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9.4 Comparison of Phosphopeptides

9. Post-Translational Modification » 9.4 Comparison of Phosphopeptides

Comparison of phosphopeptides is accomplished through the identification©Compare tool, as shown below: Figure 9.5.1: identification©Compare link The identification©Compare tool can be used to assess the overlap of phosphopeptides between analyses, filter high…

9.5 Quantitation of Phosphopeptides

9. Post-Translational Modification » 9.5 Quantitation of Phosphopeptides

The following procedure should be followed to calculate the relative ratios of phosphopeptides between isotopically-labeled samples (i.e. SILAC or 15N). For phosphopeptides with only one phosphorylated residue, this corresponds to quantitation of the phosphorylation…

9.3 Validation of Phosphopeptides and Phosphorylation Sites

9. Post-Translational Modification » 9.3 Validation of Phosphopeptides and Phosphorylation Sites

DTASelect is used to filter modified and unmodified peptide sequences from IP2 search results. Ascore can be used for phosphorylation site localization. After selecting the DTASelect filter, run “PhosphoAnalysis” by clicking “Run” as shown below: Figure…

10.1 Identification Compare

10. Statistical Analysis With Multiple Data Sets » 10.1 Identification Compare

The identification Compare tool is used to compare search results in a peptide or protein level. This tool does not calculate any statistical values (e.g. p- or q- values). This is a legacy tool, and we recommend users to try the Identification Stat Compare tool…

7.3 Protein Identification Results

7. Protein Identification » 7.3 Protein Identification Results

This section describes how to understand the search results In the experiment page, click “Whole view” to display protein database search results in one page. Or, click the “page view” to display protein database search results in split pages.…

7. Protein Identification

7. Protein Identification

7.1 ProLuCID Search Engine 7.2 Comet Search Engine 7.3 Protein Identification Results 7.4 Delta mass Plotter 7.5 PSM Score Distribution 7.6 Search a protein throughout a project 7.7 Correct mass in spectra 7.8 Batch search 7.9 Download sqt…

9. Post-Translational Modification

9. Post-Translational Modification

9.1 Background and Description 9.2 Analysis of Localization Score 9.3 Validation of Phosphopeptides and Phosphorylation Sites 9.4 Comparison of Phosphopeptides 9.5 Quantitation of Phosphopeptides 9.6 Kinase Enrichment Analysis with…

10.2 Identification Stat Compare (label-free analysis using spectral count)

10. Statistical Analysis With Multiple Data Sets » 10.2 Identification Stat Compare (label-free analysis using spectral count)

Identification Stat Compare statistically compares samples by using spectral counts to find regulated proteins. To start, you can click the “Project” tab to view all existing projects with a set of experiments. Figure 10.2.1: Home Page. Click…

1.3 Points of Contact

1.General Information » 1.3 Points of Contact

Mailing address : 9920 Pacific Heights Blvd #150 San Diego, CA 92121 Tel: +1-858-263-3260 Fax: +1-858-794-1450 Email: support@integratedproteomics.com

9.2 Analysis of Localization Score

9. Post-Translational Modification » 9.2 Analysis of Localization Score

IP2 currently provides two localization scoring tools – AScore (Sean A. et. al., 2006) and Luciphor (Fermin et al. 2014). AScore is for phosphopeptides, while Luciphor is for any PTM peptides. To run Luciphor, find PTM localization analysis box in the search…

13.2 Batch Generation of Fragment ion Annotation Images

13. Additional Tools » 13.2 Batch Generation of Fragment ion Annotation Images

Users can generate all spectra for a project/experiments as either image or pdf files. Figure 13.1.2 An image file generated by the batch tool.

1.General Information

1.General Information

IP2 updated info: 3/21/19 Fractional data analysis tool was added. 2/26/19 isobaric quant analysis (e.g. TMT) updates progress bar in the job status page 2/22/19 Molecular weight and protein length are added to the protein-level identification page 2/20/19 Batch…

14.1 For your Publications

14. For your Publications » 14.1 For your Publications

SAMPLE METHODS FOR PUBLICATIONS Please customize any of the following sections for your publication: Version 1 Protein identification and quantification analysis were done with Integrated Proteomics Pipeline (IP2, Integrated Proteomics Applications, Inc. San Diego,…

9.1 Background and Description

9. Post-Translational Modification » 9.1 Background and Description

As with all shotgun proteomics experiments, global quantitative post-translational modification (PTM) proteomics relies heavily on appropriate bioinformatics analyses. The relevant bioinformatics methods associated with global quantitative post-translational…

10. Statistical Analysis With Multiple Data Sets

10. Statistical Analysis With Multiple Data Sets

10.1 Identification Compare 10.2 Identification Stat Compare 10.3 Quantification Compare 10.4 Quantification Compare Isobaric 10.5 Quantitative Regression 10.6 Label-free Analysis using XIC

1.1 System Overview

1.General Information » 1.1 System Overview

The Integrated Proteomics Pipeline (IP2) is a proteomics data analysis platform that has been designed by a core bioinformatics team. We understand that you want to receive your results fast without trading accuracy. Consequently, the IP2 has been designed to easily…

13.4 Fractional analysis

13. Additional Tools » 13.4 Fractional analysis

IP2 can compare fractionated biological samples in two different ways. Users can use either identification compare tool (see manual section 10) or the fractional analysis tool. With identification compare tool, users need to upload each fractional data to separate…

7.6 Search a protein throughout a project

7. Protein Identification » 7.6 Search a protein throughout a project

On the Project View page, there is a link for “Search Protein” at the top of the page, underneath the “More” dropdown button. Click it to view the protein search result list. Figure 7.6.1: Project View with “Search Protein”…

8.1 MS1-based Analysis(SILAC, 15N, Dimethyl, etc)

8. Quantification » 8.1 MS1-based Analysis(SILAC, 15N, Dimethyl, etc)

Quantitative analysis can be largely divided into two different categories – label-free and labeling analysis. In this chapter, we will cover both labeling analysis and label-free analysis using peak area. For label-free analysis using spectral count, see…

8.3 Isobaric labeling(e.g.TMT)

8. Quantification » 8.3 Isobaric labeling(e.g.TMT)

Result 1. After protein identification is done, click the ‘run now’ link underneath the quantitative analysis column on the experiment page. Figure 8.3.1 2. Select isobaric labeling. If you have less than or equal to 6-plex, select “Isobaric…

8.5 Site Specific tag Analysis

8. Quantification » 8.5 Site Specific tag Analysis

This option is for site-specific chemical probe tagging (e.g TEV) or metabolomic labeling (e.g. AHA) analysis. 1. Protein Identification In the ProLuCID search page, differential modification search options are recommended as below: Please note that if you consider…

13.7 Thermal Proteome Profile (TPP) Temperature Range (TR)

13. Additional Tools » 13.7 Thermal Proteome Profile (TPP) Temperature Range (TR)

The Cellular Thermal Shift Assay (CETSA) is based on the discovery that protein melting curves can also be generated in intact cells and that drug binding leads to very significant thermal stabilization of proteins. It quantifies the changes in the thermal stability of…

7.2 Comet Search Engine

7. Protein Identification » 7.2 Comet Search Engine

An open-source tandem mass spectrometry (MS/MS) sequence database search tool. The comet is a search engine developed by Jimmy Eng, who is an author of SEQUEST. In the IP2, we have added additional features such as heavy/medium search, graphical user interface, cluster…

7.1 ProLuCID Search Engine

7. Protein Identification » 7.1 ProLuCID Search Engine

ProLuCID is a sensitive tandem mass spectra-based protein identification program developed in Yates laboratory at The Scripps Research Institute. From the experiment page, click the ProLuCID search button to open search parameter page. Figure 7.1.1: link to start…

11.1 Spectral Quality Control

11. Quality Control Tool » 11.1 Spectral Quality Control

To evaluate spectral quality, we recommend users to upload both MS1 and MS2 files to general all graphs (Uploading MS2 files only will generate graphs only for MS2 files). Uploading Thermo experiment log file will help generate additional information. Type in purity…

12.1 Gene ontology

12. Gene ontology » 12.1 Gene ontology

After running the Protein Identification STAT Compare tool, you can find the gene ontology column to run GO analysis. Click ‘run’ or ‘re-run’ to start gene ontology analysis. In the backend analysis, the IP2 uses Ontologizer9 Figure 12.1.1: Gene Ontology run…

11.4 Additional Quality control

11. Quality Control Tool » 11.4 Additional Quality control

QUALITY CONTROL AFTER PROTEIN/PEPTIDE IDENTIFICATION Click the “View” link under the Misc quality check column. Figure 11.4.1: Search result page Figure 11.4.2: Charge state distribution Figure 11.4.3: Molecular Weight Histogram Figure…

8.4 Isobaric labeling N-plex Analysis

8. Quantification » 8.4 Isobaric labeling N-plex Analysis

1. To run N-plex Isotope Labeling (e.g. 10-plex TMT), click ‘Run Now’ on an experiment page. Figure 8.4.1 2. Select TMT N-plex Figure 8.4.2 3. Type the number of plex such as 10, and click the button. Figure 8.4.3 4. Make sure all parameters…

6.1 Project management

6.Project management » 6.1 Project management

After login, click the “Projects” tab to view the list of experiments. Then, click the “view” link to list the DTASelect search result in the table; Figure 6.1.1 Home page. From the list of project, click the “view” to display…

5.2 IP2Daemon for batch file uploading

5. Upload Spectral Files » 5.2 IP2Daemon for batch file uploading

CONVERT RAW TO SPECTRAL FILES USING IP2DAEMON CLIENT SOFTWARE IP2Daemon is a software to automatically convert raw files into ms1/ms2/ms3 files and upload to the ip2 server. Currently, IP2Daemon supports the following features. RAW file to spectral files WATERS…

10.6 Label-free Analysis using XIC

10. Statistical Analysis With Multiple Data Sets » 10.6 Label-free Analysis using XIC

In the project page, click the Label-Free Analysis button on the menu Figure 10.6.1: Link for Quantitative Comparison. The Label-Free Analysis Page will show previous analysis results. To run a new Label-Free Analysis, click on “Run Label-Free…

10.4 Quantification Compare Isobaric (TMT, iTRAQ, etc)

10. Statistical Analysis With Multiple Data Sets » 10.4 Quantification Compare Isobaric (TMT, iTRAQ, etc)

In the list of experiments, click “quantitativeCOMPARE” to list all existing quantitation comparisons. Figure 10.4.1: Link for Quantitative Comparison. Click “run new quantitative ©COMPARE” to build a new comparison. Figure 10.4.2 view…

11.3 PSM Score Distribution

11. Quality Control Tool » 11.3 PSM Score Distribution

1. Click the plot link in PSM score column at search result page. Figure 11.3.1 2. The plot shows PSM score distribution. Figure 11.3.2

7.5 PSM Score Distribution

7. Protein Identification » 7.5 PSM Score Distribution

1. Click the plot link in PSM score column at the search result page. Figure 7.5.1 2. The plot shows PSM score distribution. Figure 7.5.2

16.1 IP2 FAQ

16. Appendix » 16.1 IP2 FAQ

Q1. In QuantCompare option, what is the difference between the protein ratio from Linear Regression and the protein composite ratio? A. The protein ratio from linear regression is conventional Census ratio using linear regression of light and heavy peptides in…

15.1 Server

15. IP2 Troubleshooting » 15.1 Server

IP2 troubleshooting Q. IP2 page is not accessible. A. Check that tomcat server is running (as ip2 user) : killtomcat (this command will restart the IP2 server) Check that apache server is running (as root) : /etc/init.d/httpd status or /etc/init.d/httpd restart…

10.3 Quantification Compare (SILAC, 15N, Dimethyl, etc)

10. Statistical Analysis With Multiple Data Sets » 10.3 Quantification Compare (SILAC, 15N, Dimethyl, etc)

In the list of experiments, click “quantCOMPARE” to list all existing quantitation comparisons. Figure 10.3.1: Link for Quantitative Comparison. Click “run new quantitative ©COMPARE” to build a new comparison. This section shows how to…

11.2 Delta mass Plotter

11. Quality Control Tool » 11.2 Delta mass Plotter

Plot delta mass in ppm. 1. Click the “Plot” link in Delta Mass column on the search result page. Figure 11.2.1 2. The plot shows delta mass distribution. The x-axis is delta mass in ppm, and the y-axis is peptide count. There are three different…

7.4 Delta mass Plotter

7. Protein Identification » 7.4 Delta mass Plotter

PLOT DELTA MASS Plot delta mass in ppm. 1. Click the “Plot” link in Delta Mass column on the search result page. Figure 7.4.1 2. The plot shows delta mass distribution. The x-axis is delta mass in ppm, and the y-axis is peptide count. There are three…

13.5 Reactome Pathway Analysis

13. Additional Tools » 13.5 Reactome Pathway Analysis

1. Click the “Tools->reactome pathway analysis” in the menu. 2. It opens “Reactome Pathway Analysis” page. 3. To run a new pathway analysis, click “Run new reactome analysis” link. 4. Users can upload an input file by…

10.5 Quantitative Regression

10. Statistical Analysis With Multiple Data Sets » 10.5 Quantitative Regression

We have introduced the definition of data analysis methods in chapter 5. In this chapter, you will find the steps to execute the analysis in IP2. Figure 10.5.1: Link for quant ©Regression of the desired project Figure 10.5.2: List of samples for each experiment…

13.6 Clustering Analysis

13. Additional Tools » 13.6 Clustering Analysis

User can run a clustering analysis for grouping protein/genes/peptides upon quantitative value changes. The tool uses the k-means clustering algorithm. 1. Click the “Tools->reactome pathway analysis” in the menu. 2. It opens the “Clustering…

13. Additional Tools

13. Additional Tools

13.1 Proteome Discoverer Compatibility 13.2 Batch Generation of Fragment ion Annotation Images 13.3 Export data for other tools 13.4 Fractional Analysis 13.5 Reactome Pathway Analysis 13.6 Clustering Analysis 13.7 Thermal Proteome Profile (TPP) Temperature…

15.2 Search

15. IP2 Troubleshooting » 15.2 Search

Q. DTASelect failed after search A. Login to the IP2 server by ssh command line. Type ‘jobstatus’ or open $HOME/ip2_tomcat/webapps/ip2/config/jobstatus.txt file to find the path of the failed job. Check # of sqt and ms2 files. If they are the same, the…

17.1 API Usage

17. API » 17.1 API Usage

Objectives The IP2 platform creates multiple types of quantitative output files as analysis of the data and uses tabular or graphical tools for display in web browser. The graphical web user interface is a great tool to easily access data for scientists. Meanwhile,…

2.1 System Summary

2. System Summary » 2.1 System Summary

The IP2 has been developed as a server-side platform, so it is operating system independent. Users need a web browser (e.g. firefox, IE, safari, Chrome, etc.) to access the IP2 system. However, some of the graphical user interfaces require the Java be installed on the…

7.9 Download sqt files

7. Protein Identification » 7.9 Download sqt files

The SQT file contains matching information between MS/MS spectra and a sequence database. See more information at McDonald, W.H. et al. “MS1, MS2, and SQT-three unified, compact, and easily parsed file formats for the storage of shotgun proteomic spectra and…

3.5 Administration Account

3.Getting Started » 3.5 Administration Account

The administration account is different from other user accounts. This account is not for data analysis. It is used to manage user accounts in your IP2 server, including creating new users, enabling or disabling users, and updating user information. Each instance of…

4.1 Database View

4.Protein Database Management » 4.1 Database View

After logging in to your account, the menu bar will be displayed at the top of the browser window. Move the mouse to “Database” and click to navigate to the page which will show all the existing databases. Figure 4.1.1 By default, the table is sorted by…

13.1 Proteome Discoverer Compatibility

13. Additional Tools » 13.1 Proteome Discoverer Compatibility

Users can upload any pepxml files to the IP2 platform for downstream data analysis (e.g. the result file from the Proteome discoverer). In the pepxml file, you need to make sure the fasta database name is available in the IP2 database page. You can change the database…

3.2 System Menu

3.Getting Started » 3.2 System Menu

Through the Navigation menu at the top of the page, it is easy to view system information by categories. Home: Click this tab to return to home page from any page. Database: By clicking this tab, user can view all existing databases, with data source, date and…

9.6 Kinase Enrichment Analysis with Phosphosites

9. Post-Translational Modification » 9.6 Kinase Enrichment Analysis with Phosphosites

In the experiment page, there are two download buttons in a PTM-Gene download column – search results and quant results buttons. The quantitative result button may not appear if the quantitative analysis is not done yet. Figure 9.7.1 When you click the…

3.1 Logging On

3.Getting Started » 3.1 Logging On

Enter your URL in your browser’s navigation bar, and the login page shown below will load. URL can be your local IP2 server, a remote one, or cloud one depending on your IP2 license type. Figure 3.1.1 Login page for IP2 Enter your username and password and then…