3 - GE Healthcare Life Sciences
Contents
1. 50 50 or 0 1 M DHB in water acetonitrile 50 50 Note that the protein samples are dissolved in the buffers and then mixed with the appropriate lyophilized enzyme Table 3 8 Examples of reagents and chemicals Use sequencing grade chymotrypsin and endoproteinase Asp N Use the dried droplet method to apply samples to the sample slide and leave the crystals to air dry for 5 minutes before beginning an analysis Suggested Evaluation Procedure 1 Filter spectra 2 Perform external calibration of each protein mass spectrum 3 Perform internal recalibration of each protein mass spectrum if peaks for internal calibration are present Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA Sample Preparation E Extract peak masses from protein mass spectrum 5 Compare measured mass with protein sequence 6 Perform external calibration of each tryptic peptide mass spectrum 7 Perform internal recalibration of tryptic peptide mass spectrum using trypsin autolysis peaks 8 Extract peak masses from the tryptic peptide mass spectrum 9 Remove background peak masses in tryptic peptide mass spectrum using a list of trypsin autolysis peptide masses 10 Compare measured tryptic peptide masses with unmodified protein sequence 11 Compare measured tryptic peptide masses with protein sequence allowing for common modifications 12 Steps 6 to 11 are repeated for chymotrypsin and endoproteinase Asp N respectively Results will p2. O A A CalBra die AE AD OR NE OE N N ENIGE E EP oran daa A DE RO GR ee OORSEE mees GE N oe RE ER Ee Ee Coomassie BIUS ESE Ge ai COS IR OE IE RE EE TOT DHB Ete Ee ie E rev is dried droplet method mairena E electrophoresis gel nia E A ge ENCOPIOLSIMaSe susan ia detal OD a EG LT MALDE TOE erro a Ge aA F fragmentation analysis taeng OROUCHIIS os G Bel IE A A RA COCO SR NA ie ta Ne Eie IG es SE RN H ii A a AG K ETES PE A se de ae ie n Se DE DR DR EE Ge E Ge ES ETE L linear detector mode HEESE GE EE GE EG Ee low mass rejection gate Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA Index O M ii EE PU II LI DO N EE IE N EEN 8 MATO sn ia 10 20 jie AO N N ER RE EE OO 29 P peptide mass fingerprinting ccooocccooccnnnccnnncnnoncnnoncnnononanarnnnnos 30 esse dIE SA EE OE EE 36 PROS ene M Eie ASe AE OE N Oe ed ON 36 post source decay anaconda 13 OO Po CR OO E OO E OE ai 7 DODANO sirep irene E PU A OE EE Ai 71e protein characterization omiso daa 32 33 o e Ue ER PO POPE EGO EE AA 9 R ento a A e O PES PO A LS reverse phase chromatography eie ee ee ee ee ee ee ee ee ee 18 sample extraction EO dla 19 o RR N EE RE OE EN 10 24 o N NE N N EE EN 30 seeded microcrystalline film method ee ee ee ee ee 28 SES No EE N RE EE RA 19 SOON CIO GE N EO EE 2 Slow crystallization method LL 27 sodium dodecyl sulphate es EA oe Geo oi oon ie ee 20 T thin layer method RR _POO0O
3. de book proteomics O Ettan MALDI TOF Method Handbook gb 18 1144 03 Amersham Biosciences All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group which supplies them A copy of these terms and conditions is available on request Ettan is a trademark of Amersham Biotech Limited Amersham is a trademark of Nycomed Amersham plc ZipTip is a trademark of Millipore Inc Amersham Biosciences AB 2000 All rights reserved Office Addresses Amersham Biosciences AB SE 751 84 Uppsala Sweden Amersham Biosciencse UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Biosciences Inc 800 Centennial Avenue P O Box 1327 Piscataway N J 08855 1327 USA Amersham Biosciences Europe GmbH Munzinger Strasse 9 D 79111 Freiburg Germany Amersham Biosciences KK Sanken Building 3 25 1 Hyakunincho Shinjuku ku Tokyo Japan Contents 1 Introduction 1 1 Symbols and common abbreviations ciaci n 8 2 An introduction to MALDI ToF MS 2 1 Principles of MALDI ToF mass spectrometry 10 2 2 Applications for MALDI ToF mass spectrometry 12 3 Sample Preparation S L Removal or contamina tds 17 3 2 Sample Extraction from Electrophoresis Gels 19 3 3 Choosing the A Ge De De tees 20 34 Preparing Solutions for Crystallization ua
4. 18 1144 03 Edition AA Sample Preparation E AER 35 3 Slow Crystallization Method The slow crystallization method may require some practice to achieve a successful result Useful when samples contain involatile solvents e g 30 glycerol high salt concentrations e g 4M NaCl low protein concentrations lt 1uM or if the dried droplet approach has failed Suitable for use with 4 HCCA and sinapinic acid 1 Add 1 2 ul of protein to 20 30 ul of saturated matrix solution Leave vial open in the dark for a minimum of 1 hour 00 id to allow microcrystals to form but note that the crystallization process may take several hours 3 Remove supernatant Wash twice with cold 4 C Step 2 0 1 aqueous TFA solution N 4 Add 1 ul of water Carefully make a slurry of crystals y do not crush Step 3 Apply slurry to sample slide N 6 Allow to dry 7 Insert into mass spectrometer Acquire mass spectrum Step 4 IN Step 5 Step 6 Step 7 AA Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA 27 ED Sample Preparation 3 5 4 Seeded Microcrystalline Film Method The seeded microcrystalline film method produces very uniform samples and the crystals are well adhered to the surface of the sample slide It is a direct replacement for the dried droplet technique May improve the mass resolution and mass accuracy obtained in reflectron mass spectrometers Suitable for use with 4 HCCA and sinapinic acid
5. 728187220070 278 e 2 7700 2 Identified proteins 210 721201 e Abundance up 007 oS SE SSS O Abundance down 2120 712 70le00 0 Unidentified proteins ee 200ee7e Data evaluated and potential protein markers and drug targets identified Biological experiments to elucidate function Target proteins identified Fig 2 1 The role of mass spectrometry in proteomic studies Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA 9 2 An introduction to MALDI ToF MS S 2 1 Principles of MALDI ToF mass spectrometry 10 A mass analysis begins by careful sample preparation Most frequently the source material originates from an SDS electrophoresis gel and samples must be extracted and cleaned before preparation for the MS analysis The cleaned protein or peptide is mixed with a matrix The matrix ensures that the surroundings of a protein or peptide are such that when subjected to the intense light pulse from a laser the molecules become ionized As the matrix crystallizes on the surface of the sample slide Fig 2 2 the proteins or peptides become incorporated into the crystals The sample slide is used to carry the samples into the flight tube of the mass spectrometer Fig 2 2 Sample slide from Ettan MALDI ToF Fig 2 3 illustrates the process that follows as soon as the samples are loaded into the mass spectrometer It is at this stage that Matrix Assisted Laser Desorption and Ionization MALDI begins Laser puls
6. Method Handbook 18 1144 03 Edition AA An introduction to MALDI ToF MS E However for mass analysis of a peptide mixture as required for protein on by peptide mass fingerprinting a MALDI ToF instrument is used in reflectron mode The masses in a peptide mixture are smaller and likely to be distributed over a narrow range of mass values The ions generated must be made to travel longer distances in order to enhance the differences in flight times between these ions The reflectron acts as an ion mirror which corrects for variations in energy of ions of equivalent mass charge m z ratio The reflectron increases the flight path of the ions by applying a stepwise increase in voltage across the ion flight path as illustrated in Fig 2 5 A longer flight path results in a greater separation between the different masses seen as higher resolution in the mass spectrum The overall result is a better time focus at the detector and high resolution mass spectra with excellent mass accuracy Peptides can be studied further by creating conditions under which the peptides themselves are broken apart within the mass spectrometer and the resulting fragments are analysed This analysis not only provides more information about the original protein but if initial identification has been unsuccessful it can also be used for database comparisons To perform fragmentation analysis a MALDI TOF instrument is used in reflectron mode but now the peptides are
7. Single high resolution result and ProFound identification report AAN Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA 7 ED Introduction Symbols and common abbreviations general advice that can improve procedures or recommendations for action under specific situations warning when special care should be taken in a procedure y MS mass spectrometry MALDI ToF matrix assisted laser desorption and ionization time of flight 4 HCCA a cyano 4 hydroxycinnaminic acid DHB 2 5 dihydroxybenzoic acid also known as gentisic acid 8 Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA An introduction to MALDI ToF MS 2 2 An introduction to MALDI ToF MS MALDI ToF mass spectrometry has become one of the most important tools for the identification of proteins and peptides and is particularly important in proteomic studies where it plays a key role in the identification of potential target proteins as illustrated in Fig 2 1 This chapter gives an overview of the key steps in mass analysis by MALDI ToF MS Normal cell Transformed cell D Extract protein fraction of interest 2 D PAGE PEIES Key Differential Abundance up display Abundance down Excise spots of interest Trypsin digest and MS analysis es sequence database searched
8. aqueous TFA solution Wait 2 10 seconds and remove washing solution by vacuum suction sos aa M Do not heat the droplet to speed up the drying process this will alter matrix crystallization Homogeneity of the film can be improved by adjusting the TFA concentration Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA 25 ED Sample Preparation AE 3 5 2 Thin Layer Method The thin layer method produces a uniform layer of small crystals that are mechanically well adhered to the surface of the sample slide allowing contaminants to be washed away Useful when samples contain non peptide contaminants such as buffering substances 4M urea or salts Suitable for use with 4 HCCA and sinapinic acid spread thinly Xx 1 Apply matrix solution diluted with isopropanol 2 Dry at ambient temperature to provide a very thin barely visible layer of matrix Step 1 3 Apply 0 5 ul of peptide protein in saturated matrix solution er A Leave to stand for 1 2 minutes until a milky white surface is visible Remove remaining droplet and Cd allow to dry N N 6 Wait up to 15 seconds and then remove washing solution by vacuum suction INN y 7 Insert into mass spectrometer Dry sample A thoroughly 1 2 minutes Step 5 9 Step 2 h Step 3 nN Wash with cold 4 C 0 1 aqueous TFA solution Step 4 Acquire mass spectrum Step 6 Step 7 es Step 8 26 Ettan MALDI ToF Method Handbook
9. prepared to be resistant to autodigestion for example trypsin that has undergone lysine alkylation Trypsin should not be protected by polyoside derivatisation of lysines as this can dramatically decrease efficiency when handling elycoproteins Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA 29 ED Sample Preparation 30 Identification by peptide mass fingerprinting Starting material is often a protein gel band isolated by SDS PAGE The protein is digested in gel with trypsin and the proteolytic peptides the digest are extracted from the gel and analysed The protein is identified by comparing the digest peaks with a computer generated database of tryptic peptides from known proteins Protein band cut out SDS PAGE gt F Enzymatic Peptide digestion extraction SO Fa Database search Protein m Fig 3 5 Identification by peptide mass fingerprinting Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA Sample Preparation E AA Table 3 6 gives examples of the chemicals and reagents that can be used as a basis for development of an optimized protocol Trypsin digestion Trypsin in 50 mM NH4C03 5 mM CaClo pH 8 0 0 2 0 5 ug trypsin CaCl is not required if trypsin has undergone lysine digestion alkylation Extraction solvent 0 1 0 5 aqueous TFA acetonitrile 30 70 or 40 60 Peptide mass hACTH fragment 18 39 2464 191 amu calibrants hACTH fragment 7 38 3656 923
10. tada 22 3 41 Dissolving the protein or peptide occonncoocnosoconccnernonanesss 23 SE BISSONE IET 23 3 5 Applying Sample to the Slide Surface ese ee see ee ee ee ee 24 HO Dried Droplet MOOD SEE ED EG A dose 25 3 92 Faour EV OF AVI CLOG ad Mantes N 26 SONS SIOW CIYVSTAUIZATON MONO os EER er ren 27 354 Seeded Microcrystalline Film Method ee ee ee 28 3 6 Preparation for Specific Applications usina dit 29 Ol Identification by peptide mass fingerprinting 30 DE Fotireharacterza ION Ti watts tees ES ns nt nel 32 0 Froti CATACTCIIZAUON MS ne ne nes 33 3 6 4 Identification of protein phosphorylation sites 36 Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA V Introduction ET 1 Introduction Ettan MALDI TOF combines the protein knowledge of Amersham Biosciences that of experts in protein identification Proteometrics LLC and mass spectrometry Scientific Analysis Instruments Ltd to offer a MALDI ToF mass spectrometer for fast reliable protein identification Ettan MALDI TOF is particularly suited for high throughput work requiring rapid identification and high sensitivity detection as in proteomic studies In order to obtain an accurate mass analysis and so achieve a reliable identification the correct sample extraction and preparation procedures are as crucial as the interpretation and evaluation of mass spectra The purpose of this handbook is to familia
11. 0R7E005UUC0 50 0 0 CQMQOOPPAS a 26 PP a eo 11 LS ies AR AE nn AE OE aeniays 79 30 DL 39 U dt RICO CO N A O OE Eo PO N EE 26 Z A A hea cays ne 18 Amersham Biosciences 40 Ettan MALDI TOF Method Handbook 18 1144 03 Edition AA
12. 1 Step 1 2 y 3 Step 2 y 4 lt 4 5 Step 3 6 y Step 4 G SS Step 5 Step 6 AA 28 Add matrix to acetone containing 1 2 water until the solution is nearly saturated Place a droplet e g 0 5 ul onto the sample slide and leave to spread out and dry forming a uniform deposit Dissolve the sample of interest in an aqueous solution containing 20 30 organic solvent Place approximately 0 5 ul onto the sample slide and leave to dry at room temperature Wash the dried deposit with pure water or 0 1 TFA for 2 10 sec and remove excess water Insert into mass spectrometer cquire mass spectrum Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA Sample Preparation E 3 6 Preparation for Specific Applications Sample preparation methods may need to be slightly adjusted in order to achieve the most satisfactory result for an application The general recipes described here can be used as a basis to develop optimized sample preparation methods protocols for some of the most common applications of mass analysis by MALDI ToF mass spectrometry Proteins that are already in solution should be prepared following the recommendations given earlier in this chapter Always use HPLC quality solvents reagent grade chemicals and the highest quality HPLC grade water Always use siliconized microtubes for sample processing to avoid loss of material by non specific adsorption Use sequencing grade trypsin that is
13. amu mix 50 50 with tle l Angiotensin 111 896 5232 amu matrix solution Dissolve lyophilized material in water 200 pmol ul dilute 10 fold with 0 5 TFA 50 acetonitrile Store at 20 C human adrenocorticotropic hormone Peptide matrix 4 HCCA in 0 5 TFA acetonitrile 50 50 or solution 0 1 M DHB in water acetonitrile 50 50 Table 3 6 Examples of chemicals and reagents used for peptide mass finger printing Use gloves to prevent keratin contamination Excised gels and extracted dried down sample pellets can be stored at 20 C or used immediately Always excise a small strip of gel from a background area to use as a control sample during analysis Use the dried droplet method to apply samples to the sample slide and leave the crystals to air dry for 5 minutes before beginning an analysis Suggested Evaluation Procedure 1 Filter spectra 2 Perform an external calibration of each protein tryptic digest mass spectrum and background control spectrum 3 Perform an internal recalibration of each protein tryptic digest mass spectrum using trypsin autolysis peaks 4 Extract peak masses 5 Remove background peak masses Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA 31 ED Sample Preparation AAA 6 Begin database search Results will provide a list of proteins that are identified at the selected significance level 3 6 2 Protein characterization Proteins of known sequence are characteriz
14. ate to the most intense peak However it should be noted that charge states depend not only on the matrix used but also on the concentration and nature of the acid present in the matrix solvent and on the nature and respective concentrations of peptides and proteins in the sample Selection of the most suitable matrix for a particular sample may be a case of trial and error However experience has shown that 4 HCCA is a versatile and easy to use matrix particularly for peptides and smaller proteins Table 3 4 gives guidelines as to the advantages and disadvantages of the matrix compounds for protein and peptide mass analysis First choice for peptides and smaller proteins lt 5 000 Da Intense signals for proteins and peptides No adduct peaks Protein ions may undergo metastable decay at very low laser energies and low protein concentration Sinapinic acid First choice for proteins Peptides lt 3 000 Da may give weak signals Alternative matrix for peptides Suitable when looking at metastable ions e g when performing PSD analysis on peptides May not always work with proteins Table 3 4 Guidelines for matrix selection Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA 21 ED sample Preparation TEE 3 4 Preparing Solutions for Crystallization Both the sample and the matrix should be dissolved separately before mixing and beginning the crystallization process Fig 3 2 to Fig 3 4 shows the crystalliza
15. ctor reflectron mode Laser N Accelerating 671 77 voltage Sample slide Fig 2 5 Quadratic field reflectron of Ettan MALDI TOF This unique quadratic field reflectron provides time focusing of all ions irrespective of energy over the entire range of m z Hence a complete fragment on mass spectrum is obtained under the same experimental conditions with each pulse of the laser as shown in Fig 2 6 Ser mee ss T Angiotensin III RVYIHPI Detection mode PSD intensity Ion gate mass 897 10000 Ion polarity Positive Acceleration potential e Ee Mass ii Dew 391 29 504 3 641 39 Pulsed extraction On EE Focus mass 897 6000 419 24 532 32 669 38 50 shots per spectrum ao E ia in Manual tuning ha B5 Fig 2 6 Example of a MS spectrum of Angiotensin II using PSD AAA 14 Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA An introduction to MALDI ToF MS E In all Ettan MALDI TOF modes a low mass rejection gate eliminates interference from low molecular weight compounds such as those generated by components of the matrix to further improve the detection and resolution of peaks in the mass spectrum Ettan MALDI ToF is designed to switch easily between linear and reflectron mode to give the performance required for a specific application Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA 15 Sample Preparation E 3 Sample Preparation 3 1 Removal of contaminants To achieve the accurate mass analysi
16. d to carry them into the flight tube of the mass spectrometer The dried droplet method is a suitable sample application method for the majority of applications For completeness three alternative methods are also described Thin layer and microfilm methods produce more noise than the dried droplet method and a lower signal intensity for peptides gt 3 000 Da Consequently the sensitivity may not be sufficient when working in reflectron mode However in linear mode the advantage of signal uniformity over the entire spot may outweigh any disadvantage of low sensitivity particularly when requiring high throughput and when acquisition is automated Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA Sample Preparation E E 35 1 Dried Droplet Method y The dried droplet method is the first choice for most applications Do not use non volatile solvents e g glycerol polyethylene glycol DMSO Triton X with this method Step 1 i ES 1 Mix peptides or proteins with saturated matrix NX solution A final concentrations peptide approximately 40 nM and protein approximately 10 uM Step 2 ED ca 2 Apply 0 5 ul of solution to the sample slide Step 3 3 Leave to dry at ambient temperature y 4 Insert into the mass spectrometer Dry sample thoroughly 1 2 minutes Acquire the mass spectrum Step 4 a 5 If heavy salt or buffer contamination is suspected remove from the vacuum and wash with cold 4 C XQ 2 0 1
17. e o Detector E ef ED c e E et gt gt L length of drift tube Fig 2 3 Matrix Assisted Laser Desorption lonization and Detection The sample slide and laser are together referred to as the ion source used for sample irradiation Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA An introduction to MALDI ToF MS E Short duration nanosecond pulses of laser light are directed at the crystals The energy absorbed causes a portion of the crystal s surface to be volatilized and ionized creating an ion cloud The application of an electrostatic field prompts the ions to move through the vacuum of the flight tube towards the detector If the ion cloud contains protein or peptide molecules they will also move The energy deposited by the laser can be adjusted to optimize conditions for this ion ablation step The gas phase ions are accelerated to a fixed energy i e a common kinetic energy and after passing through the flight tube they impact onto an ion detector By measuring the time between the application of the accelerating electrostatic field and the arrival of the ion at the detector A tm the mass of the ion can be determined by Time of Flight ToF mass analysis m 2gVA 1 m ion mass q ion charge V potential through which ion is accelerated length of flight tube The complete derivation of this equation is given in the Ettan MALDI ToF Instrument Handbook In simple t
18. ecipitation Always keep tubes closed when not in use Use fresh matrix solutions whenever possible Solutions will decompose under normal laboratory conditions Keep the concentration of non protein materials to a minimum It may be possible to wash the crystals to remove non protein materials Maintain the pH of the crystal growing solution below pH 4 by adding 0 1 1 0 TFA This avoids ionization of the matrices The optimal amount of TFA depends upon the specific matrix and analyte Aim for 5 10 pmoles protein or 10 fmoles of peptide on the sample slide Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA 23 ED Sample Preparation 24 y y LA MOST IMPORTANT There must be enough protein or peptide dissolved in the crystal growing solution to give a signal of sufficient intensity although reducing protein concentration can sometimes increase signal intensity Analyse as soon as possible after crystallization Do not leave matrix crystals in the protein matrix solution this causes loss of signal Remove by centrifugation if necessary Take care during sample handling protein adsorption to surfaces such as pipettes or Eppendorf tubes can significantly reduce the true sample concentration particularly if the original concentration is low 3 5 Applying Sample to the Slide Surface After mixing with the selected matrix solution the samples are applied onto the surface of the sample slide the stage use
19. ed i e the most common modifications identified and located after proteolysis in solution The starting material is the purified protein lyophilized or in solution The protein is digested with trypsin and proteolytic peptides are analysed by mass spectrometry Modifications are identified by comparing the proteolytic peptide masses with peptide masses calculated from the sequence of the unmodified versus modified protein Table 3 7 gives examples of the chemicals and reagents that can be used as a basis for development of an optimized protocol Trypsin digestion Trypsin in 50 mM NH4CO3 5 mM CaCl pH 8 0 CaCl is not required if trypsin has undergone lysine alkylation Protein mass calibrants Apomyoglobin 20 ng dissolve directly into protein carbonic anhydrase 70 ng matrix solution enolase 280 ng Protein matrix solution 0 04 M sinapinic acid 67 of a 0 1 aqueous TFA solution 33 acetonitrile Peptide mass calibrant solution hACTH fragment 18 39 2464 191 amu mix 50 50 with peptide matrix hACTH fragment 7 38 3656 923 amu solution We7l_Angiotensin 111 896 5232 amu Dissolve lyophilized material in water 200 pmol ul dilute 10 fold with 0 5 TFA 50 acetonitrile Store at 20 C human adrenocorticotropic hormone Peptide matrix solution 4 HCCA in 0 5 TFA acetonitrile 50 50 or 0 1 M DHB in water acetonitrile 50 50 Table 3 7 Examples of chemicals and reagents Use the dried droplet method to a
20. erms when all ions are equally charged a lighter mass will travel faster than a heavier mass As each ion reaches the detector a peak is generated creating a mass spectrum i e a plot of signal intensity generated against time as shown in Fig 2 4 The mass to charge ratio m z that corresponds to a particular peak is calculated using simple equations and the time to intensity data is converted into an m z to intensity histogram Intensity Medium mass Fig 2 4 Example of a mass spectrum Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA 11 2 An introduction to MALDI ToF MS 12 Each peak in a mass spectrum is now assigned a specific mass By recording time of flight mass spectra as intensity versus time of flight tables the mass to charge m z data is calculated as follows 12 at b miz Although the constants a and b are related to the geometry of the ion source and mass analyser they are most commonly determined by the simple and accurate method of selecting two peaks of known m and z and deriving the constants from their measured flight times With the constants a and b established the values can be applied to calibrate other spectra produced under the same experimental conditions Further details on calibration methods are outlined in the Ettan MALDI ToF User Manual It should be noted that a two point calibration is generally satisfactory when working with peptides However in some instruments this calibra
21. ic hormone Peptide matrix solution A HCCA in 0 5 TFA acetonitrile 50 50 or 0 1 M DHB in water acetonitrile 50 50 Table 3 9 Examples of chemicals and reagents Use gloves to prevent keratin contamination Excised gels and extracted dried down sample pellets can be stored at 20 C or used immediately Always excise a small strip of gel from a background area to use as a control sample during analysis Use the dried drop method to apply samples to the sample slide and leave the crystals to air dry for minutes before beginning an analysis Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA Sample Preparation E AA suggested Evaluation Procedure 1 Filter spectra 2 Perform an external calibration of each protein tryptic digest mass spectrum phosphorylated dephosphorylated and of the background control spectrum 3 Perform an internal recalibration of each protein tryptic digest mass spectrum phosphorylated dephosphorylated using trypsin autolysis peaks 4 Extract peak masses 5 Remove background peak masses 6 Compare measured mass of the phosphorylated and non phosphorylated tryptic peptide with protein sequence Results will provide a list of contaminant masses coverage of the protein sequence and localization of phosphorylation sites E Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA 97 Index Numerics O se a a seek ee tn altel Duta Via GE DE EG A OOPTE RS A A IE AS A C
22. ionized at an increased laser power thus giving them more internal energy Bonds are broken apart giving rise to a product ion and a neutral fragment This phenomenon is known as post source decay PSD The linear section of the Ettan MALDI TOF is used to separate ions of different m z ratio and bring them into time focus at the entrance to the reflectron A timed ion gate selects a precursor mass for PSD analysis in the reflectron In PSD experiments the energy of ionization is increased by the application of high laser power this generates ions of higher energy and promotes fragmentation When fragmentation occurs the fragment ions continue to travel with the same velocity as the precursor ion but have lower energy Thus fragments from a precursor ion and the intact precursor ion arrive at the timed ion gate simultaneously The flight times of the fragment ions in the reflectron vary by virtue of their differing m z ratio and a fragment ion spectrum is obtained In this way the fragments of a single specific peptide can be detected and analysed Ettan MALDI ToF contains a unique reflectron based on patented z technology that applies an increasing voltage non linearly to create a perfect quadratic field as shown in Fig 2 5 Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA 13 2 An introduction to MALDI ToF MS es Detector Reflectron linear mode voltage 72 Timed ion gate Dete
23. n The protein is digested in parallel with trypsin chymotrypsin and endoproteinase Asp N Proteolytic peptides are analysed Modifications are identified by comparing the proteolytic peptide masses with peptide masses calculated from the sequence of the unmodified versus modified protein Table 3 8 gives examples of the chemicals and reagents that can be used as a basis for development of an optimized protocol Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA 33 ED Sample Preparation 34 Trypsin or chymotrypsin digestions Trypsin or chymotrypsin in 50 mM approximately 0 2 ug trypsin and NH CO3 5 mM CaCl pH 8 0 0 2 ug chymotrypsin per digestion CaCls is not required if trypsin has undergone lysine alkylation Endoproteinase Asp N digestion Endoproteinase Asp N in 50 mM NH4 5PO3 pH 7 0 Protein mass calibrants Apomyoglobin 20 ng carbonic dissolve directly into protein matrix Janhydrase 70 ng enolase 280 ng solution Protein matrix solution 0 04 M sinapinic acid 67 of a0 1 POS mAn agur FA soluto 33 acetone Peptide mass calibrant solution hACTH fragment 18 39 2464 191 mix 50 50 with peptide matrix amu solution hACTH fragment 7 38 3656 923 amu le Angiotensin 111 896 5232 amu Dissolve lyophilized material in water 200 pmol ul dilute 10 fold with 0 5 TFA 50 acetonitrile Store at 20 C human adrenocorticotropic hormone Peptide matrix solution 4 HCCA in 0 5 TFA acetonitrile
24. oes not usually interfere with subsequent mass analysis Coomassie Blue R 350 is the most sensitive form of the dye available from Amersham Biosciences code no 17 0518 01 The highest quality HPLC grade water should be used for every step after the sample has been excised from the gel Although silver staining is 100 times more sensitive than Coomassie Blue 1t may interfere with mass analysis However a recent publication outlines a method that avoids this problem by using a modified silver staining protocol from which glutaraldehyde is eliminated Gharahdaghi et al Electrophoresis 20 p601 605 1999 Using the modified protocol while improving mass analysis results may reduce the sensitivity of the silver staining Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA 19 ED Sample Preparation 20 Detergents and excess salts are usually eliminated during normal staining procedures Strong ionic detergents such as sodium dodecyl sulphate SDS must be completely removed as they will interfere with crystallization and so disrupt the mass analysis 3 3 Choosing the Matrix The surroundings of a protein or peptide should ensure that when subjected to the intense light pulse of a laser the molecule is transferred in an ionized state into the flight tube To achieve this effect the sample is incorporated into crystals of a second material the matrix The sample containing crystal absorbs the light pulse and the abs
25. orbed energy causes desorption of material from the crystal s surface creating an ion cloud If the desorbed material contains protein or peptide molecules they will also enter the flight tube It should be noted that in earlier MALDI TOF instruments matrix compounds were seen to produce adduct peaks These are satellite signals resulting from the chemical breakdown of the matrix into more reactive species that bind to the protein or peptide They have slightly higher mass than the analyte molecule peaks However this phenomenon occurred when very high laser energies were required to compensate for lack of sensitivity Adduct peaks are rarely seen in today s more sensitive MALDI ToF instruments If salts are present in the sample then satellite peaks containing Na or K that replace protons as the charge donor may be seen Similar satellite peaks will also occur when using poor quality matrix substances Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA Sample Preparation E AA Table 3 3 shows the most commonly used matrix compounds and indicates the ionization states Matrix Molecular mass Peptides a cyano 4 hydroxycinnaminic Da acid 4 HCCA Sinapinic acid sinapic acid all Da trans 3 5 dimethoxy 4 hydroxycinnamic acid 2 5 dihydroxybenzoic acid 154 Da SE A A Table 3 3 Properties of commonly used matrices An increased ionization state increases the signal intensity and gives a higher charge st
26. pply samples to the sample slide and leave the crystals to air dry for 5 minutes before beginning an analysis 32 Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA Sample Preparation E Suggested Evaluation Procedure 1 Filter spectra 2 Perform external calibration of each protein mass spectrum 3 Perform internal recalibration of each protein mass spectrum if peaks for internal calibration are present 4 Extract peak masses from protein mass spectrum 5 Compare measured mass with protein sequence 6 Perform external calibration of each tryptic peptide mass spectrum 7 Perform internal recalibration of tryptic peptide mass spectrum using trypsin autolysis peaks 8 Extract peak masses from tryptic peptide mass spectrum 9 Remove background peak masses in tryptic peptide mass spectrum using a list of trypsin autolysis peptide masses 10 Compare measured tryptic peptide masses with unmodified protein sequence 11 Compare measured tryptic peptide masses with protein sequence allowing for common modifications Results will provide a comparison of measured protein mass with protein sequence a list of contaminant masses coverage of protein sequence and indicate possible modifications 3 6 3 Protein characterization Il Proteins of known sequence are characterized i e the most common modifications identified and located after proteolysis in solution The starting material is the purified protein lyophilized or in solutio
27. rize the reader with the key practical aspects that should be considered for successful protein identification Specific protocols are supplied for applications performed on Ettan MALDI TOF systems together with general background information that may be of use to those new to MALDI ToF mass spectrometry Intensity E File OneR2 16 Spectrum 1 Laser 0 Position 16 32 Date Wed Feb 16 11 56 32 2000 Shots 250 1500 1400 1300 12003 11003 10003 9004 800 7004 6004 5004 4004 3004 2004 1004 0 800 1000 1200 1400 160 1 800 2 000 2 200 2 400 2 600 Mass m z Version 4 10 7 ProFound Search Result Summary 1997 2000 ProteoMetrics Probability Protein Information and Sequence Analyse Tools T gi1351907 sp PO2769ALBU_BOVIN SERUM ALBUMIN 1 1 0e 000 2 41 PRECURSOR 34 33 729 Q gij 399493 gb 4 4 F30907 114 E002147_3 4E002147 unique E ne Da hypothetical membrane lipoprotein Ureaplasma urealyticum EE Sn 3 1 2e 051 gil6323487 refINP_013559 1 Yir454wp Saccharomyces cerevisiae 7 84 30641 T 37e 052 i 2il6319695 reffNP 009777 1 pyruvate carboxylase Pyc2p 9 61 13141 0 Saccharomyces cerevisiae 5 3 5e 052 i gil 7304192 eb 4 4F39228 1 AEDO3340 CG2093 gene product 6 55 36822 0 Drosophila melanogaster 6 1 9e 052 gl1173565lebl A 4051791 1 31906 golgin 245 Homo sapiens 10 53 24619 Fig 1 1 The Ettan MALDI ToF system
28. rovide a comparison of measured protein mass with protein sequence a list of contaminant masses coverage of protein sequence and indicate possible modifications Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA 35 ED Sample Preparation 36 Identification of protein phosphorylation sites Protein phosphorylation sites are identified by in gel trypsin digestion and comparison of phosphorylated dephosphorylated peptide maps The proteolytic peptides are analysed by Ettan MALDI TOF and the phosphorylation sites are identified by comparing the peptide masses with masses calculated from the protein sequence Table 3 9 gives examples of the chemicals and reagents that can be used as a basis for development of an optimized protocol Destaining solution for 50 mM NH4CO3 acetonitrile 50 50 or Trypsin bacterial alkaline Trypsin phosphatase in 50 mM NH COsz phosphatase digestion 5 mM CaCl pH 8 0 approximately 0 2 ug trypsin CaCls is not required if trypsin has digestion 0 01 ug phosphatase dephosphorylation undergone lysine alkylation Extraction solvent 0 1 aqueous TFA acetonitrile 30 70 or 40 60 Peptide mass calibrants hACTH fragment 18 39 2464 191 amu mix 50 50 with matrix solution IhACTH fragment 7 38 3656 923 amu We7l_Angiotensin 111 896 5232 amu Dissolve lyophilized material in water 200 pmol ul dilute 10 fold with 0 5 TFA 50 acetonitrile Store at 20 C human adrenocorticotrop
29. s needed for reliable identification the correct sample preparation is crucial The protein or peptide should be transferred intact into the flight tube of the mass spectrometer The presence of contaminants from previous techniques the solutions used to dissolve the sample and the type of matrix used to contain the sample on the slide surface may all affect this process The MALDI ToF technique is quite tolerant of certain contaminants Table 3 1 However to achieve a high quality mass analysis it is advisable to remove as many contaminants as possible or at least reduce them to acceptable levels When working with low amounts of material e g an extract from a weak 2D spot the removal of salt will improve the signal to noise ratio Potential contaminant Phosphate buffers Alkali metal salts Ammonium bicarbonate Table 3 1 Tolerance levels for contaminants in a MALDI ToF analysis A range of solvents and chemicals are frequently used for protein and peptide dissolution Although necessary they provide a source of unwanted contaminants during sample preparation and mass analysis The solvent chosen to dissolve the sample must be compatible with the matrix Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA 17 ED Sample Preparation A Table 3 2 18 shows the most commonly used solvents and chemicals together with the actions required to avoid any interference in a subsequent mass anal
30. the sample since the peptides bind in the presence of 0 1 TFA and are eluted in a small volume 1 4 ul of 50 acetonitrile 0 1 TFA Columns are often handmade by packing the RPC medium into microtips or obtained as commercial products such as Ziplip Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA e e y y Sample Preparation E 3 2 Sample Extraction from Electrophoresis Gels Samples are often extracted from an electrophoresis gel before being prepared for mass analysis For example in proteomic studies potential target proteins are selected during expression analysis a process that compares protein profiles between normal and altered tissue Two dimensional electrophoresis separations generate patterns of protein spots that can be visualized and compared by image analysis The proteins of interest are then cut from the gel The gel pieces are destained subjected to an in gel digest and the peptides extracted and cleaned as illustrated in Fig 3 1 Transfer gel piece Extract peptides to microtube desalt and analyze by MALDI TOF MS gt gt gt gt Destain Dehydrate Rehydrate Incubate and digest Fig 3 1 Preparation of protein spots for MS identification Coomassie Blue is recommended for visualization of protein spots The dye binds stoichiometrically to proteins a significant advantage when determining relative amounts of protein Staining is simple and the Coomassie Blue d
31. tion may not be completely linear so that higher terms will be required in the equation Under these circumstances an accurate calibration over a wider mass range should be obtained using more than two points multi point calibration Depending upon the matrix solution as well as other specific sample conditions the MALDI TOF spectrum may contain single or double charged species Evaluation software is used to identify the peaks that have originated from a single mass with different charges Ettan MALDI TOF uses the most powerful data analysis software available for the identification process The list of masses for example a peptide mass fingerprint generated from mass analysis of a proteolytic digest is compared with real or theoretically generated data stored in databases until a match is found or a new protein discovered 2 2 Applications for MALDI ToF mass spectrometry The diagram shown in Fig 2 3 illustrates a MALDI ToF mass spectrometer used in linear detector mode ions under the influence of an electric field travel in a straight line towards the detector and lighter masses arrive before the heavier masses In the simplest terms the mass relates to the time of flight and a mass spectrum shows the separation between the masses according to their flight time to reach the detector For mass analysis of full length proteins it is usually sufficient to use a MALDI Tof instrument in linear detection mode Ettan MALDI ToF
32. tion states of three of the most commonly used matrices These examples show pure crystals Their appearance may change according to the type of sample being analysed Fig 3 2 Crystals of a cyano 4 hydroxycinnaminic acid Fig 3 4 Crystals of 2 5 dihydroxybenzoic acid 22 Ettan MALDI ToF Method Handbook 18 1144 03 Edition AA Sample Preparation E AE 34 1 Dissolving the protein or peptide Please refer to Sections 3 1 and 3 2 3 4 2 Dissolving the matrix Typically a water organic solvent solution is used to dissolve the matrix compounds Use reagent quality acetonitrile methanol or propanol and the highest quality HPLC grade water Table 3 5 shows suggested water solvent ratios for solubilization of the matrix compounds Water Solvent 4 HCCA 22110 Lil Sinapinicacd eon DB Bros Table 3 5 Recommended water solvent ratios for matrix solubilization Conditions may need to be adjusted according to the type of sample application method see next section or if precipitation occurs Although the final crystallization mixture may contain acids salts lipids detergents and organic solvents it is essential that neither the matrix nor the protein or peptide precipitate when the two solutions mix There are several points to note for successful crystallization The protein must be truly dissolved in the solvent a slurry is not acceptable Avoid inadvertent changes in solvent composition that may cause pr
33. ysis Methods for the application of sample to the slide surface are also referred to in this table and are described in detail further on in this chapter Sodium azide MUST be removed Suppresses on CT fomaten the mass Spectrometer 1 2 3 v v and proteins High concentration formic acid Avoid long term exposure if mass accuracy is essential forms formyl derivatives of N terminal amino acids High concentration salts buffers Remove if possible May interfere with crystallization If present use thin layer method Strong ionic detergents MUST be removed Prevent incorporation of samples into growing crystals Non ionic detergents e g Triton X May interfere with crystallization avoid or N octylglucoside if possible Effects vary according to specific detergent e g N octylglcoside may have a beneficial effect at low concentrations If present use slow crystallization method N B this requires some practice Table 3 2 Potential contaminants Low signals calibration peptides cannot be seen and poor resolution noisy background spectrum indicate that the sample should be cleaned Run the first analysis on only half of the sample This will allow a second analysis on a cleaned up sample if required Reverse phase chromatography RPC is frequently used to remove salts and other low molecular weight substances Peptides are bound to an RPC column containing 10 20 um C18 medium This process desalts and concentrates
Download Pdf Manuals
Related Search