window named ARawDSC from the standard DSC routines In addition to the Auto DSC routines this window also known as a menu Microcal LLC level contains all the functions of the regular DSC data analysis Auto DSC ET Before starting this lesson you should have Origin up and running The routines for e To launch the Auto DSC menus double click on the MicroCal LLC Autosampler icon on the Desk Top Or alternatively select Sta
98. reater detail e Select Step at Peak from the Baseline menu The Step at Peak baseline will provide a quick and easy method to determine the ACp At this point your graph should look similar to what is shown below 1 12000 10000 B EBD 4000 2000 Y Axis Title 2000 4000 BUOU 10 20 30 AQ 50 B 70 B a 100 A Axis Title 78 Lesson 7 Other Useful Details e Click OK then click NO when asked Subtract baseline e Select the Data Reader tool then use the left and right arrow keys to read the difference of EB Data Reader Tool baseline y values at the step of the peak for this example determined by where the baselines were set there was measured to be a high value of 1960 and a low value of 3040 at the step for a ACp to be approximately 1100 cal deg mole Note Your ACp value may be different depending on where you placed the lower and higher temperature baseline segments during the baseline session To determine AH by calculating the area under the curve e d ab Data Selector Tool Select the Data Selector tool and using the mouse or arrow keys position the left data marker to the approximate position as shown below This will eliminate the effect of the aberration located at the beginning of the scan D I Hat x T 40 50 BO 70 80 gg A Axis Title To set the markers press enter or double click on one of the markers Select the Peak Integrate from Baseline menu ite
99. retty well the low peak did not This could mean that the low peak is not really a single peak but two smaller overlapping peaks that combine and obscure each other To test this hypothesis see if you can get a better fit using model 1 and three transitions 1 Click on the Close button X located in the upper right corner of the Fitting Session dialog box to abort the current fit 2 Select 2 State Cursor Init from the DSC menu 3 Type 3 in the Number of peaks dialog box and click OK 29 Parameter Value Vay Error Dependency Tml 53 30 M 0 0784 0 0578 H1 1 10955 lv 1167 0 01923 E Tm2 63 75555 mo oos 006637 H2 fi 427E5 M 11065 0 02942 is jes ecd 2 Press Esc key to stop fitting iterations Bm 1 Levenberg M arquardt E Successfully progressed 1 rounds E in Reduced Chi sqr 149587520471 m Total 5 rounds in this session be 2 Levenberg M arquardt Successfully progressed 1 rounds c Reduced Chi sqr 1495806 14851 C g Total 6 rounds in this session o 2 A 4 O en TH sn 3n 1m Chi Sqr 1Iter f B Tier 100 Simplex Iter Done a Ji Temperature i C Basic Mode Lesson 5 Curve Fitting 4 Double click to set each of the three Tm s one for each transition Place the first Tm just to the left of the low peak the second just to the right and center the third on the high peak ew o O m o e
100. right endpoint until the graph looks approximately like the figure below you can also use the arrow keys to move an endpoint Use the Up and Down arrows to move up or down Control Left and Control Right arrows to move left and right and Right and Left arrows to select a new end point un Cp kcal mole C Temperature C 4 Now use the cursor to adjust the right linear segment as follows 48 Lesson 5 Curve Fitting mn e Za 9 o s o o O Temperature C 5 Select Baseline Progress Baseline Origin plots a progress baseline 6 Select OK from the menu bar 7 Click Yes in the dialog box to subtract the progress baseline Your graph should now look like this S n D a E n eo A c p 5n Bn T an 100 110 Temperature C Step 3 Fit to the least complex model Generally you will want to use the simplest model e g with the fewest floating parameters that provides a good fit to the data You can then add complexity if necessary Begin the fitting session by using model 2 state with zero ACp to fit the data To do so 1 Select 2 State Cursor Init from the DSC menu This starts the fitting session with model 1 selected The Cursor Init option allows you to initialize the parameters rather than accept any existing parameters 49 DSC Data Analysis Tutorial Guide 2 Origin prompts you to enter the number of transitions The simplest fit wou
101. rt Programs OriginLab MicroCal LLC Auto DSC When you start Origin the program automatically opens the ARawDSC graph window Note that this window contains several buttons in addition to the regular DSC routines These buttons let you execute certain multiple data file manipulations fay Origin 7 UNTITLED ARawDSC File Edit View Graph Data Math Peak DSC Tools Format Window Help 81 x okeee S 8 2 se S aE A eA e Aee l Ee EE AETA ma 1 Ha A Aa EEE er E Subtract Reference 10 Subtract Baseline 6 Find One Tm Find Two Im a Find Delta Tm Find One Tm Find Two Tm Cp cal C TY Offset 2 Hormalize Concentration 0 0 2 i g Auto Binding Constants Temperature C Kb Threshold Value X N x rib ame m re oe x AStart G 17 W 5E ES Ko 3J Exploring My Doc Inbox Outlook Ex 3 Exploring New2 ffi Origin 7 UNT BA 1226 PM 59 DSC Data Analysis Tutorial Guide The buttons displayed in the gray area to the left of the Origin graph and described below are exclusive to the ARawDSC template and allow easy manipulation of the multiple Auto DSC files Examples of the use of each button are presented in the following pages Subtract Baseline Allows you to select a baseline to be used to subtract from all other scans plotted in the active layer Find One Tm Will determine a single Tm for all scans plotted in the active layer Find Two Tm
102. s appear Notice that Page View is checkmarked showing it to be the selected view mode Page View uses the screen driver to display font information e Select Print View from the menu Print View is now the selected view mode Print View gets font information directly from the printer driver and is a true WYSIWYG view mode The plot window text is now sized and positioned correctly 0 0014 c Sampleidse_cp SAMPLE1DSC_CP BASE o 0012 stag of Sample 1dic of from SAMFLEIDZC DCF BAZE Q 00 TU en ae T Range 4 80103 gt 7 3 0 0258 Are oe 0789 i Tm P3 INI TIEI HAR TU wl NN Ga 0 0006 O E s G oO 0 nna D OD OOO BL Temperature C e Before proceeding with this lesson re select the View Page View command For more about view mode see Chapter 12 Plotting Customizing the Page Display in the Origin User s Manual or select the Index tab from the Help Origin menu option and enter View modes into the upper text box 33 DSC Data Analysis Tutorial Guide To remove the data markers Click Data Markers from the Data menu to remove the check mark Origin hides the data markers Plotting the Integration Area Data Shortcut Click on the New Graph button 34 The integration area data is saved as a function of X which is temperature for calorimetric data into a data set named _integ area The leading underscore signifies that integ area is a temporary data set which will be deleted when
103. s useful for offsetting repetitive scans so they do not overlap on the Y axis Click once on the number 0 000 in the offset text box to open the offset dialog box Enter the offset you wish in the Step Size box enter the offset in mcal even though the data are plotted in cal and click OK Click on either scroll arrow so the proper positive or negative Y offset for the next data set appears in the offset field For each successive data set the offset will advance automatically by the same increment This is used for removing small amounts of peak broadening due to using a feed back mode which has a response time too slow to resolve a peak adequately Refer to pages 88 91 for more information This is used to calculate Absolute heat capacity from raw DSC data Refer to pages 54 57 for more information Clicking this button will open dialog boxes to allow the user to input the parameters used to calculate binding constants from DSC data Using three experimental parameters of a protein scan midpoint temperature Ty enthalpy change at Tm AH Ty and heat capacity change at Ty ACp the Gibbs free energy AG T may be obtained at temperature T Lesson Basic Data Analysis fa Origin 7 UNTITLED RawDSC El Fie Edit View Graph Data Math Peak DSC Tools Format Window Help 8 x El hiat Cs sasae eiiis S IS A a R e Suis E s B T Arial I gt B z U x x xi xg A amp mm ji za E i FE
104. sed in units of 1 point This is 9 DSC Data Analysis Tutorial Guide 92 usually the most satisfactory representation of the fit curve but may exhibit an excursion from the actual fit curve if there 1s a sharp corner in the dataa B Spline The B spline curve can be described by parametric equations Unlike spline curves which pass through the original data points the B spline curve winds around the original data points without passing through them Thus this curve may not produce a satisfactory representation of the fit curve For a complete discussion of the B spline connection see the Origin User s Manual Inserting an Origin graph into Microsoft Word There are two ways to include your Origin graph into Word or other applications you may import your graph into Word or you may share your graph with Word When you import your graph Word will display the graph as an object and it cannot be edited by Origin tools although it may be resized or reposition in the Word document When you share your graph Word displays the graph as an object which can be edited by Origin or linked to Origin and updated when the Origin graph changes Please refer to the Origin manual for more information about Creating a Graphic Presentation Importing your graph into Word e Create your graph in Origin and when you are satisfied with its appearance select Edit Copy page e Open your Word document and click at the location where you want the g
105. ssssseeeeeeeennnnrrnnrnes 101 VI Calculating Protein Protein Binding Constant from DSC Dat ccc cccccccccccccccseeeeeeeeeeeeeeeeeeeeseeeeeeees 102 VII Using DSC data to calculate AG at temperature T by extrapolating from the midpoint Ty 103 ln qu rrr ner 105 a Introduction Introduction to DSC Data Analysis MicroCal Origin is a general purpose scientific and technical data analysis and plotting tool In addition Origin can carry add on routines to solve specific problems Analyzing Differential Scanning Calorimetric data from the MicroCal MC 2 MCS or VP DSC instruments is one such specific application This version of Origin includes routines designed to analyze DSC data Most of the DSC routines are located in the Peak and DSC Differential Scanning Calorimeter menus in the Origin menu display bar A number of routines are implemented as buttons in plot windows This tutorial will show you how to use all of the DSC routines When working through this tutorial 1t is recommended that you do not save changes to the original files but use a different filename so that the next person may follow this tutorial Lesson 1 provides an overview of the entire DSC data analysis and fitting process Please work through this lesson first The subsequent lessons each look in more detail at particular aspects of DSC data analysis and may be read in whatever order you see fit I
106. t outs Only on screen display is affected Note About Data Import MicroCal offers four versions of the DSC instrument the MC 2 the MCS VP DSC and VP CAPILLARY DSC All together there are five different data collection software packages available for use with these instruments 1 a DOS based and 2 a Windows based package for the MC 2 3 a Windows based package for the MCS 4 a Windows based version for the VP DSC and 5 a Windows based version for the VP CAPILLARY DSC This version of Origin with the optional autosampler module will accept data files from any MicroCal DSC instrument and from any of the five versions of the data collection software To import a data file generated by the MC 2 DOS based data collection software you click on the Read Data button in the RawDSC plot window and select MC 2 data dat from the List Files of type box To import a data file generated by the OSC Data dsc Windows based data collection software either EM from the MC 2 the MCS or the VP DSC you click on the Read Data button in the RawDSC plot window and select DSC Data dsc from the List Files of type box PLEASE NOTE Data DSC Data Analysis Tutorial Guide file names should not begin with a number nor should they contain any hyphens periods or spaces Once a data file is called into Origin all further operations on the data are identical regardless of the original source of the data Note that in this t
107. tasaiasavabaxesesabatedseaseissatabaiossnats 77 Ligand Protein Binding Constants from Standard DSC Data ccccccccccccceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeees LT Protein Protein Binding Constants from Standard DSC Data ssssssssssseeeeeeeeennennrennnnns 83 Protein Protein Binding Constants from Standard DSC Data T lt Tm lt To cccccccccccccccccccccccceeeeeeeeeeeeeeeees 83 Protein Protein Binding Constants Tm Los Tyrion bt ache E a E AEEA 84 CalculitesAGr ab temperate T3 demos e i A AE 87 Chiesquare ebr 2 Mii ZAG OM eniin T TRA EEA O AA ESE 88 Response Mme VPSDSC asus ipu a A I AN 88 Line Types for EIE Cr VCS iia a rs eect Dei eM ELM M ie Seal Reta la alas ti SEA 91 Inserting an Origin graph into Microsoft Word cccccecccscscceecceeeeececccececceccceeeecceececeeeeeeeceeeeeeeeeeeeeeeeeeeees 92 Appendix Equations Used to Deconvolute DSC Data e eee eee eee eee eee eee eene eene teet ettet teet eese e eee aa 95 Indeperdent Mansions eii obe Eri ip irn t icu sinibus uu iesu inii M eO eM I diu erede ud 95 H Sequenttal le 2 afc HONS sapin re ee Ue er E ee re ener eee on ore eee 97 III Single Two State Transition with Subunit Dissociation 00 ccccccccccsscceccceeeeceeeeeeceeeeeeeeeseeseeeeeessesseeess 99 IV Absolute Heat Capacity of Proteins ioo ESTHER E I pel HR b etel tuts 100 V Calculating Protein Ligand Binding Constant from DSC Doata eeeeeesess
108. ter the following values as determined in the preceding pages DSC Ligand Protein Binding Constants x Farameters T 61 C f k n 1 0 n ii sites per mole ARH at T 110000 cal mole ACp 1100 cal mole deg 110000 cal male cal male deq Cancel e Click OK The binding constants are calculated for all data sets that are plotted in the active layer and listed in the summary table 74 Lesson 6 Autosampler Data Optional Accessory pe209040D1 DSC amp h1 1 1 Q0 Baseline pc20904002DSC 1 3 1 U0 Baseline pe20904003DSC 1 5 1 D0 Baseline neSDDDADSC 1 2 109 10 61 0 Rnase onb no liaand pc20904005 DSC 1 893 0 09 10 J 827 134 Rnase Phosphate bea0904006 08C 1 1l oos 10 65 154 RNase 3 CMP pc20904007 DSC 1 13 oog 10 70 10900 RNAse 2 CMP c20904008 DSC 1 15 oos 10 5097 0 Rnose only no ligand pc20904009 05C 1 17 0 09 10 82 63 128 Rnase Phosphate L mwwmDDs 1 19 009 10 865 M90 Hase TCMP pc20904011 DSC 1 21 oog 10 70 6 11200 RNAse CMP weona nse 1 23 099 10 5033 Rave ony o ligand pc20904013 DSC 1 25 0 09 10 8275 139 Rnase Phosphate bea0904014DSC 1 2 oos 10 654 1510 RNase 3 CMP pe2
109. the equation 99 DSC Data Analysis Tutorial Guide 100 AH Dp Tf C T B B T f T AC E E F T p T The above equations are general for any value of n so long as the bulk concentration C is expressed as n mer equivalents For systems which associate when unfolding occurs 1 e 26 l n 4 rep 27 the same equations are valid providing the bulk concentration C is expressed as monomer equivalents and that n in the above equations is equated to J IV Absolute Heat Capacity of Proteins The equation used to calculate Absolute Heat Capacity of Proteins is as follows AC g p t V 0 000020 C2 v 1 a c o Where AC cal deg is the sample buffer baseline minus the buffer buffer baseline g is the concentration of protein gm ml in the solution p t is the relative density of water stored in Origin as a polynomial in t V is the nominal volume ml of the sample cell t is the temperature in C Cp t is the absolute heat capacity cal deg gm of the protein in solution v is the partial specific volume of the protein ml gm a is the coefficient of thermal expansion of the protein and Cp t is the unit volume heat capacity of water cal deg ml Cp t is calculated from literature data on the specific heat and density of water as a function of temperature by fitting to a polynomial in temperature The thermal coefficient of cubical expansion of tantalum 1s 00002 The above equa
110. the low temperature and high temperature segments to approximately what 1s shown below then double click an endpoint or press enter to set the baseline segments See Lesson 4 Baseline Determinations for greater detail e Select Step at Peak from the Baseline menu The Step at Peak baseline will provide a quick and easy method to determine the ACp At this point your graph should look similar to what is shown below E a lt gt 40 50 BU TO g0 gg 100 Axis Title 69 DSC Data Analysis Tutorial Guide B Data Reader Tool Click OK then click NO when asked Subtract baseline Select the Data Reader tool from the tools toolbar then use the left and right arrow keys to read the difference of baseline y values at the step at the peak for this example determined by where the baselines were set there was measured to be a high value of 2030 and a low value of 3160 at the step to determine the ACp to be approximately 1100 cal deg mole Note Your ACp value will be slightly different depending on where you placed the lower and higher temperature baseline segments during the baseline session To determine AH at T by calculating the area under the curve Es Data Selector Tool 70 Select the Data Selector tool and using the mouse or arrow keys position the left data marker to the approximate position as shown below This will eliminate any effect of the aberration located at the beginning of the scan 2
111. the six options for creating a baseline You will find the baseline options in the Baseline menu To select an option for creating the baseline Click on the Baseline menu option Choose a baseline option from the menu When you are satisfied with the baseline click on the OK command in the menu bar to exit the baseline session Origin gives you the opportunity to subtract the baseline then returns you to the RawDSC window or if you had normalized the data the NormDATA plot window The six baseline options are described in detail below Progress Baseline In this option the baseline in the peak region is created such that each point reflects the extent of progress of the reaction That is the placement of a baseline point at any temperature in the transition region 1 e relative to the left and right baseline extrapolated to the same temperature 1s determined by the fraction of the total area that has been completed at that temperature The progress baseline for the SAMPLE data is shown below Lesson 4 Baseline Determinations For a single transition such as this the calculated baseline then approximates the molar heat capacity for the mixture of the states as they exist at any temperature in the transition region The area between this baseline and the data then approximates the total heat change due to the transition Note that only the slope and intercept of the linear segments are used in the calculation for t
112. tion is solved for Cp t from the experimental data file AC using the stored polynomial expressions for Cp t and p t as well as the operator input parameters Zo V Qt and Vo This procedure assumes the relative density and the unit volume heat capacity Cp of the buffer solution can be replaced by the corresponding properties of pure water This should be a very good approximation at electrolyte concentrations commonly used in buffer solutions but would be less accurate at very high electrolyte concentrations 21M or in the presence of high concentrations of organic additives Also the value obtained for Cp depends strongly on the partial specific volume of protein so it is desirable to have an experimental value of v for the protein in question at a single temperature at the least The coefficient of thermal expansion is typically small 10 10 and exerts only a small effect on the Cp value over a moderate temperature range Appendix Equations Used in Deconvolution V Calculating Protein Ligand Binding Constant from DSC Data Ligands frequently bind much stronger to the native folded form of a protein than they do to the thermally unfolded form The treatment below assumes no binding to the unfolded form This being the case the thermal midpoint of the unfolding transition will necessarily occur at a higher temperature Ty when the ligand is present in solution than when the ligand is absent T For reversible transitions th
113. to subtract the reference data from the sample data To subtract the REF1 reference data from the SAMPLE1 sample data Click on the Subtract Reference button in the RawDSC window The Subtract Reference Data dialog box opens The first file opened in this case Sample1 DSC will appear in both the Data and Reference drop down list box Note that the data set listed in the Reference box will be subtracted from the data set listed in the Data box e Select Sampleldsc cp from the Data drop down list Sampleldsc_cp becomes highlighted and will be entered as the Data e Select Refldsc_cp from the Reference drop down list Refidsc cp becomes highlighted and will be entered as the Reference Subtract Reference Data Baie xl Data Reference Sampleldsc c Data Reference Ref dec cp CEN Click OK Every point in Refldsc cp is subtracted from the corresponding point in Sampleldsc cp The result is plotted as Sampleldsc cp in the RawDSC plot window 13 DSC Data Analysis Tutorial Guide 14 Sample1dsc cp THHE O o Em o Q O 60 Temperature C Viewing Worksheet Data The series of values from which Origin creates a data plot is called a data set Each data set is contained in a unique worksheet column The data set is named after its worksheet and worksheet column separated by an underscore When you opened the Sample1 DSC data file the data were placed in a worksheet named Sam
114. to the Basic Mode which contains its own menu bar but with fewer options for fitting procedures Done Exits the Fitting Session dialog box returns to the NormData window the fit curve is plotted and the fitting results are plotted in the Results text window and to a text box in the Normdata window Use the 1 Iter amp 100 Iter buttons to control the iteration of the fitting cycles as described above Watch the fitting parameters and Chi 2 update in the Fitting Session dialog box When the Chi 2 value no longer reduces you may click the Done button to exit the session Sam pleidsc_o Model Mza E chiz TICS T GT 0 02445 oH B51E4 272 TM e m a E ni e co i 5n 60 T Temperature THES To fit a curve with the non 2 state model model 2 e Select Non 2 State Cursor Init from the DSC menu e Repeat the steps you followed above when fitting with the 2 state model The fitting procedure is identical for the two models e Click Done when finished A fit curve is generated and the fitting results copy to the Data Sampleldsc_cp ter text box in the Normdata ee eee Pee Chi 2 DoF 5 897E4 Window Note the difference in parameter Tm 60 79 30 0529 values between the two models AH 9 180E4 503 AH 9 947E4 675 To fit a curve with the 2 state w dCp model model 3 Model 3 is designed for use with data having a non zero ACp Model 3 requires that a baseline be associated with the data so
115. us version of the document you must update the document to version 7 0 To update a previous version of Origin Document project select File Update to Origin 7 0 Interface All templates will be updated to new templates compatible with version 7 0 You may then analyze the data with version 7 0 Please note when you update the Origin templates to Origin 7 0 most of the text labeling on the graphs will be lost including the fitting parameters If you want to save the old fitting parameters text you must copy the text before you update to Origin 7 0 To copy the fitting parameter text or any other text right click anywhere in the text box and select copy After you update to Origin 7 0 you may right click in any of the 7 0 templates and select paste Lesson 1 Basic Data Analysis Lesson 1 Basic DSC Data Analysis This first lesson presents an overview of basic DSC data analysis procedures You will learn how to start Origin read a DSC data file into Origin open multiple DSC data files subtract reference data from sample data normalize data by concentration create and subtract baseline data and use Origin s curve fitting models to fit a curve to your data We will also briefly discuss instrument calibration After working through this lesson you will know all of the basic procedures involved in analyzing DSC data Subsequent chapters look at specific procedural elements in more detail Starting Origin Ej Before starting this less
116. utorial all data were generated by a Windows based data collection software and so we will be using only the DSC data file opening procedure If your own data files are generated with DOS data collection programs you must open them via the MC 2 procedure Refer to Lesson 1 for more information about data import An isothermal mode of collecting experimental data 1s available for the VP DSC This mode may be used when it is desirable to use the DSC to monitor a process at a constant temperature for varying periods of time such as when determining kinetics of a chemical reaction at several different temperatures e g estimates of shelf life For this mode data is plotted Vs time rather than temperature Selecting Isoscan iso will read in data that has been collected in the VP DSC isothermal mode and will plot the data mcal min Vs time seconds Analyzing Previous Versions of Origin ORG Documents and Projects OPJ Shortcut Saved Origin documents or projects org or opj may be opened from explorer by double clicking on the file name To open a previous version of an Origin document project select File Open This menu command opens the Open dialog box Select Old version ORG from the List Files of type drop down list Select the desired file from the list box and click Open to close the dialog box and open the document You may then make formatting changes and print the graph If you wish to analyze the previo
117. ve data set then click the Find Peaks button The peak temperature should be ca 66 6 C for the protein with ligand 1 Close the Pick Peaks dialog box Pick Peaks Fe E Pick Peaks W Positive Search Rectangle Width Height Minimum Height E Display Options i Show Center W Show Label Find Peaks Lesson 7 Other Useful Details To calculate the ligand protein binding constant e Return to the RawDSC window i e select the menu item Window RawDSC Binding Constants from DSC fa x e Click the Binding Constant button and the Binding Constants from DSC dialog box will C Protein protein interations Tos T me Tor open up C Protein protein interactions Tm T a T 6 Cancel e Click the Ligand protein interactions radio button then click OK The Ligand protein interactions dialog box will open up as shown below e Enter the following values as determined in the Ligand Frotem I HES aaa preceding pages pages 78 80 Identification Ligand Protein Binding Constants Parameters To 61 C Tm 66 6 C To n 1 AH at T 110000 cal mole me ACp 1100 cal mole deg n sites per male Protein Conc 00009 moles l BEST Moo m Ligand Conc 010 moles l Cp 100 cal male deg Protein Conc f 00009 males l Ligand Conc o moles e Click OK The binding constant at Tm will be calculated and the Binding Results dialog box will open listing the Cancel

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