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ProtoArray Prospector v3.1 User`s Manual

1. To format the data open the results file in Excel and delete all columns except the Name column from the ProtoArray gal file ID column from the ProtoArray gal file and the columns containing the feature and background signals of interest Also delete any rows above the column header rows If necessary rearrange the columns and replace the header names as described in Table 1a For applications that require signal scatter compensation KSI and radioactive SMI applications the X and Y coordinate of the spots must be included A column with the protein description can be included as an option Append the columns with X and Y coordinate information as described in Table 1b immediately after the required data columns Note that the text you use as column headers must be identical to those noted in Table 1a and Table 1b including capitalization Save the file as a text txt file ProtoArray Prospector is then able to open and read the resulting file and analyze your data 10 Table 1a Required Data Column Header The column header must be identical to those shown below Contents Name ID Signal Background The name The position The values for the The values for the associated with a associated with a signal intensities background microarray feature microarray feature associated with intensities as defined in the Name field of the ProtoArray gal file as defined in the ID fie
2. 34 Analyzing Positive Control Data from ProtoArray Control Protein Microarrays 35 Analyzing Experimental Data from a Single ProtoArray Human Protein ae 37 Comparing Results from ProtoArray Human Protein Microarrays eee 41 Small Molecule Protein Interaction Mode cessere 42 o ucadgiz Mr CM uH C ees 42 Analyzing Positive Control Data from ProtoArray Control Protein Microarrays SMI Fluorescent copie SE ries Oe URN ot Seen RA exp Ene fut DRBREA C SAET Eeee MEA A UR DAR RENE 43 Analyzing Positive Control Data from ProtoArray Control Protein Microarrays SMI RadioactiVe E 45 Analyzing Experimental Data from a Single ProtoArray Human Protein Mictoartay 5 MI PIHOFeSCent ME 47 Analyzing Experimental Data from a Single ProtoArray Human Protein EE Ee E 48 Comparing Results from ProtoArray Human Protein Microarrays eee 49 Immune Response Profiling Mode eeeeeeeeeeeeeeeeeeeeee nennen nnn 54 OYOEVIe WV phe pred Geer 54 Analyzing Data from a Single ProtoArray Human Protein Microarray s sessssie seses 55 Comparing Results from ProtoArray Human Protein Mieroarras eee 56 Program el TE 57 SEV LE agitated a eu MI oN MID UU MEE UE 57 Analysis EE 58 bob ici T 60 Web Parameters KE 62 File Formals iia cda dnd akin i a a Ll ni ER CUR RARO LR RR NUR RES RE 63 Single Microarray Analysis Pile Fortitudine te eei e p
3. ProtoArray Prospector automatically opens data files performs analyses appropriate for selected applications creates files containing results that can be opened in Microsoft Excel and displays a summary of results in the application user interface ProtoArray Prospector is designed to identify any protein features present on a ProtoArray microarray with signal intensities indicating that the feature binds an exogenous probe or serves as a substrate for phosphorylation How the Software Functions There are three basic approaches embodied in the software e To normalize all signals from the protein features to their population mean creating a new set of normalized values expressed in terms of the population standard deviation the standard Z Score This method identifies features with signals that lie at the upper most end of the distribution of all signals from the protein features e To measure the signals of individual protein features relative to the background on the array calculated on a per subarray basis The Z Factor measures the signal to noise ration with a Z Factor gt 0 5 corresponding to a signal gt 2 fold above the noise e To compare individual values of the protein features to those from the negative control features on the same chip the CI P Value described in Appendix I and identify protein features least likely to be representative of the negative controls The use of these approaches is described in great
4. C Program Files Invitrogen Prosp Lot Number HA20169 Array ID Block Row Column Signal Used CV Z Score GenePix Flags pamiscis ol 18 EI A e4sriscio sl 18 mal ProtoArray Prospector is a freeware tool that performs a default analysis of data coming from the ProtoArray Human Protein Microarrays Please select the application Protein Protein Interaction Kinase Substrate Identification Immune Response Profiling or Small Molecule Profiling you are working with The first step is to use Load amp Analvze function to process GenePix or 4 column output files The processing results are saved into Single Microarray Results subfolder of the program working directory The Summary function shows the list of proteins that were found significantly interacting You can then use Compare function to compare two or more analysis results Please Register the program to receive information about ProtoArray updates and news Figure 1 Main Menu File gt Main Menu Command Action Load and Analyze Opens a dialog box for navigating to data files and prompts ProtoArray Prospector to perform an analysis of the data from a single microarray The results are displayed in the Result Display Pane Corresponding Result files are created and stored in the respective subfolders in the main Prospector folder and an entry is made in the Single Microarray Results section of the R
5. Z Roe where o signal sample standard deviation for the positive control features c signal sample standard deviation for the negative control features L mean signal for the positive control features A mean signal for the negative control features 78 Z Score A value that indicates how far and in what direction a signal from a specific protein feature deviates from the mean of the signals of all protein features The value is expressed in terms of standard deviations Specifically Zi X H Oo A where X the signal value from the k protein feature A mean signal for all protein features o signal sample standard deviation for all protein features References Zhang J H Chung T D Y and Oldenburg K R 2000 Confirmation of Primary Active Substances from High Throughput Screening of Chemical and Biological Populations A Statistical Approach and Practical Considerations J Com Chem 2 258 265 Sboner Andrea et al 2009 Robust Linear Model Normalization to Reduce Technical Variability in Functional Protein Microarrays Journal of Proteome Research 8 5451 5464 79 For support visit www lifetechnologies com support or email techsupport dlifetechn com www lifetechnologies com technologies 2 March 2015
6. data are available by selecting the experiment in the Result Navigation Tree and clicking the Show button The results will be contained in the control block of the Excel spreadsheet that opens Table 4 Test Return Negative Control Range Z Score A measure of the distance that the negative control features e g buffer lie below a constant value chosen to identify a reasonable lower point in the expected dynamic range of the system The value should preferably be less than 6 and the mean should be close to zero and much less than the positive controls Positive Control Gradient Control Z value A measure of the separation band between the positive and negative control features based on the measured dynamic range of the assay The value should be greater than 2 and the mean should be much greater than the negative controls Value The probability that the values from the control features present at different amounts are behaving as expected e g a differential gradient of positive controls giving differential levels of signal that are proportional to the gradient concentrations The value should be much smaller than 0 05 3l Procedure for Experimental Data Using a ProtoArray Human Protein Microarray 1 To begin the analysis open ProtoArray Prospector and set the application in the Mode Selection Menu to Ubiquitination Ligation Profiling mode 2 Select the Analyze button from the
7. 29 Analyzing Data from a Single ProtoArray Human Protein Microarray Procedure for a Positive Control Using a ProtoArray Control Protein Microarray 1 To begin the analysis open ProtoArray Prospector and set the application in the Mode Selection Menu in the upper right corner to Ubiquitination ligation profiling mode 2 Select the Analyze button from the Tool Bar and navigate to the file containing the data for the microarray assayed with the Array Control Protein Select the file and then click the Open button on the navigation window to import the data into Prospector and perform the analysis 3 ProtoArray Prospector automatically opens the file identifies the type of data contained in the file c performs the needed calculations i subtracts background if toggled on ii removes outliers from controls if toggled on iii calculates Z Scores iv performs control calculations see Appendix I d creates and saves files containing 1 all the results the Analysis file and 2 a summary of the results the Summary file ProtoArray Prospector saves these files to the respective subfolders in the main Prospector folder e inserts an entry into the Single Microarray Results section of the Result Navigation Tree f presents a summary of the result in tabular form in the Result Display Pane 30 Interpreting the Control Data If your system is working properly the results should have the pattern noted in Table 4 These
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9. Appendix I Calculate Z factor on per Sub Array When selected Z factors are calculated based on the negative control signals in the corresponding subarray otherwise all array negative control signals population is used 58 Significant Hits Identification Parameter Group Box This group box is used to change the criteria for identifying proteins that score as hits i e positive in the system The pull down text box allows you to choose one of three different metrics the Z Score the Z Factor or CI P Value e The Z Score is the default metric for the PPI mode and will return protein features with values greater than the cut off value default value of 3 e The Z Factor is the default metric for the SMI radioactive and fluorescent KSI IRBP and IRBP with Plasma modes and will return protein features with values greater than the cut off value default value of 0 4 e The CI P Value will return protein features with values less than the cut off limit default value of 1 number of proteins In all cases unchecking the default check box will reveal a text box in which you can enter the specific cut off level you desire Note that each metric can be used for any application if desired A brief description of the metric is included in the associated text pane Default Options button Resets the option to the default Load Options button Restores the program options to the last set saved Save Options button
10. Factors of all the corrected intensities of the human protein features 3 Identify the features that have a Z Factor greater than the user defined cut off value 0 4 by default These are the protein features that ProtoArray Prospector scores as positive for binding the exogenous test molecule When first using the ProtoArray system for identifying small molecule protein interaction we recommend that you start by performing a positive control assay employing a known small molecule protein pair and the ProtoArray Control Protein Microarray For fluorescently labeled small molecules Alexa Fluor 647 coupled staurosporine can be used to bind CAMK2A printed in each subarray For radiolabeled small molecules radiolabeled estradiol can be used to bind estrogen receptor printed in each subarray ProtoArray Prospector can analyze data based on control and experimental microarrays For details refer to the ProtoArray Applications Guide available from www lifetechnologies com Once the performance of the system is established you can then be confident that the experimental conditions are appropriate for identifying binding of new or uncharacterized proteins using the ProtoArray Human Protein Microarray In addition to identifying the proteins that score as positive on a single microarray ProtoArray Prospector also allows you to compare the results across several microarrays This will allow you to identify human proteins on the ProtoArray mic
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12. Tool Bar and navigate to the file containing the data for the microarray Select the file and then select the Open button on the navigation window to bring the data into ProtoArray Prospector and perform the analysis ProtoArray Prospector automatically a b C opens the file identifies the type of data contained in the file performs the needed calculations i subtracts background if toggled on ii calculates Z Scores iii performs control calculations see Appendix I iv identifies hits see Chapter 1 creates and saves files containing 1 all the results the Analysis file and 2 a summary of the results the Summary file ProtoArray Prospector saves these files to the respective subfolders in the main Prospector folder inserts an entry into the Single Microarray Results section of the Result Navigation Tree presents a summary of the results in tabular form in the Result Display Pane 32 Comparing Results from ProtoArray Human Protein Microarrays Overview ProtoArray Prospector also allows you to compare the results across several microarrays This allows you to identify human proteins on the ProtoArray microarrays that are ubiquitinated by different exogenous E3 ligases We recommend performing experiments with arrays from a single lot If different lot numbers are selected and the lot numbers have different array designs ProtoArray Prospector shows a warning message and asks if you would like
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14. after installation Please have an Administrator follow the required steps listed below to assign Write permission to all regular users Open Windows Explorer and go to C Program Files x86 Invitrogen Right click on the folder Prospector and select Properties In the Prospector Properties panel select the Security tab and click the Edit button In the Permissions panel select Users in the list under Group or user names a F Ye NY e In the dropdown list under Permissions for Users find the Write row click and select the corresponding checkbox in the Allow column 6 Click Apply and OK to complete r Permissions for Prospector m Security Objectname C Program Files x86 Invitrogen Prospector Group or user names S CREATOR OWNER Z SYSTEM M 82 Administrators CBDLH10BDN1 Administrators LU Zi lsers CBDLH10BDN1 Users 82 Trustedinstaller Add Remove Permissions for Users Allow Deny ery Read amp execute v D Listfolder contents iv Read y E 3 Jd Special permissions Learn about access control and permissions Cancel Apply 14 Some regular users may still encounter problems with the Prospector Analyzer application after this fix specifically around running Group Characterizations and Two Groups Comparisons under the Tools menu bar If these two options do not show up under the Tools menu bar then please follo
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16. less than the positive controls Positive Control Gradient Control Z value A measure of the separation band between the positive and negative control features based on the measured dynamic range of the assay The value should be greater than 2 and the mean should be much greater than the negative controls Value The probability that the values from the control features present at different amounts are behaving as expected e g a differential gradient of positive controls giving differential levels of signal that are proportional to the gradient concentrations The value should be much smaller than 0 05 26 Procedure for Experimental Data Using a ProtoArray Human Protein Microarray 1 To begin the analysis open ProtoArray Prospector and set the application in the Mode Selection Menu to Protein Protein Interaction mode 27 Select the Analyze button from the Tool Bar and navigate to the file containing the data for the microarray Select the file and then select the Open button on the navigation window to bring the data into ProtoArray Prospector and perform the analysis ProtoArray Prospector automatically a b C opens the file identifies the type of data contained in the file performs the needed calculations i subtracts background if toggled on ii calculates Z Scores iii performs control calculations see Appendix I iv identifies hits see Chapter 1 creates and saves fil
17. of variation for the signals from the two replicates is less than 0 5 The default setting of 1 number of proteins can be changed to any positive real number using the Analysis tab of the Program Options dialog box called by the Options command By default the background corrected signal is used for these calculations Operating System and Hardware Requirements ProtoArray Prospector runs on a computer that uses the Windows XP or Windows 7 operating systems and is connected to the Internet While it may run on previous versions of Windows those configurations are not supported The hardware requirements for ProtoArray Prospector are minimal and any computer capable of running Windows XP or Windows 7 should be adequate An Internet connection is required to download lot specific microarray configuration files from the Life Technologies website for the microarrays you analyze The recommended computer configuration is e A Pentium 4 processor 800 MHz or faster e 128 MB RAM e Atleast 50 MB of free hard drive space for storing the application and Result files If you decide to save the output from ProtoArray Prospector the amount of needed hard drive space will increase with the number of microarrays analyzed Data Files and Formats Input Files ProtoArray Prospector is designed to utilize three types of data files gpr files generated by GenePix Pro from Molecular Devices Corporation To generate gpr files from Ge
18. opens the file b identifies the type of data contained in the file c performs the needed calculations i subtracts background if toggled on ii removes outliers from controls if toggled on iii calculates Z Factor Values iv performs control calculations see Appendix I d creates and saves files containing 1 all the results the Analysis file and 2 a summary of the results the Summary file ProtoArray Prospector saves these files to the respective subfolders in the main Prospector folder e inserts an entry into the Single Microarray Results section of the Result Navigation Tree f presents a summary of the result in tabular form in the Result Display Pane 55 Comparing Results from ProtoArray Human Protein Microarrays Overview ProtoArray Prospector also allows you to compare the results across several microarrays This will allow you to identify human proteins on the ProtoArray microarrays that are bound by antibodies present in different serum samples and help you establish patterns of binding specificity Procedure 1 To begin the analysis open ProtoArray Prospector and set the application in the Mode Selection Menu to Immune Response Profiling or Immune Response Profiling with Plasma 2 Select the Compare button from the Tool Bar In the dialog box that opens select the checkboxes next to the single microarray analyses you would like to include in the comparison Click on the Next button 4 In
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20. should be much greater than the negative controls Value The probability that the values from the control features present at different amounts are behaving as expected e g a differential gradient of positive controls giving differential levels of signal that are proportional to the gradient concentrations The value should be much smaller than 0 05 Substrate Control Z value A measure of the separation band between the positive and negative control features based on the measured dynamic range of the assay The value should be greater than 2 and the mean much greater than the negative controls 36 Analyzing Experimental Data from a Single ProtoArray Human Protein Microarray Overview The objective of a kinase substrate identification experiment employing ProtoArray microarrays is to identify the proteins on the microarray human protein features that can be specifically phosphorylated by a user supplied kinase added to the system An important consideration in interpreting the results of these experiments is distinguishing between autophosphorylation of a protein feature or other possibilities such as specific y P ATP binding and the specific phosphorylation of that protein feature by the exogenous kinase While not absolutely required it is strongly recommended that you set up at least one experiment with a ProtoArray Human Protein Microarray to identify human proteins on the microarray that may non
21. specifically score as positive This single assay may then serve as a reference for all other assays employing the same buffer system Creating a reference data set for your system can be done by omitting all exogenous kinases from the assay ProtoArray Prospector is able to include this data in its analysis and exclude human proteins that score as false positive from your final results For a protein to score as positive on the experimental microarray the corrected feature intensity must be greater than 1 5 times that noted on the reference microarray In addition to identifying the proteins that score as positive on a single microarray ProtoArray Prospector also allows you to compare the results across several microarrays This will allow you to identify proteins on the microarrays that are phosphorylated by different exogenous kinases and help you establish patterns of substrate specificity 37 Analysis with a Reference Buffer Only Microarray 1 To begin the analysis open ProtoArray Prospector and set the application in the Mode Selection Menu to Kinase Substrate Identification mode Select the Analyze button from the Tool Bar and navigate to the file containing the data for the reference e g buffer only microarray Select the file and then select the Open button on the navigation window to import the data into Prospector and perform the analysis In the Control assay parameters dialog box that appears ensure the Buffer onl
22. the Open button You can select multiple files at once and ProtoArray Prospector opens each file and processes the data from each file ProtoArray Prospector v5 2 3 contains the required support files which are now included with the installation of the software 4 ProtoArray Prospector automatically a opens the file b identifies the type of data contained in the file and if needed obtains the required application specific information from ProtoArray Central c performs the needed calculations i applies signal scatter correction if toggled on ii removes outliers if toggled on iii subtracts background if toggled on iv performs control calculations see Appendix I d creates and saves files containing 1 all the results the Analysis file and 2 a summary of the results the Summary file ProtoArray Prospector saves these files to the respective subfolders in the main Prospector folder e inserts an entry into the Single Microarray Results section of the Result Navigation Tree f presents a summary of the result in tabular form in the Result Display Pane 45 Procedure Using a ProtoArray Control Protein Microarray continued 5 To view the results for all proteins on a microarray along with values for the control features on the array select the corresponding entry in the Single Microarray Results node in the Results Navigation Pane and then click on the Show button Note this function requires that you
23. the signals from the two replicates is less than 0 5 The default setting of 1 number of proteins can be changed to any positive real number using the Analysis tab of the Program Options dialog box called by the Options command By default the background corrected signal is used for these calculations CI P Value continued Small Molecule Interaction Profiling SMI Mode fluorescent and radioactive In the optional CI P Value analysis a hit in the SMI mode fluorescent or radioactive is defined as any protein where 1 CIP Values for both replicates are less than 1 number of unique proteins on the microarray AND 2 The coefficient of variation for the signals from the two replicates is less than 0 5 The default setting of 1 number of proteins can be changed to any positive real number using the Analysis tab of the Program Options dialog box called by the Options command By default the background corrected value is employed for these calculations in the SMI fluorescent mode and the background corrected value after the application of the signal scatter correction is employed for these calculations in the SMI radioactive mode Immune Response Biomarker Profiling IRBP and IRBP with Plasma Mode In the optional CI P Value analysis a hit in the IRBP and IRBP with Plasma mode is defined as any feature with 1 CIP Values for both replicates are less than 1 number of unique proteins on the microarray AND 2 The coefficient
24. uninstall ProtoArray Prospector go to Control Panel and run Add Remove Programs Select ProtoArray Prospector in the list and click Remove Note that only files installed during ProtoArray Prospector setup and not modified since then will be removed All analysis results will be retained If you do not need these files you will have to manually remove files from the ProtoArray Prospector folder Changing Installation Options To add more features to ProtoArray Prospector already installed on your computer run the installation again Options are given to modify installation repair and uninstall Prospector 16 ProtoArray Prospector User Interface Overview The ProtoArray Prospector user interface contains the tools required to analyze your data using built in analyses and comparisons To start the application open Windows Start menu and select Programs gt Invitrogen gt Prospector gt Prospector Analyzer The interface consists of six elements that include the 1 Main Menu 2 Tool Bar 3 Mode Selection Menu 4 Result Navigation Tree 5 Result Display Pane and 6 Help Pane see Figure 1 Each is described in detail below Main Menu Tool Bar Result Navigation Tree Result Display Pane Help Pane 17 ProtoArray TM Prospector 5 2 Mode Selection Menu Summary Results EE X I E Y Application Protein Protein Interaction f G2 0031377 txt Result File
25. you have a licensed version of Microsoft Excel installed on your computer To view the summaries from previous comparisons double click the corresponding entries in the Microarray Comparison Results section in the Results Navigation Pane Comparing Data Across Multiple Microarrays WITHOUT Using an Unlabeled Competitor SMI Fluorescent i To begin the analysis open ProtoArray Prospector and set the application in the Mode Selection Menu to Small Molecule Profiling Fluorescent mode Select the Compare button from the Tool Bar In the dialog box that opens select the checkboxes next to the single microarray analyses you would like to include in the comparison Click on the Next gt button In the Compare Order Files dialog box that opens adjust the order in which the information will appear in the output for the different data sets you have chosen To change the name select the corresponding lt file gt entry in the Alias column and enter the name in the text box that appears Select the Finish button ProtoArray Prospector automatically a opens the file b identifies the type of data contained in the file and if needed obtains the required application specific information from ProtoArray Central c performs the needed calculations i removes outliers if toggled on ii subtracts background if toggled on iii performs control calculations see Appendix I d creates and saves files containing 1 all the
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27. Excel A user interface providing the tools needed to select the appropriate set of analyses or comparisons based upon the biochemical application of interest the Application Mode is selected from the pull down menu on the upper right tools for navigating to files changing options relating to individual pre defined analyses etc viewers for display of summaries and listings of previously performed analyses and a variety of other functions ProtoArray Prospector is capable of automatically recognizing the specific type of ProtoArray microarray employed e g human or control and automatically applying the correct analysis within a biochemical application ProtoArray Prospector will prompt the user for additional input to successfully run the analysis In addition the pre defined analyses contain a variety of methods for evaluating and employing the signals from the controls features e g the Buffer features that are typically present on ProtoArray microarrays and used differently depending on the selected application Software Workflow The general workflow consists of the following steps 1 Open ProtoArray Prospector and set the Application Mode using the pull down menu in the upper right of the user interface 2 Use the Analyze button to open single microarray data files in ProtoArray Prospector and the software analyzes the data ProtoArray Prospector automatically identifies features scoring as a hit in the assay base
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29. Microarray Summary IRP or Single Microarray Summary IRP_Plasma subfolder depending upon the application selected and are created after the user clicks on the Analyze button The file content is different for control and experimental arrays e for control array analysis Summary file shows results of controls quality check and distribution parameters of different control groups e for experimental array analysis Summary file shows the list of significant hit proteins Comparison File Formats Two files are created as a result of the Compare function The Result file is put into Comparison Results PPI Comparison Results KSI Comparison Results ULP Comparison Results SMF Comparison Results SMR Comparison Results IRP or Comparison Results IRP_Plasma subfolders depending on the application selected This file contains complete report of the comparison completed in the tab separated text format The Summary file is put into Comparison Summary PPI Comparison Summary KSI Comparison Summary ULP Comparison Summary SMF Comparison Summary SMR Comparison Summary IRP or Comparison Summary IRP_Plasma subfolder depending on the application selected This file contains a user friendly presentation of the most significant analysis results in HTML format The Summary file is automatically shown in the Result Display Pane after the comparison is completed 64 Comparison File Formats continued Comparison Result File This file has the comparison re
30. Navigation Tree serves the following functions e Helps keep track of multiple analyses and comparisons The software creates a unique name for each of the analyses or comparisons that it performs In general the name displayed in the Result Navigation Tree corresponds to the name ProtoArray Prospector assigns to the data file created for each analysis e Changes the results displayed in the Result Display Pane Double click the entry or select the entry and press the Summary button in the tool bar to display the summary results from that analysis You can remove an entry in the tree by selecting it and clicking the Delete button Note that this action also deletes the underlying data files from the subfolders in the main Prospector folder e Shows contents reflecting the specific Application Mode that was used to analyze the data The listing in the pane changes depending on the setting of the Application Mode Asa convenience the two nodes in the Result Navigation Tree are collapsible by simply clicking on the box next to the node If collapsed they can be expanded by clicking the box Result Display Pane ProtoArray Prospector displays a summary of single microarray analyses or multiple microarray comparisons in the Result Display Pane The displayed results can be changed by selecting the desired analysis or comparison in the Result Navigation Tree and pressing the Summary button The results displayed in this pane are the summaries of th
31. Saves any changes to the program options for later use 59 Warning Tab Program Options Analysis Wamings Web Parameters File Overwriting Options Do not overwrite files with the same name always append a version number C Always overwrite file names and give no waming Misc IV Inform user of the SMI competition option when selecting compare Figure 4a File Overwriting Options Group Box The series of radio buttons alters the manner that ProtoArray Prospector names new files that might be created when reanalyzing data from the same source file see Figure 4a Default Options button Resets the option to the default Load Options button Restores the program options to the last set saved Save Options button Saves any changes to the program options for later use 60 Warning Tab continued Helpful Tip Competition Analysis For assays performed with and without an unlabeled compound a competitive analysis can be performed See the option available under Tools gt Competition Analysis Hide this tip for subsequent selections Figure 4b Misc Group Box When checked a pop up will appear to inform the user of options when comparing data across multiple microarrays for the SMI application see Figures 4a and 4b 61 Web Parameters Tab Program Options Analysis Wamings Web Parameters URL to update ProtoAray Application Information PAI files Aools invitrogen com conte
32. USER GUIDE ProtoArray Prospector v5 2 3 Publication Number MANO0013674 Revision A 0 For Research Use Only Not for use in diagnostic procedures novex oy Life technologies technologies For Research Use Only Not for use in diagnostic procedures Information in this document is subject to change without notice DISCLAIMER TO THE EXTENT ALLOWED BY LAW LIFE TECHNOLOGIES AND OR ITS AFFILIATE S WILL NOT BE LIABLE FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING YOUR USE OF IT IMPORTANT LICENSING INFORMATION These products may be covered by one or more Limited Use Label Licenses By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses TRADEMARKS 2015 Thermo Fisher Scientific Inc All rights reserved All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified GenePix is a registered trademark of Molecular Devices Corporation Microsoft is a registered trademark of Microsoft Corp ii Contents Il HE HOER em 1 OVeDVIe Wesen dese ee Rude EA EE 1 Definitions for ES oec ee uke resins inns wha Ber itane ue vases dep UE 3 Operating System and Hardware Requirements 2e teer teer eee re tk 9 Data Files and ee 10 NET EE 13 Installing ProtoArray Prospector weeder tdeo iius re ante EE 13 Installation on Computers with the M
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34. an analyze data based on both control and experimental microarrays The recommended workflow is summarized in Table 5 For details refer to the ProtoArray Applications Guide available from www lifetechnologies com Table 5 Step Description Perform positive control to confirm that the system works by probing a 1 ProtoArray Control Protein Microarray with a control kinase MAPK14 p38 alpha 2 Perform positive control to confirm that the system works for your kinase of interest by probing a ProtoArray Control Protein Microarray with your kinase Perform a control to identify any arrayed proteins that autophosphorylate bind 3 ATP etc by probing a ProtoArray Human Protein Microarray using all reagents exceptthe kinase Perform experiment to identify potential kinase substrates on experimental 4 ProtoArray Human Protein Microarray These targets should score as positives with the users kinase of interest but not score positive in Step 3 34 Analyzing Positive Control Data from ProtoArray Control Protein Microarrays Overview The recommended first steps in using ProtoArray microarrays for identifying kinase substrates is ensuring that your system is working properly using ProtoArray Control Protein Microarrays The positive control for the system may employ a control kinase described below as Step 1 in ProtoArray Prospector or a different user supplied kinase described below as Step 2 in Pro
35. ary of the results in the Results Display Pane on the right side of the application s main window The summary is a list of proteins that score as hits see manual for details You can view the summaries of hits in other single microarray analyses by double clicking the entry in the Single Microarray Results node in the Results Navigation Tree To view the results for all proteins on a microarray along with values for the control features on the array select the corresponding entry in the Single Microarray Results node in the Results Navigation Pane and then click on the Show button Note this function requires that you have a licensed version of Microsoft Excel installed on your computer Analyzing Experimental Data from a Single ProtoArray Human Protein Microarray SMI Radioactive Procedure for SMI Radioactive Application 1 To begin the analysis open ProtoArray Prospector and set the application in the Mode Selection Menu to Small Molecule Profiling Radioactive mode 2 Select the Analyze button from the Tool Bar 3 In the Open dialog box that appears select the type of file gpr or txt from the Files of type pull down list and navigate to your data file s Select the file s for analysis and click the Open button You can select multiple files at once and ProtoArray Prospector opens each file and processes the data from each file ProtoArray Prospector v5 2 3 contains the required support files which are now included w
36. ate fluorescent signal values taking into account corrections for background and the negative control features on the microarray 2 Calculate Z Factors for all the corrected intensities of the human protein features Identify the features that have Z Factors greater than the user defined cut off value 0 4 is the default These are the protein features that ProtoArray Prospector scores as positive for binding the primary antibody In addition to identifying the spotted proteins that score as positive for antibody binding on a single microarray ProtoArray Prospector also allows you to compare the results across several microarrays This will allow you to identify human proteins on the ProtoArray microarrays that are bound by antibodies in different serum samples and helps you establish patterns of binding specificity 54 Analyzing Data from a Single ProtoArray Human Protein Microarray Procedure for Experimental Data Using a ProtoArray Human Protein Microarray 1 To begin the analysis open ProtoArray Prospector and set the application in the Mode Selection Menu to Immune Response Profiling or Immune Response Profiling with Plasma 2 Select the Analyze button from the Tool Bar and navigate to the file containing the data for the microarray Select the file and then select the Open button on the navigation window to import the data into ProtoArray Prospector and perform the analysis 3 ProtoArray Prospector automatically
37. ay Prospector and set the application in the Mode Selection Menu to Small Molecule Profiling Fluorescent mode Select the Analyze button from the Tool Bar In the Open dialog box that appears select the type of file gpr or txt from the Files of type pull down list and navigate to your data file s Select the file s for analysis and click the Open button You can select multiple files at once and ProtoArray Prospector opens each file and processes the data from each file ProtoArray Prospector v5 2 3 contains the required support files which are now included with the installation of the software ProtoArray Prospector automatically a opens the file b identifies the type of data contained in the file and if needed obtains the required application specific information from ProtoArray Central c performs the needed calculations i removes outliers if toggled on ii subtracts background if toggled on iii performs control calculations see Appendix I d creates and saves files containing 1 all the results the Analysis file and 2 a summary of the results the Summary file ProtoArray Prospector saves these files to the respective subfolders in the main Prospector folder e inserts an entry into the Single Microarray Results section of the Result Navigation Tree f presents a summary of the result in tabular form in the Result Display Pane After processing the data ProtoArray Prospector displays a summ
38. by selecting the Show button When this file is created a corresponding file is also created in HTML format and its contents are displayed in the Results Display Pane Single Microarray Summary lt PPI KSI ULP SMI or IRP gt Files containing a summary of the analysis performed on data from a single microarray in HTML format The contents of this file are displayed in the Results Display Pane when the corresponding name is selected in the Result Navigation Tree Comparisons Results lt PPI KSI ULP SMI or IRP gt Files containing all results from comparing two or more microarrays These files are standard text txt files and can be opened and viewed in any text editor or spreadsheet application This file is opened by selecting the Show button When this file is created a corresponding file is also created in HTML format and its contents are displayed in the Results Display Pane Comparison Summary PPI KSI ULP SMI or IRP gt Files containing a summary from a comparison of two or more microarrays in HTML format The contents of this file are displayed in the Results Display Pane when the corresponding name is selected in the Result Navigation Tree 12 Installation Installing ProtoArray Prospector v5 2 3 Download and extract the zip archive Prospector_v5 2 3 zip with the installation executable from the Online Tools page accessible from the BioMarker Discovery Resources section a
39. cant Hits Identification Parameters Z Factor The Z Factor for a pair of protein features Use standard cut off a Z Factor gt 0 4 associated with that feature pair and negative controls You may choose to use all the negative controls on the array or only those in the same sub array as that feature pair Default Options Load Options Save Options Cancel Apply Figure 3 Analysis to be Performed Group Box Background Subtraction Used in the PPI SMI fluorescent IRBP or IRBP with Plasma mode to subtract the background values in the source file from the signal values In the KSI or SMI radioactive mode it subtracts the median signal value for the all the negative control features set in the PAI file typically buffer and all GST features in each subarray from the signal values of all other features spots in that subarray This is applied after Signal Scatter Compensation is applied if selected and any control features with signals judged as outliers are identified and removed from the analysis if selected Signal Scatter Compensation Only available in the KSI and SMI radioactive mode The algorithm adjusts a feature s signal value to compensate for scatter from any neighboring features The calculations are described in Appendix I Outlier Detection Applies an algorithm to detect control feature signals that are outliers and remove them from the analysis The calculations are described in
40. ch file and processes the data from each file ProtoArray Prospector v5 2 3 contains the required support files which are now included with the installation of the software 4 ProtoArray Prospector automatically a opens the file b identifies the type of data contained in the file and if needed obtains the required application specific information from ProtoArray Central c performs the needed calculations i removes outliers if toggled on ii subtracts background if toggled on iii performs control calculations see Appendix I d creates and saves files containing 1 all the results the Analysis file and 2 a summary of the results the Summary file ProtoArray Prospector saves these files to the respective subfolders in the main Prospector folder e inserts an entry into the Single Microarray Results section of the Result Navigation Tree f presents a summary of the result in tabular form in the Result Display Pane 43 Procedure Using a ProtoArray Control Protein Microarray continued 5 After processing the data ProtoArray Prospector displays a summary of the results in the Results Display Pane on the right side of the application main window The summary is a list of proteins that score as hits see page 3 for details You can view the summaries of hits in other single microarray analyses by double clicking the entry in the Single Microarray Results node in the Results Navigation Tree 6 To view the r
41. d on the default or user selected criteria presents a summary of the results and stores the results in a series of files 3 To compare results across microarrays that have already been analyzed select the Compare button and then select the results you wish to compare When started the software performs the selected comparison presents a summary of the comparison and stores the results as files in a series of subfolders located within the ProtoArray Prospector directory Definitions for Hits ProtoArray Prospector utilizes a series of pre defined analyses to identify protein features that score as hits in the system Three different approaches are taken by the software e A standard Z Score normalization that compares each probe signal to that of all the signals from the entire protein feature population e A Z Factor calculation to measure the signal to noise ratio e Anovel algorithm that compares the signal from each protein feature to a control distribution derived from the signals that comprise the negative controls on the microarray the Chebyshev s Inequality P Value or CI P Value Z Score A Z Score indicates how far and in what direction the value of an individual data point in a population falls from the mean in units of standard deviations ProtoArray Prospector collects all the signals from the proteins on a microarray calculates the mean value and standard deviation and then calculates a Z Score correspondin
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43. e analyses and comparisons showing only the protein features on the microarray s that score as positive The results for all the features in the selected entry can be viewed by selecting the Open button that opens the underlying file in Microsoft Excel Alternatively the corresponding files can be found in the series of Analysis subfolders in the Prospector main folder and opened using a spreadsheet program or text editor that handles text files The Result Display Pane provides a mechanism to obtain additional information about your features that score as hits The features listed in the Database ID column are hyperlinked to an appropriate public database and when selected will open the results in your default web browser For human microarrays the contents of the name field are linked to the nucleotide database at NCBI or the Ensembl transcript database at EMBL EBI the Sanger Institute The Result Display Pane also provides a similar mechanism to obtain additional information about any features from human microarrays that have an Ultimate ORF Card entry with In Life Technologies Hyperlinks to the corresponding features are found in the Ultimate ORF ID column and the ORFCard also opens in your default browser The Result Display Pane is not opened when you first start ProtoArray Prospector 22 Help Pane The Help Pane contains useful information to help you get started using ProtoArray Prospector 23 Protein Protein Interaction M
44. e the order for displaying the information or rename the way the information will be displayed Select the Finish button 5 ProtoArray Prospector automatically a opens the file b identifies the type of data contained in the file and if needed obtains the required application specific information from ProtoArray Central c performs the needed calculations i removes outliers if toggled on ii subtracts background if toggled on iii performs control calculations see Appendix I d creates and saves files containing 1 all the results the Analysis file and 2 a summary of the results the Summary file ProtoArray Prospector saves these files to the respective subfolders in the main Prospector folder e inserts an entry into the Microarray Comparison Results section of the Result Navigation Tree f presents a summary of the result in tabular form in the Result Display Pane 6 ProtoArray Prospector displays a summary of the proteins that score as positive across the selected microarrays in the Results Display Pane 49 Comparing Data Across Multiple Microarrays Using an Unlabeled Competitor SMI Fluorescent continued 7 Proteins are identified as specific hits if they score as positive in the assay without a competitor and the signal is reduced by a user defined amount default setting 30 in the presence of an unlabeled competitor 8 To view the results for all the proteins on all the microarrays you compar
45. ed select the corresponding entry in the Microarray Comparison Results section of the Result Navigation Tree and select the Show button Note this function requires that you have a licensed version of Microsoft Excel installed on your computer 9 To view the summaries from previous comparisons double click the corresponding entries in the Microarray Comparison Results section in the Results Navigation Pane 50 Comparing Data Across Multiple Microarrays Using an Unlabeled Competitor SMI Radioactive 1 51 To begin the analysis open ProtoArray Prospector and set the application in the Mode Selection Menu to Small Molecule Profiling Radioactive mode Select the Competition Analysis from the Tools pull down menu In the Competition Analysis dialog box that opens select the checkboxes next to the microarray results from assays without unlabeled competitor that you would like to include in the comparison Click on the upper gt gt button to move them into the Assay without Competitor field Next if any assays were run in the presence of unlabeled competitor select the checkboxes next to the microarray results from assays with unlabeled competitor that you would like to include in the comparison Click on the lower gt gt button to move them into the Assay with Competitor field Select the Next button If desired rearrange the order for displaying the information or rename the way the information will be displayed Select t
46. ein where 1 The average Z Factor is greater than 0 4 AND 2 The coefficient of variation for the signals from the two replicates is less than 0 5 The default setting of 0 4 can be changed to any positive number less than or equal to 1 0 using the Analysis tab of the Program Options dialog box called by the Options command By default the background corrected signal is used for these calculations Cl P Value The CI P Value approach is an option in the PPI ULP KSI SMI both fluorescent and radioactive and IRBP modes However calling hits with the CI P Value is not recommended with the KSI mode Protein Protein Interaction PPI Mode In the optional CI P Value analysis a hit in the PPI mode is defined as any protein where 1 CI P Values for both replicates are less than 1 number of unique proteins on the microarray AND 2 The coefficient of variation for the signals from the two replicates is less than 0 5 The default setting of 1 number of proteins can be changed to any positive real number using the Analysis tab of the Program Options dialog box called by the Options command By default the background corrected signal is used for these calculations Ubiquitin Ligase Profiling ULP Mode In the optional CI P Value analysis a hit in the ULP mode is defined as any protein where 1 CI P Values for both replicates are less than 1 number of unique proteins on the microarray AND 2 The coefficient of variation for
47. elected and are created after the user clicks on the Analyze button The file contains three blocks header probes list and controls list The file is a tab delimited text file The header block contains information about analysis parameters and control results e source file name and path e lot number array type and application e creator version and date e negative controls test e positive controls test e gradient controls test e substrate control test e population parameters of each control group and probes e significant hit detection parameters used 63 Single Microarray Result File continued The array content block contains a table of all protein features on the array with a number of columns identifying protein position on the array and content analysis results and significant hit flag e ahuman array protein is identified by the database type ID and Ultimate ORF ID e acontrol array does not contain protein features and this block is omitted The controls list block contains a table of all controls grouped by control type each row contains control feature content identification and array location and analysis results Single Microarray Summary File This file contains an abstract of the most significant single microarray analysis results These files are found in the Single Microarray Summary PPI Single Microarray Summary KSI Single Microarray Summary ULP Single Microarray Summary SMF Single Microarray Summary SMR Single
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49. er detail in the next section In addition to the fundamental analyses of the protein features ProtoArray Prospector also provides a number of analyses for interpreting the behavior of the on chip control features Software Elements ProtoArray Prospector consists of the following three general elements A set of pre defined numeric analyses that are automatically applied to the data derived from an individual ProtoArray microarray assay These data are imported from an image processing program in the form of a data file e 2 a gpr file After executing the pre defined analyses a Single Microarray Result file automatically named Analysis of source file name gt txt is created for each microarray This file can be conveniently opened in Microsoft Excel Executing the pre defined analyses also presents a general summary in the ProtoArray Prospector user interface of proteins from each microarray that score as a hit and the average values used to make the call Analyses allowing a series of cross chip comparisons of two or more ProtoArray microarrays using the Single Microarray Result files created by the output of the analyses described above These comparisons collect and tabulate the different sets of features that score as hits on individual ProtoArray microarrays and create a Comparison Results file automatically named Comparison Results a textual description of the comparison txt that can also be conveniently opened in
50. es containing 1 all the results the Analysis file and 2 a summary of the results the Summary file ProtoArray Prospector saves these files to the respective subfolders in the main Prospector folder inserts an entry into the Single Microarray Results section of the Result Navigation Tree presents a summary of the results in tabular form in the Result Display Pane Comparing Results from ProtoArray Human Protein Microarrays Overview ProtoArray Prospector also allows you to compare the results across several microarrays This allows you to identify human proteins on the ProtoArray microarrays that are bound by different exogenous proteins and help you establish patterns of binding specificity We recommend performing experiments with arrays from a single lot If different lot numbers are selected and the lot numbers have different array designs ProtoArray Prospector shows a warning message and asks if you would like to continue or stop the comparison Arrays from different array versions i e v4 0 v4 1 or v5 0 cannot be compared within the software ProtoArray Prospector shows an error message and stops the comparison Procedure 1 To begin the analysis open ProtoArray Prospector and set the application in the Mode Selection Menu to Protein Protein Interaction mode 2 Select the Compare button from the Tool Bar 3 In the dialog box that opens select the checkboxes next to the single microarray analyses you wou
51. es web site at http www lifetechnologies com termsandconditions If you have any questions please contact Life Technologies 74 Appendix I Calculations Chebyshev s Inequality CI P Value An algorithm that calculates the p value for the null hypothesis that a given signal belongs to negative control features distribution 1 X SU O CI P Value o j m X gt He oO X k He where X the signal value from the k protein feature A mean signal for the negative control features o signal sample standard deviation for the negative control features Leave One Out Chebyshev s Inequality LOOCI Outlier Detection A series of algorithms applied to sets of control features to identify and then exclude features with signals that appear to be outliers Specifically the algorithm first defines X asthesignal for thei replicate in the g control group X asthefor thei smallest signal in the g control group where g 1 2 representing the control group number e g buffer i 1 2 n where n is the number of replicate features for the g group Next the algorithm determines X and X gng To determine signals that indicate they are outliers the algorithm then calculates the minimum and maximum mean leave out values along with the corresponding sample standard deviations SD Ng 2 X i X a n l D minimum mean X g 0 1a 75 Zu H 5 v ic 8i Sin maximu
52. esult Navigation Tree Summary Opens the results from a single microarray analysis or comparison in the Result Display Pane The command is only active when an entry in the Result Navigation Tree is selected Compare Opens a dialog box for choosing previously performed single microarray analyses for comparison and prompts ProtoArray Prospector to perform the comparison The results are displayed in the Result Display Pane Corresponding Result files are created and stored in the respective subfolders in the main Prospector folder and an entry is made in the Microarray Comparison Results section of the Result Navigation Tree Delete Options Deletes the selected entry in either the Single Microarray Results or Microarray Comparison Results section of the Result Navigation Tree This action also deletes the underlying files ProtoArray Prospector stores in the respective subfolders in the main Prospector folder Opens the Program Options dialog box Exit Exits ProtoArray Prospector 18 Tools gt Main Menu Command Action Group Characterization Two Group Comparison These items will appear in the Tools gt menu Menu items are only enabled when Immune Response Profiling or Immune Response Profiling with Plasma application is selected See IRBP Toolbox manual for more information Vector Xpression Stores the contents of the selected results entry to a Vector Xpres
53. esults for all proteins on a microarray along with values for the control features on the array select the corresponding entry in the Single Microarray Results node in the Results Navigation Pane and then click on the Show button Note this function requires that you have a licensed version of Microsoft Excel installed on your computer 44 Analyzing Positive Control Data from ProtoArray Control Protein Microarrays SMI Radioactive Overview The recommended first steps in using ProtoArray microarrays for identifying small molecule interacting proteins is ensuring that your system is working properly using ProtoArray Control Protein Microarrays The control microarrays contain a positive control feature that you can use to check that your system can detect an interaction with a known control feature e g H estradiol as a ligand for the Estrogen Receptor The ProtoArray Control Protein Microarrays also contain a number of negative control features to evaluate the specificity of your system Procedure Using a ProtoArray Control Protein Microarray 1 To begin the analysis open ProtoArray Prospector and set the application in the Mode Selection Menu to Small Molecule Profiling Radioactive mode 2 Select the Analyze button from the Tool Bar In the Open dialog box that appears select the type of file gpr or txt from the Files of type pull down list and navigate to your data file s Select the file s for analysis and click
54. f significant hits has exactly the same column set as the Comparison Results File but only probes with corresponding hit count are listed and only values selected for hit identification Z Score Z Factor or CI P Value are shown 65 ProtoArray Application Information File Format The ProtoArray Application Information file contains information about array type and control features that ProtoArray Prospector uses to correctly analyze experimental results This file is application specific the file extension is PAI and the name is composed of the application abbreviation e g PPI PAT All PAI files are located in the Application Lot Specific Files subfolder These files are included with the ProtoArray Prospector application for v5 0 and v5 1 microarrays ProtoArray Prospector automatically downloads PAI files from the Life Technologies web portal if the required file is not found in the subfolder The file is a tab delimited text file It contains a header and a control table The header of the file contains general information about the array and application Value Description ProtoArray Application Information File Version xxx ID of this file type Version of this file ArrayType Human Experimental Array Known array types Human Experimental Array Control Array Application Protein Protein Interaction Known applications Protein Protein Interaction Ubiquitin Ligase Profil
55. from the Tool Bar and navigate to the file s containing the data for the microarray s assayed with your unknown or uncharacterized kinase s Select the file s and then select the Open button on the navigation window to import the data into Prospector and perform the analysis 3 In the Control assay parameters dialog box that appears ensure the Customer kinase step 4 radio button is selected 4 ProtoArray Prospector automatically a opens the file b identifies the type of data contained in the file c performs the needed calculations i applies signal scatter correction if toggled on ii removes outliers from controls if toggled on iii subtracts background if toggled on iv calculates Z Factors v performs control calculations see Appendix I vi identifies hits see Chapter 1 d creates and saves files containing 1 all the results the Analysis file and 2 a summary of the results the Summary file ProtoArray Prospector saves these files to the respective subfolders in the main Prospector folder e inserts an entry into the Single Microarray Results section of the Result Navigation Tree f presents a summary of the result in tabular form in the Result Display Pane 40 Comparing Results from ProtoArray Human Protein Microarrays Overview The software compares results across multiple arrays to identify human proteins that are phosphorylated on more than one microarray with different unknown or uncharacter
56. g to the signal from each feature see Appendix I for details The Z Score approach is set as the default in the PPI mode and is available as an option in the KSI ULP SMI both fluorescent and radioactive IRBP and IRBP with Plasma application modes Protein Protein Interaction PPI Mode By default a hit in the PPI mode is defined as any protein where the two replicate features of the protein 1 Have an average Z Score of greater than three AND 2 The coefficient of variation for the signals from the two replicates is less than 0 5 The default setting of three standard deviations can be changed to any positive real number using the Analysis tab of the Program Options dialog box called by the Options command By default the background corrected value is employed for these calculations Ubiquitin Ligase Profiling ULP Mode By default a hit in the ULP mode is defined as any protein where the two replicate features of the protein 1 Have an average Z Score of greater than three AND 2 The coefficient of variation for the signals from the two replicates is less than 0 5 Note that by default the background corrected value is employed for these calculations in the SMI fluorescent mode and the background corrected value after the application of the signal scatter correction is employed for these calculations in the SMI radioactive mode The default setting of three standard deviations can be changed to any positive real
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58. have a licensed version of Microsoft Excel installed on your computer Interpreting the Control Data If your system is working properly the results should have the pattern noted in Table 7 The details for the calculations of the returned values are described in Appendix I Table 7 Test Return Range Z Score A measure of the distance that the negative control features e g buffer lie below a constant value chosen to identify a reasonable lower point in the expected dynamic range of the system The value should preferably be less than 6 and the mean should be close to zero and much less than the positive controls Negative Control Z value A measure of the separation band between the positive and negative control features based on the measured dynamic range of the assay The value should be greater than 2 and the mean should be much greater than the negative controls Positive Control Value The probability that the values from the control features present at different amounts are behaving as expected e g a Gradient Control differential gradient of positive controls giving differential levels of signal that are proportional to the gradient concentrations The value should be much smaller than 0 05 46 Analyzing Experimental Data from a Single ProtoArray Human Protein Microarray SMI Fluorescent Procedure for SMI Fluorescent Application ib 47 To begin the analysis open ProtoArr
59. he noise The Z Factor approach is set as the default in the KSI SMI both fluorescent and radioactive IRBP and IRBP with Plasma modes and is available as an option in the PPI mode Small Molecule Interaction Profiling SMI Mode fluorescent and radioactive By default a hit in the SMI mode fluorescent or radioactive is defined as any protein where 1 The average Z Factor is greater than 0 4 AND 2 The coefficient of variation for the signals from the two replicates is less than 0 5 The default setting of 0 4 can be changed to any positive number less than or equal to 1 0 using the Analysis tab of the Program Options dialog box called by the Options command By default the background corrected value is employed for these calculations in the SMI fluorescent mode and the background corrected value after the application of the signal scatter correction is employed for these calculations in the SMI radioactive mode Kinase Substrate Identification KSI Mode By default a hit in the KSI mode is defined as any feature where 1 The average Z Factor is greater than 0 4 AND 2 The coefficient of variation for the signals from the two replicates is less than 0 5 AND 3 The mean signal of the replicates is at least 1 5 times that of the mean signals from the corresponding features on the reference array when data from a reference microarray is available The default setting of 0 4 can be changed to any positive number less tha
60. he Finish button ProtoArray Prospector automatically a opens the file b identifies the type of data contained in the file and if needed obtains the required application specific information from ProtoArray Central c performs the needed calculations i applies signal scatter correction if toggled on ii removes outliers if toggled on iii subtracts background if toggled on iv performs control calculations see Appendix I d creates and saves files containing 1 all the results the Analysis file and 2 a summary of the results the Summary file ProtoArray Prospector saves these files to the respective subfolders in the main Prospector folder e inserts an entry into the Microarray Comparison Results section of the Result Navigation Tree f presents a summary of the result in tabular form in the Result Display Pane ProtoArray Prospector displays a summary of the proteins that score as positive across the selected microarrays in the Results Display Pane Proteins are identified as specific hits if they score as positive in the assay without a competitor and the signal is reduced by a user defined amount default setting 30 in the presence of an unlabeled competitor To view the results for all the proteins on all the microarrays you compared select the corresponding entry in the Microarray Comparison Results section of the Result Navigation Tree and select the Show button Note this function requires that
61. icrosoft Windows 7 Operating System 14 Upgrading from a Previous Version of ProtoArray Drospechor 16 Uninstalling ProtoArray Prospector s ssiatebodcsp te oe VIDT o OR RR DU MS 16 Changing Installation e iecit S Dni bd eren d dp t HR eda dy 16 ProtoArray Prospector User Interface eesseeeeeeeee reete tette nnns 17 RIVE V LOW um 17 E DE a cer eet EEE pence tp ESEE EESK EENEN wos ana E e Ere En E STEE ok 18 Tool Bat oa ieee op EM MIC Oh pe dE MU EE 20 EE 21 Result Navigation RE 22 Result Display ANC eneen 22 Help BARN cusses chokes 23 Protein Protein Interaction MOdEC cssssssssessssseesseesseesneeseeeseseseeeseenseenasenseenaes 24 Keier C 24 Analyzing Data from a Single ProtoArray Human Protein Microarray s sessssiesesesee 29 Comparing Results from ProtoArray Human Protein Microarrays eee 28 Ubiquitination Ligation Profiling Mode eeeeeeeeeeeeeeeeeernnnnnnne 29 usd M TTE 29 Analyzing Data from a Single ProtoArray Human Protein Microarray s sessssiesesesee 30 Comparing Results from ProtoArray Human Protein Microarrays eee 33 iii Kinase Substrate Identification Mode ee KEN KEREN KEN RENE KEREN EEN RENE KENNEN KEREN 34 CIV CEV LOW scsi
62. ing Kinase Substrate Identification Immune Response Profiling Immune Response Profiling with Plasma SMlI Radioactive SMlI Fluorescent Column name The control table contains following columns Description Control Name The character string that control names starts with All features with names starting from this string are grouped together Type Known types Negative Positive Gradient Substrate Ignore Comment A description of the control 66 If a control has two functions for example it is both positive and a gradient the PAI file will contain two lines for the control All features with names not listed in Control Names are considered by the program as probes Input File Formats GenePix Tab Delimited File To import a GenePix GPR file this tab delimited file must start with the signature ATF and have the following column headings Feature Column Name in GenePix Protein Name Name Spot ID ID Spot Signal F lt wavelengths gt Median Spot Background B lt wavelengths gt Median Note that the ProtoArray Prospector will identify the wavelength you have set in GenePix ProtoArray Prospector ignores all other columns and column headings The file must be formatted to use ASCII text Tab Delimited Microarray Data File To import data generated by an image processing program that does not have a GPR output option it must be con
63. ing features on the reference array 2 When data from a reference microarray is NOT available a hit is defined as any protein where the two replicate features of the protein a Have an average Z Score of greater than three AND b The coefficient of variation for the signals from the two replicates is less than 0 5 The default setting of three standard deviations can be changed to any positive real number using the Analysis tab of the Program Options dialog box called by the Options command By default the background corrected value after the application of the signal scatter correction is employed for these calculations Immune Response Biomarker Profiling IRBP and IRPB with Plasma Modes In the optional Z Score analysis a hit in the IRBP and IRBP with Plasma mode is defined as any protein where the two replicate features of the protein 1 Have an average Z Score of greater than three AND 2 The coefficient of variation for the signals from the two replicates is less than 0 5 The default setting of three standard deviations can be changed to any positive real number using the Analysis tab of the Program Options dialog box called by the Options command By default the background corrected value is employed for these calculations Z Factor The Z Factor calculation measures the dynamic range and the variation associated with the control and sample features A Z Factor gt 0 5 indicates a signal greater than 2 fold above t
64. ion An algorithm available only when ProtoArray Prospector is in the Kinase Substrate Identification or SMI radioactive modes that adjusts the signal value of a feature to compensate for signal scatter from neighboring features Specifically X new X oa up where i defines all the neighbors within a N x N square around this feature but the feature itself R is the distance to the i feature in logical units A 0 078 0 2 K 4 N 5 B is a background value which is delimited by the signal intensity from the feature which falls at the 10 percentile of all features of the subarray TI Z Factor A metric that takes into account the signal dynamic range and the variation associated with the control and sample features to measure the quality of a screening assay It is defined by Zhang and colleagues Zhang et al 2000 as Z wp ees SET di u he l where o signal sample standard deviation for the protein features o signal sample standard deviation for the negative control features A mean signal for the protein features A mean signal for the negative control features Z Factor The ratio of the overall variability of the positive and negative control features to the dynamic range of the control values It is based on the signals of the positive and negative features as set by the content of the respective PAI file It is defined by Zhang and colleagues Zhang et al 2000 as 36 330 Ia He l
65. ith the installation of the software 4 ProtoArray Prospector automatically a opens the file b identifies the type of data contained in the file and if needed obtains the required application specific information from ProtoArray Central c performs the needed calculations i applies signal scatter correction if toggled on ii removes outliers if toggled on iii subtracts background if toggled on iv performs control calculations see Appendix I d creates and saves files containing 1 all the results the Analysis file and 2 a summary of the results the Summary file ProtoArray Prospector saves these files to the respective subfolders in the main Prospector folder e inserts an entry into the Single Microarray Results section of the Result Navigation Tree f presents a summary of the result in tabular form in the Result Display Pane 5 After processing the data ProtoArray Prospector displays a summary of the results in the Results Display Pane on the right side of the application s main window The summary is a list of proteins that score as hits see manual for details You can view the summaries of hits in other single microarray analyses by double clicking the entry in the Single Microarray Results node in the Results Navigation Tree 6 To view the results for all proteins on a microarray along with values for the control features on the array select the corresponding entry in the Single Microarray Resul
66. ithin or for any United States federal agency are provided with LIMITED RIGHTS and RESTRICTED RIGHTS as defined in DFARS 252 227 7013 and FAR 52 227 19 15 GOVERNING LAW SEVERABILITY This Agreement shall be governed in all respects by the laws of the State of California USA without regard to its conflicts of law rules or principles Any dispute arising out of or related to this Agreement shall be resolved only in the state or federal courts having subject matter jurisdiction in California Each party hereby consents to the exclusive jurisdiction and venue of such courts If any provision of this Agreement is held to be illegal or unenforceable for any reason then such provision shall be deemed to be restated so as to be enforceable to the maximum extent permissible under law the remainder of this Agreement shall remain in full force and effect 72 GENERAL This Agreement is the parties complete agreement regarding its subject matter superseding any prior oral or written communications representations or agreements You understand and agree that to the extent Licensor permits you to use a non Licensor purchase order or other form to order Software Licensor does so solely for your convenience Any terms in any such forms that purport to vary or are in addition to or inconsistent with any terms in this Agreement shall be deemed to be void and of no effect Amendments or changes to this Agreement must be in mutually executed writin
67. ive Immune Response Profiling or Immune Response Profiling with Plasma The different modes employ different analyses that are appropriate for analyzing the five different types of data Choose the appropriate mode see Figure 2 for your application to obtain the best results ls File View Tools Help X a Application Protein Protein Interaction i ions Show J KI Analyze Compare Summary ab HE Delete Op Invitrogen About Single Microarray Results amp Microarray Comparison Results ProtoArray Prospector is a freeware tool that performs a default analysis of data coming from the ProtoArray Human Protein Microarrays Please select the application Protein Protein Interaction Kinase Substrate Identification Immune Response Profiling or Small Molecule Profiling you are working with The first step is to use Load amp Analyze function to process GenePix or 4 column output files The processing results are saved into Single Microarray Results subfolder of the program working directory The Summary function shows the list of proteins that were found significantly interacting You can then use Compare function to compare two or more analysis results Please Register the program to receive information about ProtoArray updates and news Done Nmf 4 Figure 2 21 Result Navigation Tree ProtoArray Prospector is designed to analyze and compare a large number of microarrays The Result
68. ized kinases ProtoArray Prospector performs the comparisons using the results from previously analyzed single microarrays We recommend performing experiments with arrays from a single lot If different lot numbers are selected and the lot numbers have different array designs ProtoArray Prospector shows a warning message and asks if you would like to continue or stop the comparison Arrays from different array versions i e v4 0 v4 1 or v5 0 cannot be compared within the software ProtoArray Prospector shows an error message and stops the comparison Procedure 1 To begin the analysis open ProtoArray Prospector and set the application in the Mode Selection Menu to Kinase Substrate Identification mode 2 Select the Compare button from the Tool Bar 3 In the dialog box that opens select the checkboxes next to the single microarray analyses you would like to include in the comparison Note that if you have performed the recommended buffer only control experiment you do not need to include those in the comparison Select the Next button 4 In the Compare Order Files dialog box that opens adjust the order in which the information will appear in the output for the different data sets you have chosen To change the name select the corresponding file gt entry in the Alias column and enter the name in the text box that appears Select the Finish button 5 ProtoArray Prospector automatically a compares the results of the si
69. l c performs the needed calculations i applies signal scatter correction if toggled on ii removes outliers if toggled on iii subtracts background if toggled on iv performs control calculations see Appendix I d creates and saves files containing 1 all the results the Analysis file and 2 a summary of the results the Summary file ProtoArray Prospector saves these files to the respective subfolders in the main Prospector folder e inserts an entry into the Single Microarray Results section of the Result Navigation Tree f presents a summary of the result in tabular form in the Result Display Pane 35 Interpreting the Control Data If your system is working properly the results should have the pattern noted in Table 6 The details for the calculations of the returned values are described in Appendix I Table 6 Test Return Negative Control Range Z Score A measure of the distance that the negative control features e g buffer lie below a constant value chosen to identify a reasonable lower point in the expected dynamic range of the system The value should preferably be less than 6 and the mean should be close to zero and much less than the positive controls Positive Control Gradient Control Z value A measure of the separation band between the positive and negative control features based on the measured dynamic range of the assay The value should be greater than 2 and the mean
70. ld like to include in the comparison Click on the Next button 4 In the Compare Order Files dialog box that opens adjust the order in which the information will appear in the output for the different data sets you have chosen To change the name select the corresponding lt file gt entry in the Alias column and enter the name in the text box that appears Select the Finish button 5 ProtoArray Prospector automatically a compares the results of the single array analyses b identifies proteins that score as hits in all the analyses If more than two analyses have been selected ProtoArray Prospector also identifies proteins that score as hits in smaller subsets of analyses c creates and saves files containing 1 all the results the Analysis file and 2 a summary of the results the Summary file ProtoArray Prospector saves these files to the respective subfolders in the main Prospector folder d inserts an entry into the Microarray Comparison Results section of the Result Navigation Tree e presents a summary of the result in tabular form in the Result Display Pane 28 Ubiquitination Ligation Profiling Mode Overview The objective of a ubiquitin ligation profiling experiment employing ProtoArray Human Protein Microarrays is to identify protein s on the microarray human protein features that can be specifically ubiquitinated by the E3 ligase being tested in the presence of purified E1 E2 and biotinylated ubiqui
71. ld of the ProtoArray gal file the features in that row of the data matrix associated with the features in that row of the data matrix Table 1b Data for Signal Scatter Compensation Contents Column Header The column header must be identical to those shown below X Y Description The X coordinate of the spot in micrometer units The Y coordinate of the spot in micrometer units The description of the microarray feature Optional 11 Output Files When analyzing data ProtoArray Prospector automatically creates a number of files containing the results of the analyses and stores them in subfolders that are automatically created in the main Prospector folder The subfolders that are created by the application and their contents are described in Table 2 ProtoArray Prospector displays the contents of the Summary files in the program user interface All the files that ProtoArray Prospector creates can be read by either a web browser the html files or any application capable of opening and displaying a text file the txt files Table 2 Subfolder Comments Single Microarray Results lt PPI KSI ULP SMI or IRP gt Files containing all results from the analysis of the data from a single microarray These files are standard text txt files and can be opened and viewed in any text editor or spreadsheet application This file is opened
72. m mean X i l 1b 8 n n Sr EEGEN minimum SD Bee Zait E S 1c Hou pauper a X a n E DG pay maximum SD EE e 1d d n 2 D Using the results from equations 1a d the absolute standard normal values are calculated Gei E X acu Kn 2a 5g D X gn gin 8 H SR 2b Ze un If K a Jn then X is an outlier and should be ignored in future analyses Additionally this step should be repeated with n reduced by 1 and X a removed from the analysis Similarly if K Jn then X n 18 an outlier and should be ignored in future analyses and the step repeated with n reduced by 1 and X n removed from the analysis Note that if this routine needs to be repeated then the ordered values i e X needs to be adjusted accordingly as well gi 76 Range Z Score This algorithm is only applied to negative control features It is designed to measure how far below a constant value chosen to represent the lower end of the expected dynamic range of the system at which the negative controls lies in terms of the standard deviation for the control features It is calculated as C Range Z Score Ta gast e Where A mean signal for the negative control features c signal sample standard deviation for the negative control features C 65536 20 this value represents the lowest 5 of the expected dynamic range for a sixteen bit system Signal Scatter Compensat
73. more features on the ProtoArray microarray The easiest approach is to use the Array Control Protein supplied with the ProtoArray Control Protein Microarray for PPI and a ProtoArray Control Protein Microarray See the ProtoArray Applications Guide available from www lifetechnologies com for details on control experiments Once the performance of the system is established you can then be confident that the experimental conditions are appropriate for identifying binding of new or uncharacterized proteins using the ProtoArray Human Protein Microarrays In addition to identifying the proteins that score as positive on a single microarray ProtoArray Prospector also allows you to compare the results across several microarrays This will allow you to identify human proteins on the ProtoArray microarrays that are bound by different exogenous proteins and help you establish patterns of binding specificity 24 Analyzing Data from a Single ProtoArray Human Protein Microarray Procedure for a Positive Control Using a ProtoArray Control Protein Microarray 1 To begin the analysis open ProtoArray Prospector and set the application in the Mode Selection Menu in the upper right corner to Protein Protein Interaction mode 2 Select the Analyze button from the Tool Bar and navigate to the file containing the data for the microarray assayed with the Array Control Protein Select the file and then click the Open button on the navigation window
74. n or equal to 1 0 using the Analysis tab of the Program Options dialog box called by the Options command By default the background corrected value after the application of the signal scatter correction is employed for these calculations Immune Response Biomarker Profiling IRBP and IRBP with Plasma Modes By default a hit in the IRBP or IRBP with Plasma mode is defined as any protein where 1 The average Z Factor is greater than 0 4 AND 2 The coefficient of variation for the signals from the two replicates is less than 0 5 The default setting of 0 4 can be changed to any positive number less than or equal to 1 0 using the Analysis tab of the Program Options dialog box called by the Options command By default the background corrected signal is used for these calculations Z Factor continued Protein Protein Interaction PPI Mode In the optional Z Factor analysis a hit in the PPI mode is defined as any protein where 1 The average Z Factor is greater than 0 4 AND 2 The coefficient of variation for the signals from the two replicates is less than 0 5 The default setting of 0 4 can be changed to any positive number less than or equal to 1 0 using the Analysis tab of the Program Options dialog box called by the Options command By default the background corrected signal is used for these calculations Ubiquitin Ligase Profiling ULP Mode In the optional Z Factor analysis a hit in the ULP mode is defined as any prot
75. n select the OK button Note that you only need to run the reference buffer only portion of the experiment once for each human microarray assay ProtoArray Prospector allows you to repeatedly reuse those data for the respective experimental microarrays on slides with the same lot number 38 Analysis with a Reference Buffer Only Microarray continued 7 39 ProtoArray Prospector automatically a opens the file b identifies the type of data contained in the file c performs the needed calculations i applies signal scatter correction if toggled on ii removes outliers from controls if toggled on iii subtracts background if toggled on iv calculates Z Factors v performs control calculations see Appendix I vi compares the experimental to the reference data and identifies hits see Chapter 1 d creates and saves files containing 1 all the results the Analysis file and 2 a summary of the results the Summary file ProtoArray Prospector saves these files to the respective subfolders in the main Prospector folder e inserts an entry into the Single Microarray Results section of the Result Navigation Tree f presents a summary of the result in tabular form in the Result Display Pane Analysis WITHOUT a Reference Buffer Only Microarray 1 To begin the analysis open ProtoArray Prospector and set the application in the Mode Selection Menu to Kinase Substrate Identification mode 2 Select the Analyze button
76. nePix Pro v6 or later use GenePix Pro to quantify feature intensity using a scanned image and the gal file appropriate for your microarray ProtoArray gal files may be downloaded from Life Technologies ProtoArray Central Click on the Invitrogen button in ProtoArray Prospector to open this web page Follow the Online Tools link under BioMarker Discovery Resources to the Online Tools page then click the link under ProtoArray Lot Specific Information and use the lot number to download the array specific information as directed on the web page After you have quantified the feature intensity select the Save Results As command from the Files menu in GenePix Pro The output file with the gpr extension can be used directly by ProtoArray Prospector tab files generated by Prospector Imager Prospector Imager implements a complete image processing workflow for the KSI and radioactive SMI applications As in the procedure described above the input is an image from the scanner and an array layout gal file The final output of Prospector Imager is a tab delimited text file which has the same format as a gpr file Tab delimited text files txt files containing your data in a columnar format ProtoArray Prospector also analyzes numerical data from other image quantification software if you save the results as a tab delimited text file and modify the contents of that file using a spreadsheet program such as Microsoft Excel
77. ngle array analyses b identifies proteins that score as hits in all the analyses If more than two analyses have been selected the software also identifies proteins that score as hits in smaller subsets of analyses c creates and saves files containing 1 all the results the Analysis file and 2 a summary of the results the Summary file ProtoArray Prospector saves these files to the respective subfolders in the main Prospector folder d inserts an entry into the Microarray Comparison Results section of the Result Navigation Tree e presents a summary of the result in tabular form in the Result Display Pane 41 Small Molecule Protein Interaction Mode Overview The objective of a small molecule protein interaction experiment employing ProtoArray microarrays is to identify the protein s on the microarray human protein features that interacts specifically with the user small molecule added to the system SMI Profiling can be performed with either radiolabeled or fluorescent small molecule probes e Use the Small Molecule Profiling Fluorescent mode when probing with fluorescently labeled small molecules on the array e Use the Small Molecule Profiling Radioactive mode when probing with radiolabeled small molecules on the array The basic approach employed by ProtoArray Prospector is to 1 Calculate the appropriate fluorescent signal values taking into account corrections for background 2 Calculate the Z
78. nloads details aspx familyid 0856eacb 4362 4b0d 8edd aab15c5e04f5 The setup installs executables and documentation and creates shortcuts in the Start gt Programs gt Invitrogen gt Prospector menu group and on the desktop Prospector Imager The IRBP Toolbox is used internally by Prospector Analyzer and does not have a separate shortcut on the desktop When Prospector is installed we recommend that you also register the product Run Prospector and select Help gt Registration from the Main Menu 13 Installation on Computers with the Microsoft Windows 7 Operating System The ProtoArray Prospector version 5 2 3 software was designed to be compatible with the Windows 7 operating system and 64 bit machines By default the ProtoArray Prospector version 5 2 3 software is installed to the path C Program Files x86 Invitrogen Prospector After installation a user in the Administrator group will be able to run the Analyzer application for data analysis of the ProtoArray protein microarrays program without issue but regular non Administrator users will encounter errors when attempting to run the application This is due to compatibility issues with legacy applications developed in Windows XP that require administrator privileges In order for the regular users to run the program properly in Windows 7 a user in the Administrator group needs to assign Write permission for the directory Prospector to the Users group
79. nt protometrx otFiles URL to update ProtoAray Application files Auto check for version updates http Awww invitrogen com downloads URL to public databases NCBI GeneBank http www ncbi nim nih gov entrez viewer fcgi db nucleotid SGD Database bes db yeastgenome org cgi bin locus pl locus Ensembl Human bes www ensembl org Homo sapiens transview transcript Ultimate ORF http orf invitrogen com cgi bin ORFCard amp set UltimateO RF Defauit Options Load Options Save Options Figure 5 ProtoArray Prospector employs a series of Application Specific Files that define how the program employs the control features on each microarray for different applications PPI KSI SMF SMR IRBP or IRBP with Plasma as well as for each lot When entering data Prospector will automatically determine whether the appropriate file is available on your hard drive If not ProtoArray Prospector will automatically download and save the needed file The URL that Prospector uses for those actions is contained in this tab and may be changed if needed This page also allows modifying the base of URL to public databases The actual link is a concatenation result of the URL base and the corresponding accession number You will only need to modify these values if corresponding URL was changed after ProtoArray Prospector was released see Figure 5 62 File Formats This section contains a short description of the formats of
80. number using the Analysis tab of the Program Options dialog box called by the Options command Z Score continued Small Molecule Interaction Profiling SMI Mode fluorescent and radioactive In the optional Z Score analysis a hit in the SMI mode fluorescent or radioactive is defined as any protein where the two replicate features of the protein 1 Have an average Z Score of greater than three AND 2 The coefficient of variation for the signals from the two replicates is less than 0 5 Note that by default the background corrected value is employed for these calculations in the SMI fluorescent mode and the background corrected value after the application of the signal scatter correction is employed for these calculations in the SMI radioactive mode The default setting of three standard deviations can be changed to any positive real number using the Analysis tab of the Program Options dialog box called by the Options command Kinase Substrate Identification KSI Mode There are two alternatives in the KSI mode 1 When data from a reference buffer only microarray is available a hit in the KSI mode is defined as any protein where the two replicate features of the protein Have an average Z Score of greater than three AND b The coefficient of variation for the signals from the two replicates is less than 0 5 AND c The mean signal of the replicates is at least 1 5 times that of the mean signals from the correspond
81. ode Overview The objective of a protein protein interaction experiment employing ProtoArray microarrays is to identify the protein s on the microarray human protein features that are capable of binding a protein added to the system ProtoArray Prospector is designed to analyze the results from experiments using exogenous proteins of interest containing a suitable tag such as the V5 epitope tag or that have been biotinylated in vitro Binding of the tagged protein of interest to the human proteins printed on the microarray is quantified by 1 probing the processed microarray slide using a labeled antibody specific for the tag or streptavidin conjugated to a fluorescent dye and 2 quantifying the fluorescence intensity of each feature on the slide The basic approach employed by ProtoArray Prospector to analyze protein protein interactions is to 1 Calculate the appropriate fluorescent signal values taking into account corrections for background 2 Calculate the Z Scores of all the corrected intensities of the human protein features Identify the features that have a Z Score greater than the user defined cut off value 3 by default These are the protein features that ProtoArray Prospector scores as positive for binding the exogenous test molecule When first using the ProtoArray system for protein protein interactions we recommend that you start by setting up a positive control assay employing a protein known to bind to one or
82. on In the Compare Order Files dialog box that opens adjust the order in which the information will appear in the output for the different data sets you have chosen To change the name select the corresponding lt file gt entry in the Alias column and enter the name in the text box that appears Select the Finish button ProtoArray Prospector automatically a opens the file b identifies the type of data contained in the file and if needed obtains the required application specific information from ProtoArray Central c performs the needed calculations i applies signal scatter correction if toggled on ii removes outliers if toggled on iii subtracts background if toggled on iv performs control calculations see Appendix I d creates and saves files containing 1 all the results the Analysis file and 2 a summary of the results the Summary file ProtoArray Prospector saves these files to the respective subfolders in the main Prospector folder e inserts an entry into the Microarray Comparison Results section of the Result Navigation Tree f presents a summary of the result in tabular form in the Result Display Pane ProtoArray Prospector displays a summary of the proteins that score as positive across the selected microarrays in the Results Display Pane To view the results for all the proteins on all the microarrays you compared select the corresponding entry in the Microarray Comparison Results secti
83. on of the Result Navigation Tree and select the Show button Note this function requires that you have a licensed version of Microsoft Excel installed on your computer To view the summaries from previous comparisons double click the corresponding entries in the Microarray Comparison Results section in the Result Display Pane Immune Response Profiling Mode Overview The objective of an Immune Response Biomarker Profiling IRBP assay employing ProtoArray microarrays is to identify proteins on the microarray human protein features that are bound by an exogenous antibody added as an unique reagent or as part of the complex mixture of antibodies contained in serum ProtoArray Prospector is designed to analyze results from experiments where any bound exogenous antibody is detected by probing with a second Ig class specific antibody labeled with a fluorescent probe such as Alexa Fluor 647 Binding of the secondary antibody on the microarray is then quantified by measuring the fluorescence intensity of each feature on the slide e Use the Immune Response Profiling mode when profiling serum samples on the array e Use the Immune Response Profiling with Plasma mode when profiling plasma samples on the array A different set of negative control features is used for data analysis in the plasma profiling mode The basic approach employed by ProtoArray Prospector to analyze protein antibody interactions is to 1 Calculate the appropri
84. perate with the Software 5 NOSEPARATION OF COMPONENTS The Software is licensed as a single product Some Licensor software products combine separately available components into a single product e g a software suite product may be comprised of multiple component products When licensed as a combination product the component parts may not be separated for use independently of the combination product You must first purchase a license to each component of the combination product before you may use it independently of the combination product 6 TERM AND TERMINATION Unless otherwise agreed the term of this Agreement shall continue until it is terminated Licensor may terminate this Agreement or any License upon 15 days written notice if you breach this Agreement and do not cure the breach within 15 days following receipt of written notice of breach Immediately upon any termination or expiration of this Agreement you agree to a pay all amounts owed to Licensor b un install and cease use of the Software for which your rights have been terminated c upon request return to Licensor or destroy all copies of the Software and any other Confidential Information or proprietary materials in your possession for which your rights have been terminated and d upon request certify in writing your compliance with b and c above 7 CONFIDENTIALITY You agree to protect Licensor s Confidential Information with the same 69 degree of care
85. r ko CI 63 Comparison File Formats ing desereret et Debs Pte ci rad Uc Perros efr ced aga ocv EUR 64 ProtoArray Application Information File Formats iiir e epos 66 Input Pe Formate osse dva pta tcd da pen uuu VU rM NUM eM I Deos 67 End User License Agreemlielil iiuiiieiiees is cina cocta dis ra pecu ca ecd q 3d pag E aD RE a OH NEE ee 68 iv UR e nd 74 Appendix CalCulatiOns viicccicicccisesseciieciccsisessessssediewcassvieansecadencsersduencnaudeecdeneieanteendea 75 Chebyshew s Inequality CI P Val e iiie iati rbd iran feodo Ee 75 Leave One Out Chebyshev s Inequality LOOCT Outlier Detecton 75 lange EE tod plac inb a eben mano aia 77 Signal Scattet C EE E 77 Vid NO KOI EEE uM 78 e e MT 78 Eeer eege eege 79 REECH E 79 Introduction Overview ProtoArray Prospector is a software tool for analyzing numerical data from ProtoArray Human or Control Protein Microarrays used in Protein Protein Interaction PPI Kinase Substrate Identification KSI Ubiquitin Ligase Profiling ULP Small Molecule Protein Interaction Profiling SMI or Immune Response Biomarker Profiling IRBP assays You can use the software to identify arrayed human proteins that e Bind other proteins in a PPI assay e Function as a substrate for phosphorylation in a KSI system e Function as a substrate for ubiquitination in a ULP system e Bind toa radiolabeled or fluorescently labeled small molecule in the SMI assay e Bind an antibody in the IRBP assay
86. results the Analysis file and 2 a summary of the results the Summary file ProtoArray Prospector saves these files to the respective subfolders in the main Prospector folder e inserts an entry into the Microarray Comparison Results section of the Result Navigation Tree f presents a summary of the result in tabular form in the Result Display Pane ProtoArray Prospector displays a summary of the proteins that score as positive across the selected microarrays in the Results Display Pane To view the results for all the proteins on all the microarrays you compared select the corresponding entry in the Microarray Comparison Results section of the Result Navigation Tree and select the Show button Note this function requires that you have a licensed version of Microsoft Excel installed on your computer To view the summaries from previous comparisons double click the corresponding entries in the Microarray Comparison Results section in the Result Display Pane 52 Comparing Data Across Multiple Microarrays WITHOUT Using an Unlabeled Competitor SMI Radioactive 1 53 To begin the analysis open ProtoArray Prospector and set the application in the Mode Selection Menu to Small Molecule Profiling Radioactive mode Select the Compare button from the Tool Bar In the dialog box that opens select the checkboxes next to the single microarray analyses you would like to include in the comparison Click on the Next gt butt
87. roarrays that are bound by different exogenous proteins and help you establish patterns of binding specificity 42 Analyzing Positive Control Data from ProtoArray Control Protein Microarrays SMI Fluorescent Overview The recommended first steps in using ProtoArray microarrays for identifying small molecule interacting proteins is ensuring that your system is working properly using ProtoArray Control Protein Microarrays This positive control for the system may employ a control small molecule or a different user supplied small molecule The control microarrays contain a positive feature that you can use to check that your system can detect an interaction with a known control feature e g Alexa Fluor 647 coupled staurosporine to CAMK2A printed in each subarray The ProtoArray Control Protein Microarrays also contain a number of negative control features to evaluate the specificity of your system Procedure Using a ProtoArray Control Protein Microarray 1 To begin the analysis open ProtoArray Prospector and set the application in the Mode Selection Menu to Small Molecule Profiling Fluorescent mode Select the Analyze button from the Tool Bar In the Open dialog box that appears select the type of file gpr or txt from the Files of type pull down list and navigate to your data file s Select the file s for analysis and click the Open button You can select multiple files at once and ProtoArray Prospector opens ea
88. single microarray analyses for comparison and prompts ProtoArray Prospector to perform the a b comparison The results are displayed in the Result Display Pane Compare corresponding Result files are created and stored in the respective Summary subfolders in the main Prospector folder and an entry is made in the Microarray Comparison Results section of the Result Navigation Tree Opens the results from a single microarray analysis or comparison in the Result Display Pane The command is only active when an entry is the Result Navigation Tree is selected X Delete Deletes the selected entry in the Single Microarray Results or Microarray Comparison Results section of the Result Navigation Tree This action also deletes the underlying files ProtoArray Prospector stores in the respective subfolders in the main Prospector folder Opens the Program Options dialog box Options Show Opens the selected Result file in Excel You will need a licensed copy of Microsoft Excel to make use of this feature e Invitrogen Opens the ProtoArray Central web page in your browser About Opens the About Prospector box 20 Application Selection Menu ProtoArray Prospector can operate in different modes depending on the specific biochemical application Protein Protein Interaction Kinase Substrate Identification Ubiquitin Ligase Profiling Small Molecule Fluorescent Small Molecule Radioact
89. sion database Note that one needs a licensed copy of Vector Xpression installed on the same computer to make use of this feature Show Result in Excel Opens the selected Result file in Excel Note that one needs a licensed copy of Microsoft Excel installed on the same computer to make use of this feature WebUpdate View gt Prompts ProtoArray Prospector to check for the latest versions of the Application Specific Files Main Menu Command Action Toolbar Toggles the display of the Toolbar on and off Status Bar Toggles the display of the Status Bar on and off Help gt Main Menu Command Action License Agreement Registration Opens the End User License Agreement dialog box Opens the Registration Information dialog box Life Technologies on the Web Opens the ProtoArray Central web page in your browser About Prospector 19 Opens the About Prospector box Tool Bar Button Action Opens a dialog box for navigating to data files and prompts ProtoArray Prospector to perform an analysis of the data from a single microarray zm The results are displayed in the Result Display Pane Corresponding Analyze Result files are created and stored in the respective subfolder in the main Prospector folder and an entry is made in the Single Microarray Results section of the Result Navigation Tree Opens a dialog box for choosing
90. sults are retained 2 If you did not change the default setting for the location of the ProtoArray Prospector folder during installation the folder will be located at Program Files Invitrogen Prospector Result subfolders from previous analyses of arrays using ProtoArray Prospector are located in the same location and are not overwritten Automatic Update of ProtoArray Prospector ProtoArray Prospector can be configured to check for available version updates and download and install any newer versions This feature checks the Life Technologies web site for newer versions of the application If a newer version is available the version information is displayed and the user has the option to download the newer version When the newer version is downloaded the user is given the option to install the upgrade at the current time or install the upgrade the next time the application is started The user may initiate the version check manually by selecting the Tools Check for software update option To configure this feature select the Options icon on the menu bar Within the Options dialog box select the Web Parameters tab Check the box labeled Auto check for version updates This setting enables the software to automatically check for updates on a regular interval If this option is not selected the user is still able to check for newer versions via the Tools Check for software update option Uninstalling ProtoArray Prospector To
91. sults for all the proteins on an array The file contains two blocks header and array content list The file is a tab delimited text file The header block contains general comparison information e names of the files compared e lot number e parameters used to detect a significant hit The array content list block contains the table of all protein probes All replicates of each protein are consolidated into a single line The table contains a number of columns identifying protein position on the array and content e human array protein is identified by database type and ID and Ultimate ORF ID e a1 designation for significant hit a 0 designation for no hit and values used to flag significant hits one column per file compared e Z Factor is the mean value of Z Factor values of all replicates of this probe e Z Score is the mean value of Z Score values of all replicates of this probe e CIP Value is the greatest CI P Value of all replicates of a particular probe e CVisthe coefficient of variance Comparison Summary File This file contains an abstract of the most significant comparison results The file contains a header and a number of tables one with the list of those proteins which were significant hits in all files one table for those with hits in all files but one one table for those with hits in all files but two etc The header contains general comparison information e names of the files compared e lotnumber Each table o
92. t www lifetechnologies com protoarray Launch the application by double clicking the ProspectorSetup exe icon Accept the license agreement On the following screens you can change the destination folder and installation configuration if required The following components of prospector are installed Component Description Application Prospector Analyzer A basic set of analysis functions For PPI SMI and ULP fluorescent described in this document application For KSI and SMI radioactive application without using Imager Prospector Imager Used for Image processing for KSI For KSI and SMI radioactive applications and SMI radioactive applications using Imager Immune Response A comprehensive set of algorithms For IRBP application using the IRBP Biomarker Profiling for the IRBP application Toolbox IRBP Toolbox The installation option for Prospector requires the optional Windows component Microsoft NET version 2 0 or 4 0 framework installed on the computer depending on the operating system If NET 2 0 4 0 is not installed Prospector helps download and install the framework After the framework is installed you will need to restart Prospector installation The Microsoft NET framework redistributable package installs the NET Framework runtime and associated files required to run applications developed to target the NET Framework For details on the NET framework visit http www microsoft com dow
93. the Compare Order Files dialog box that opens adjust the order in which the information will appear in the output for the different data sets you have chosen To change the name select the corresponding lt file gt entry in the Alias column and enter the name in the text box that appears Select the Finish button 5 ProtoArray Prospector automatically compares the results of the single array analyses b identifies human proteins that score as positive in all the analyses If more than two analyses have been selected ProtoArray Prospector also identifies proteins that score as positive in smaller subsets of analyses c creates and saves files containing 1 all the results the Analysis file and 2 a summary of the results the Summary file ProtoArray Prospector saves these files to the respective subfolders in the main Prospector folder d inserts an entry into the Microarray Comparison Results section of the Result Navigation Tree e presents a summary of the result in tabular form in the Result Display Pane A comprehensive set of additional algorithms for analysis of Immune Response Biomarker Profiling data can be found in the Immune Response Biomarker Profiling IRBP Toolbox For detailed instructions on the use of these algorithms please see the separate Immune Response Biomarker Profiling Toolbox v5 2 3 manual 56 Program Options Overview There are a number of user defined options that alter the behavior of Pro
94. the files created or used by ProtoArray Prospector Single Microarray Analysis File Formats Two files are created as a result of Analyze function The Result file is put into the Single Microarray Results KSI Single Microarray Results ULP Single Microarray Results PPI Single Microarray Results SMF Single Microarray Results SMR Single Microarray Results IRP or Single Microarray Results IRP_Plasma subfolder depending on the application selected This file contains a complete report of the analysis in the tab separated text format The Summary file is put into the Single Microarray Summary KSI Single Microarray Summary ULP Single Microarray Summary PPI Single Microarray Summary SMF Single Microarray Summary SMR Single Microarray Summary IRP or Single Microarray Summary IRP_Plasma subfolder depending on the application selected This file contains a user friendly presentation of the most significant analysis results in HTML format The Summary file is automatically shown in the Result Display Pane after the analysis is completed Single Microarray Result File This file has the results for a single microarray analysis for all the proteins on an array These files are found in the Single Microarray Results PPI Single Microarray Results KSI Single Microarray Results ULP Single Microarray Results SMF Single Microarray Results SMR Single Microarray Results IRP or Single Microarray Results IRP_Plasma subfolder depending on the application s
95. tin The basic approach employed by ProtoArray Prospector to analyze ubiquitination ligation profiling experiments is to 1 Calculate the appropriate fluorescent signal values taking into account corrections for background 2 Calculate the Z Scores of all the corrected intensities of the human protein features 3 Identify the features that have a Z Score greater than the user defined cut off value 3 by default These are the protein features that ProtoArray Prospector scores as positive for binding the exogenous test molecule When first using the ProtoArray system for ubiquitination ligation profiling we recommend that you start by setting up a control assay employing ubiquitin E1 UBE1 100 nM ubiquitin E2 UbcH5b 50 nM and biotinylated ubiquitin The ubiquitination of Mdm2 on the ProtoArray microarray can be detected with fluorescently labeled streptavidin Once the performance of the system is established you can then be confident that the experimental conditions are appropriate for identifying new or uncharacterized ubiquitin E3 substrates using the ProtoArray Human Protein Microarrays In addition to identifying the proteins that score as positive on a single microarray ProtoArray Prospector also allows you to compare the results across several microarrays This will allow you to identify human proteins on the ProtoArray microarrays that are ubiquitinated through the activity of different exogenous E3 ligases
96. to continue or stop the comparison Arrays from different array versions i e v4 0 v4 1 or v5 0 cannot be compared within the software ProtoArray Prospector shows an error message and stops the comparison Procedure 1 To begin the analysis open ProtoArray Prospector and set the application in the Mode Selection Menu to Ubiquitination Ligation Profiling mode 2 Select the Compare button from the Tool Bar In the dialog box that opens select the checkboxes next to the single microarray analyses you would like to include in the comparison Click on the Next button 4 In the Compare Order Files dialog box that opens adjust the order in which the information will appear in the output for the different data sets you have chosen To change the name select the corresponding lt file gt entry in the Alias column and enter the name in the text box that appears Select the Finish button 5 ProtoArray Prospector automatically compares the results of the single array analyses b identifies proteins that score as hits in all the analyses If more than two analyses have been selected ProtoArray Prospector also identifies proteins that score as hits in smaller subsets of analyses c creates and saves files containing 1 all the results the Analysis file and 2 a summary of the results the Summary file ProtoArray Prospector saves these files to the respective subfolders in the main Prospector folder d inserts an entry in
97. to import the data into Prospector and perform the analysis 3 ProtoArray Prospector automatically opens the file b identifies the type of data contained in the file c performs the needed calculations i subtracts background if toggled on ii removes outliers from controls if toggled on iii calculates Z Scores iv performs control calculations see Appendix I d creates and saves files containing 1 all the results the Analysis file and 2 a summary of the results the Summary file ProtoArray Prospector saves these files to the respective subfolders in the main Prospector folder e inserts an entry into the Single Microarray Results section of the Result Navigation Tree f presents a summary of the result in tabular form in the Result Display Pane 25 Interpreting the Control Data If your system is working properly the results should have the pattern noted in Table 3 These data are available by selecting the experiment in the Result Navigation Tree and clicking the Show button The results will be contained in the control block of the Excel spreadsheet that opens Table 3 Test Return Negative Control Range Z Score A measure of the distance that the negative control features e g buffer lie below a constant value chosen to identify a reasonable lower point in the expected dynamic range of the system The value should preferably be less than 6 and the mean should be close to zero and much
98. to the Microarray Comparison Results section of the Result Navigation Tree e presents a summary of the result in tabular form in the Result Display Pane 33 Kinase Substrate Identification Mode Overview The objective of a kinase substrate identification experiment employing the ProtoArray Human Protein Microarrays is to identify protein s on the microarray human protein features that can be specifically phosphorylated by the user kinase in the presence of radiolabeled ATP The basic approach employed by ProtoArray Prospector is to 1 Calculate appropriate autoradiographic signal values taking into account corrections for background and signal scatter 2 Calculate the Z Factors for all corrected intensities of the array protein features Identify features that have a Z Factor greater than a user defined cut off value 0 4 by default These are the protein features that ProtoArray Prospector scores as positive as substrates for phosphorylation When first using the ProtoArray system for identifying kinase substrate s we recommend performing a positive control assay employing a known kinase to probe a ProtoArray Control Protein Microarray In addition we recommend that you also identify any proteins on the microarrays that autophosphorylate by performing at least one assay using a ProtoArray Human Protein Microarray with the complete buffer system including y P ATP but no added kinase ProtoArray Prospector c
99. toArray Prospector By default ProtoArray Prospector utilizes a series of default options that have been chosen as reasonable for the different application modes These options can be manually changed in the different tabs of the Program Options dialog box see Figure 3 next page The dialog box can be opened by selecting the Option button from the tool bar If you manually change the options and click the OK button ProtoArray Prospector will remember the settings and retain them if you exit and then restart the program The settings will remain active until you either change them or restore the default options using the Default Options button In addition ProtoArray Prospector will allow you to save a set of active options in each tab for future use by using the Save Options button Using this procedure you can restore the group of settings you have saved by selecting the Load Options button As above ProtoArray Prospector will also remember these settings and automatically retain them when the program is restarted until you once again change them Using the Load Options button to recall saved settings is similar to using the Default Options button to reload the default settings 57 Analysis Tab Program Options E EI Analysis Warnings Web Parameters Analysis to be Performed v Background Subtraction v Signal Scatter Compensation v Outlier Detection v Calculate Z factor on per Sub Array Signifi
100. toArray Prospector The control microarray contains several positive features that are used to verify that your system can detect 1 phosphorylation based on the behavior of control features that are known to autophosphorylate under the recommended experimental conditions the Alignment Control Kinases PKCeta and 2 phosphorylation of control features MAPKAP known to be a substrate of a kinase added to the system MAPK14 p38 alpha The ProtoArray Control Protein Microarray also contains a number of negative control features to evaluate the specificity of your system Procedure Using a ProtoArray Control Protein Microarray 1 To begin the analysis open ProtoArray Prospector and set the application in the Mode Selection Menu to Kinase Substrate Identification mode 2 Select the Analyze button from the Tool Bar and navigate to the file containing the data Select the file and then select the Open button on the navigation window to bring the data into ProtoArray Prospector and perform the analysis 3 Inthe Control assay parameters dialog box that appears select either 1 the Control Kinase step 1 radio button or 2 the Customer kinase step 2 radio button if your data is based on a kinase of your choice Click the OK button 4 ProtoArray Prospector automatically a opens the file b identifies the type of data contained in the file and if needed obtains the required application specific information from ProtoArray Centra
101. ts node in the Results Navigation Pane and then click on the Show button Note this function requires that you have a licensed version of Microsoft Excel installed on your computer 48 Comparing Results from ProtoArray Human Protein Microarrays Overview The objective of a small molecule protein interaction profiling experiment employing ProtoArray microarrays is to identify the proteins on the microarray human protein features that specifically bind to the user supplied small molecule added to the system Comparing Data Across Multiple Microarrays Using an Unlabeled Competitor SMI Fluorescent 1 To begin the analysis open ProtoArray Prospector and set the application in the Mode Selection Menu to Small Molecule Profiling Fluorescent mode 2 Select the Competition Analysis from the Tools pull down menu In the Competition Analysis dialog box that opens select the checkboxes next to the microarray results from assays without unlabeled competitor that you would like to include in the comparison Click on the upper gt gt button to move them into the Assay without Competitor field Next if any assays were run in the presence of unlabeled competitor select the checkboxes next to the microarray results from assays with unlabeled competitor that you would like to include in the comparison Click on the lower gt gt button to move them into the Assay with Competitor field Select the Next button 4 If desired rearrang
102. tted by law decompile disassemble attempt to discover the source code for create derivative works based on or sublicense any of the Software iii use any Software for the benefit of any third parties e g in an ASP outsourcing or service bureau relationship or in any way other than in its intended manner iv remove any proprietary notice labels or marks on or in Software or v disable or circumvent any access control or related device process or procedure established with respect to the Software including the license key or any other part thereof If the Software design permits modification then you may only use such modifications or new software programs for your internal purposes and otherwise consistent with the License You are responsible for all use of the Software and for compliance with this Agreement any breach by you or any user of the Software shall be deemed to have been a breach by you Licensor reserves all rights not expressly granted no right or license is granted hereunder express or implied or by way of estoppel to any intellectual property rights other than as expressly set forth herein and your receipt of a license to the Software does not by itself convey or imply the right to use the Software in combination with any other product s You agree and acknowledge that you have been provided sufficient information such that you do not need to reverse engineer the Software in any way to permit other products to intero
103. used to protect your own confidential information but in no event less than a reasonable standard of care and not to use or disclose any portion of such Confidential Information to third parties except as expressly authorized in this Agreement You acknowledge that the Software including its content structure organization and design constitute proprietary and valuable trade secrets and other intellectual property rights of Licensor and or its licensors The term Confidential Information means collectively non public information that Licensor and its licensors provide and reasonably consider to be of a confidential proprietary or trade secret nature including but not limited to Software prices and confidential elements of the Software and Licensor s and its licensors technology and know how whether in tangible or intangible form whether designated as confidential or not and whether or not stored compiled or memorialized physically electronically graphically photographically or in writing Confidential Information does not include any information which you can demonstrate by credible evidence i is as of the time of its disclosure or thereafter becomes part of the public domain through no fault of yours ii was rightfully known to you prior to the time of its disclosure or to have been independently developed by you without use of Confidential Information and or iii is subsequently learned from a third party not un
104. verted to a non Unicode tab delimited text file Note that this can be achieved in a spreadsheet program like Excel by pasting the required columns into the spreadsheet and using Save As to save the file as a Text Tab delimited txt file The file must contain the required columns of data with a one line header for each column Column Name Feature Notes Name Protein Name Required ID Spot ID Required Signal Spot Signal Required Background Spot Background Required X Spot X coordinate in um For signal scatter compensation only Y Spot Y coordinate in um For signal scatter compensation only Description Protein Description Optional Example Name ID Signal Background AlexaAntiMouseAb 33 3 100604 B01R01C01 26240 1483 AlexaAntiMouseAb 33 3 100604 B01R01C02 26760 1300 BSA1 947 100604 B01R01C03 1625 1243 BSA1 947 100604 B01R01C04 1628 1262 67 End User License Agreement This is a legal agreement between you the person or entity receiving software products and Life Technologies Corporation through its Invitrogen business unit having offices at 5791 Van Allen Way Carlsbad California 92008 USA Licensor This agreement is part of a package that includes one or more software products and certain electronic and or written materials This agreement covers your licensing of such software BY CLICKING YOUR ACCEPTANCE OF THIS END USER LICENSE AGREEMENT Agreement OR BY INSTALLING OR
105. w the steps listed below which will allow the Analyzer application to run correctly 1 Open Windows Explorer and go to C Program Files x86 Invitrogen Prospector 2 Right click on the Prospector exe application and select Properties 3 In the Compatibility panel select the checkbox next to Run this program in compatibility mode for 4 Select the Windows XP Service Pack 3 option in the dropdown box Click Apply then OK to complete E fs Prospector exe Properties General Compatibility Security Details Previous Versions If you have problems with this program and it worked correctly on an earlier version of Windows selectthe compatibility mode that matches that earlier version Help me choose the settings Compatibility mode Windows XP Service Pack 3 Settings V fe __ Run in 256 colors f mes 1 __ Run in 640 x 480 screen resolution d Disable visual themes L SS LI Disable desktop composition ld e i Disable display scaling on high DPI settings MN Privilege Level i Run this program as an administrator LU Change settings for all users x canei Aw 15 Upgrading from a Previous Version of ProtoArray Prospector 1 Install ProtoArray Prospector as described on the previous page If you had previous versions installed old executables are automatically removed while the data and re
106. y step 3 radio button is selected and then select the OK button ProtoArray Prospector automatically a opens the file b identifies the type of data contained in the file and if needed obtains the required application specific information for ProtoArray Central c performs the needed calculations i applies signal scatter correction if toggled on ii removes outliers from controls if toggled on iii subtracts background if toggled on iv calculates Z Factors v performs control calculations see Appendix I d creates and saves files containing 1 all the results the Analysis file and 2 a summary of the results the Summary file ProtoArray Prospector saves these files to the respective subfolders in the main Prospector folder e inserts an entry into the Single Microarray Results section of the Result Navigation Tree f presents a summary of the result in tabular form in the Result Display Pane Select the Analyze button again and navigate to the file s containing the data for the microarray s assayed with your unknown or uncharacterized kinase s Select the file s and then select the Open button on the navigation window to import the data into Prospector and perform the analysis In the Control assay parameters dialog box that appears ensure the Customer kinase step 4 radio button is selected use the pull down list to select the appropriate reference assay the reference data from the previous steps and the

ProtoArray Prospector v3.1 User`s Manual

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