ProSightPC 2.0 QuickStart Guide

ProSightPC 2.0 QuickStart Guide ProSightPC 2.0 is the only proteomics software suite that adequately supports high-mass-accuracy MS/MS experiments performed on LTQ FT and LTQ Orbitrap instruments. ProSightPC 2.0 builds on the capabilities of ProSightPC 1.0 in several ways. This quick start guide steps you through some of ProSightPC s key features. As you go through the steps, you can use the example data files that are installed in the following folder: \ProSightPC\data\ For complete details on how to use ProSightPC, see the ProSightPC User Guide or the Help available in the ProSightPC application. Contents Importing or Creating a Database Processing LC-MS/MS Data To Generate Reports Sample Processing from a Targeted Run Importing or Creating a Database To Import a Database You can download a proteome database from the FTP Web site for ProSightPC or manually create a proteome database of your own. To download a proteome database 1. Choose Databases > Download ProSightPC Databases. This step takes you to ftp://prosightftp:gsx1gon@prosightpc.scs.uiuc.edu. This site contains 55 databases, including top-down and bottom-up databases. 2. Click the desired proteome warehouse (.pwf) file to download to your local computer. 3. After the file finishes downloading, double-click it. The Import Database(s) dialog box appears. 4. Click Import to finish the process. Human - MD Trypsin (No SNPs).pwf Revision A XCALI-97243 2009 Thermo Fisher Scientific Inc. All rights reserved.

To Create a Custom Database Using one of the wizards included in ProSightPC, you can manually create a database. You can create a database with either a FASTA or Swiss-Prot format file. To create a custom database 1. Choose Databases > Create a Custom Database to open the Create New Database wizard. 2. Set the options on each page of the wizard, and then click Next. 3. On the last (Ready to Load) page, click Go. For a more detailed description, refer to the ProSightPC User Guide or the Help available in the application. Processing LC-MS/MS Data To Set Processing Options To process the LC-MS/MS data and search the imported database, you can use the High Throughput wizard. Click the High Throughput Wizard icon,, or choose ProSightHT > HighThroughput Wizard. To set processing options 1. On the Process a dataset page of the wizard, click Add and select a.raw data file. For future processing, an input file can be either a.raw file or a ProSightPC upload format (.puf) file. For top-down proteomics MS/MS experiment data, select the following file: ProSightPC_folder\data\Example Raw Files\Human_nuclear_extract_Top_Down_LTQ_FT_MS_MS.raw For middle-down/bottom-up proteomics MS/MS experiment data, select the following file: ProSightPC_folder\data\Example Raw Files\PheATE_126kDa_Trypsin_Hi_Hi_Top3.raw Select the.raw files that you want to process. 2

2. In the Choose a Process Algorithm area, select a method of analysis-to-infer mass (AIM), which converts the data in the.raw data file to observed neutral mass values. Two are available: Xtract and THRASH. Both work well, although Xtract tends to be faster. 3. In the Choose a Process Option area, select a processing option: Top Down (MS2), Top Down (MS3), or Middle Down. 4. Click Next. Select processing algorithm. Select default processing options or manually set the parameters (Advanced Settings). Select this check box if you would like to re-search the data without processing your.raw file again. To Select a Repository You must select a repository, which is a database that stores the search results in ProSightPC. To select a repository 1. On the Running Highthroughput Logic page, select an existing repository from the Repository list or create a new repository. 3

2. If you create a new repository, specify a name for it, for example, Demo_Repository. 3. Click OK and click Save. To create a new repository, click New Repository. Give your repository a name. ProSightPC provides two default categories, good and bad, when creating a new repository. Click Save. To Select a Search Tree A search tree can hold one or more branch points. Each branch point in the tree contains one search. The simplest search tree has one branch point; this is the default for a new tree. You can create a new search or select an existing search. To select an existing search tree 1. If you want to select an existing search and are running the example files, select Demo Search Tree from the Search Tree Name list. 2. Click Save. To create a new search tree 1. To create a search, click Add Search. 2. (Optional) Add a predefined search or edit an existing predefined search. 3. Click Save. 4

Click Add Search. Create a new predefined search. Select a predefined search or create a new search. Click Save when you are done. Edit an existing predefined search. 4. To select a condition, click Conditions. 5. Set conditions that determine if the experiment will be loaded to the good category representing high-quality data or to the bad category representing low-quality data. The default of E-value < 1e-4 filters confident hits. 6. Click Save to continue. Click Conditions. Set conditions that determine if the experiment will be loaded to the good or the bad category. The default of E-value < 1e-4 filtes confident hits. Click Save to continue. 5

7. (Optional) Select the options in the Experiment Filter box to ignore experiments that are not likely to yield matches. By default, these are: Experiments that contain less than 10 fragments Experiments whose intact mass is less than 750 Da To cancel an experiment filter, clear this check box. 8. (Optional) Specify a two-level search tree by changing load to run search, and repeat this process. To add a new branch point, change load to run search, and repeat this process. 9. Click Save to save the search tree before continuing. 10. Select a search tree name and click OK twice. A search tree is usually used more than once per project. 11. Click Next. Click Save. Select a search tree name and click OK twice. Click Next. 6

To View the Summary To View the Output To view the summary 1. Review your settings in the Summary window. 2. To begin the ProSightPC High Throughput processing, select Process at the bottom. 3. When the High Throughput wizard finishes processing the data, click Finish. Once the searching is complete (which may take some time, depending on the length of.raw file, the complexity of the database, and the search parameters), you can view the repository report. Viewing includes filter, import, and export options. For each.raw file, a table displaying all the matches found appears in ProSightPC. Each row represents the best match per MS/MS experiment (see next section). ProSightPC finishes the first job and generates a report. It opens a new page in ProSightPC while continuing to run the other jobs. Repository name on new page 7

To Generate Reports ProSightPC generates a report for each repository, per category or per file. To generate reports 1. Click Repository Report icon,, or choose ProSightHT > Repository Report. 2. Select the report type (Category or File). 3. Click Generate. ProSightPC generates a report for each repository, per category or per file. If you are interested in viewing only the good matches, select the good category. If you are interested in viewing all matches (good and bad) from a specific file, select the File option. Select the report type (Category or File). Click Generate. The Report page displays filter, import, and export information. 8

Exports selected experiments to a Microsoft Excel spreadsheet. Select experiment. Exports selected experiments back to the repository. Imports selected experiments into ProSightPC for further investigation. Displays the most common filters. For example, you can display only confident hits or hits with PTMs. Select the columns to display. Displays your own custom filters. For example, you can display only experiments with intact mass > 8685.0 Da. You might want to filter the data in the report. For example, you could set a custom filter to Theoretical Mass > 2000. You can also use any fixed modifications to filter, such as setting Mass Difference to < 5.0 Da. To Set Custom Filters You can set custom filters to filter the data in the repository report. To set custom filters 1. Select the Show Custom Filters check box. 2. Click Add Custom Filter. 3. Select the filter settings, for example, Experiment Number = 15. 4. Click Add. 5. Select the check box for the custom filter that you just added. 6. Click Apply Filters. 9

Select Show Custom Filters. Click Add Custom Filter. Select the filter settings. Click Add. Select the custom filter that you just added. Click Apply Filters. To Import and Export Experiments You can import experiments from a repository into ProSightPC for manual validation and additional database searching. You can also take a set of results from the experiments that you import to the Sequence Gazer tool to see in more detail how your MS/MS data matches a retrieved protein or peptide. Use any of the following methods to import experiments: Check experiments and click Import. Check experiments, right-click, and select Import. Double-click an experiment. Choose File > Import Data from Repository. Save any changes that you made by exporting the experiments back to the repository or to a different repository. Use any of the following methods to export experiments: Select the experiments that were imported and click Export to Repository. Select the experiments that were imported, right-click, and select Export to Repository. Right-click the experiment in the ProSightPC Data Grid, and select Export Experiment to Repository. Choose File > Export data to repository. 10

Sample Processing from a Targeted Run To Import MS/MS Data If you want to import a targeted.raw file as input or if you want to enter data manually into ProSightPC, you cannot use the High Throughput wizard. You can use either of two methods to import.raw files into a ProSightPC MS/MS experiment: THRASH and Xtract. (For information on these two algorithms, refer to the ProSightPC User Guide or the online Help available in the ProSightPC application.) You can also enter data manually. To Process a.raw File with the THRASH Algorithm To process a.raw file with the THRASH algorithm 1. Click the Import Profile icon,, or choose File > Import.raw > Profile. 2. In the Browse box, select the.raw file to be imported using the THRASH use algorithm. In the example file, use ProSightPC\data\Example Raw Files\Ubiq_CID_857_50sc_100K_1_8559.616.raw. 3. In the Thrash Options area, enter the desired parameters. 4. In the Precursor Mass/Type box, select the precursor type. 5. In the box below Type, enter the precursor mass. For the example file, enter 8559.616. 6. In the Fragmentation Method box, select the fragmentation method. 7. (Optional) Select a predefined search in the Predefined Search box. For the example file, select Demo Search. Select the.raw file to be imported with the THRASH use algorithm. Enter the desired parameters. Select the precursor mass type. Enter the precursor mass. For the example file, enter 8559.616. (Optional) Select a predefined search. ProSightPC creates a new MS/MS experiment with the data processed by the THRASH algorithm. 11

To Process a.raw file with the Xtract Algorithm To process a.raw file with the Xtract algorithm Use Xtract (one of the Qual Browser tools) to create a.raw file containing the neutral monoisotopic mass values (refer to the ProSightPC User Guide or the Help available in the ProSightPC application for more information). 1. Click the Import Xtract icon,, or choose File > Import.raw > Post Xtract. 2. In the Browse box, select a Post Xtract.raw file (in the example file, ProSightPC\data\Example Raw Files\Ubiq_CID_857_50sc_100K_1_XT_00001_M_8559.616.raw). 3. In the Precursor Mass area, enter the precursor mass in the box next to m/z. Enter 8559.616 in the example. Select the mass type of the precursor ions that each Post Xtract file contains, either Average Mass or Monoisotopic Mass. 4. In the Fragmentation Ion Data area, select the mass type of the fragment ions that each Post Xtract file contains, either Average Mass or Monoisotopic Mass. 5. (Optional) Select a predefined search in the Predefined Search box. For the example file, select Demo Search. Select a Post Xtract.raw file. Enter the precursor mass. Select the precursor ion mass type. Select the fragment ion mass type. (Optional) Select a predefined search. ProSightPC creates a new MS/MS experiment with the data processed by the Xtract algorithm. To Manually Enter the Data You can manually enter the data (precursor and fragment masses) if you want to import a targeted.raw file as input. To manually enter the data 1. Click the Add Experiment icon,, or choose Tools > Experiment Adder to open the Experiment Adder dialog box (see next figure). 2. In the Fragmentation Methods area, select the fragmentation method. 3. In the Precursor Ion Data/Type box, select the precursor mass type, either Manual or Upload. 4. If you select Manual in the Precursor Ion Data/Type list, select the mass type of the precursor ion in the Mass Type box (either Monoisotopic or Average) and enter the precursor mass. If you select Upload in the Precursor Ion Data/Type list, enter the path and name of the ASCII text file or files containing the precursor ion data in the Text File box, or click Browse to browse for them. 5. (Optional) Select a predefined search in the box near the bottom of the dialog box. 12

Select the fragmentation method. Select the precursor mass type and enter the precursor mass or use a text file. (Optional) Select a predefined search. All three options for importing MS/MS data into ProSightPC create new MS/MS experiments in the Data Grid (with or without searches, depending on whether you added one or more predefined searches). MS/MS experiment in the Data Grid To Add Searches ProSightPC supports five different search modes. Each search mode represents a specific method used to query a proteome database within the proteome warehouse (you can find more information on search modes in the ProSightPC User Guide or the Help available in the ProSightPC application). To Add a New Search You can add a single search to a specific MS/MS experiment when an experiment requires you to adjust parameters to identify the protein. You can add one of several different types of searches to an experiment. For a description of these searches, refer to the ProSightPC User Guide or the Help available in the ProSightPC application. To add a new search 1. Add a new search by using one of the following methods: Click the Add Search icon,. Select the experiment and choose Experiment Tools > Add Search. Right-click the experiment and choose Add Search from the shortcut menu. 2. In the New Search in Experiment X dialog box, select the search type. 3. Select the proteome warehouse. 13

Select the search type. Select the proteome warehouse To Add a Predefined Search Predefined searches are a strategy to simplify repeating identical searches on different sets of MS/MS data instead of using Add Search to manually append the same search to multiple experiments. To add a predefined search 1. Add a predefined search by using one of the following methods: Click Append Predefined Search. Select the experiment, and choose Experiment Tools > Append Predefined Search. Right-click the experiment and select Append Predefined Search from the shortcut menu. 2. In the Append Predefined Searches to Experiment X dialog box, select the searches that you want to append. 3. Select Append. 14

Click here to add a new predefined search. Select the searches that you want to append. Select Append. To Perform Searches You have two options for searching: performing searches manually or performing them in batch mode. To Perform Searches Manually If you have a limited number of searches that you want to perform, you can manually search the database. To perform searches manually Right-click the experiment and select Run Search 1. If you select more than one experiment or search, right-click and select Run Searches. To perform searches in batch mode With batch processing, you can queue and perform a large number of searches over any number of experiments in a single action. 1. Select all the desired experiments, right-click, and select Append Predefined Search. 2. Click Save in all the New Search in Experiment X dialog boxes. 3. Choose Tools > Batch Run. To Interpret Results After performing the searches, double-click a search. A new tab opens with the experiment number. Experiment Information Click the display control arrows to reveal or conceal the various displays. These arrows indicate that information related to a search is ready for display. Click the down arrow to reveal search information. 15

Display control arrows Search Information Each result list displays the number of protein isoforms found. Click the side arrow next to the results list to display the results. Results list 16

Protein Isoform Information Graphical representation of the protein isoforms (location of PTMs and matching fragment ions) Statistics table Short description of the protein isoform Sequence Gazer The Sequence Gazer is an interactive environment for querying MS/MS data against a known protein sequence. Use the Sequence Gazer to characterize identified proteins by selectively adding or removing PTMs or custom masses to amino acids within a protein sequence. Click Take to Sequence Gazer. In the Sequence Gazer, you can examine the current experiment information (for precursor and fragment masses) to be queried against this sequence. 17

Trademarks ProSightPC is a trademark of Thermo Fisher Scientific Inc. in the United States. Microsoft and Excel are registered trademarks of Microsoft Corporation in the United States and other countries. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. 18