IP2 Phosphorylation Analysis Manual

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Background and Description: As in all shotgun proteomics experiments, global quantitative phosphoproteomics relies heavily on appropriate bioinformatics analyses. The relevant bioinformatics methods associated with global quantitative phosphoproteomics are confident identification and validation of thousands of phosphopeptides from MS/MS spectra, determination of phosphorylation stoichiometry of phosphopeptides, localization of phosphorylation sites, and measurement of the ratio of phosphorylated peptides. Each one of these steps is of equal importance. That is, if any one of these steps is inaccurate or of low confidence, the entire quantitative analysis is equally inaccurate and of low confidence. Identification of phosphopeptide sequences and measurement of phosphorylated peptides leverages the accuracy of global quantitative proteomics (i.e. SEQUEST, ProLuCID, DTASelect, and CENSUS). When the appropriate filtering methods are used, these two steps are already of high confidence. The remaining essential steps of phosphosphopeptide validation, determination of phosphorylation stoichiometry of phosphopeptides and phosphorylation site localization have been addressed by the introduction of Debunker and AScore. This manual describes the integration and usage of Debunker and Ascore in the quantitative pipeline (Phospho Quant) of the web-based Integrated Proteomics Pipeline (IP2) using Orbitrap MS data and 15N isotopic labeling. These methods can be applied to other high mass accuracy instruments (i.e. TOF, FTIRC, etc.) and labeling strategies (e.g. SILAC).

Identification of phosphopeptides using SEQUEST or ProLuCID

  1. Use RAWExtract (downloadable at https://fields.scripps.edu) to convert .RAW files to MS1 and MS2 files.
  2. Upload files .RAW, MS1, and MS2 files to an experiment within a project on IP2.
  3. Click either SEQUEST or ProLuCID to search the MS/MS spectra against a protein database. The following steps will describe the use of ProLuCID, but the SEARCH MANUAL can be consulted for searching using SEQUEST. The following screen should appear:
    Pic48.png
  4. Select the location for the computational search to be performed, either “Cluster” or “Cloud”, depending on your available resources.
  5. Make the appropriate selections within the “Basic parameters” section:
    • Select a “Protein database” for the organism your sample originated. For this example analysis, mouse was the sample origin so we select a mouse database.. See the DATABASE manual for uploading databases if one is not present for your sample organism.
    • Select the “Fragmentation/activation method” for which MS/MS spectra were acquired. For this example analysis, multistage-activation CID was used so we selected “CID”.
    • Select the “Precursor/peptide mass tolerance”. An Orbitrap was used for these MS analyses, so we select “High resolution”. Although not as confident for phosphorylation analysis, if low resolution precursor data was acquired (i.e LTQ) select “Low resolution”. Please see the SEARCH MANUAL for a description of the precursor and fragment mass tolerance default values.
  6. Choose the appropriate options within the “Enzyme specificity” section. Trypsin was used for this sample, but see the SEARCH MANUAL for a description of settings for other proteases:
    • Select a “Specificity” of “one end” and a “Max num internal miscleavage” of “unlimited”. Phosphorylation events near lysine or arginine residues can inhibit trypsin cleavage, so leaving the option for only one tryptic end and more than 3 missed cleavages is advantageous to identifying more phosphopeptides. If adequate computational resources are available, a “Specificity” of “none” can be used to potentially find all phosphopeptides.
    • Leave the defaults of “Protease name” of “trypsin”, “Residues” of “KR”, and “Cut position” of “C-term”.
  7. If cysteine residues were carbamidomethylated with iodoacetamide or chloroacetamide, then “57.02146 C” should be added in the “Amino acid specific static modifications” box.
  8. Continue adding search parameters in the “Differential/variable modifications” section as shown below:
    • Enter the number of phosphorylation modifications expected or desired to find per phosphopeptide; 3 is commonly used for most phosphopeptide enrichment methods.
    • Add the mass and residues (79.9663 STY) for differential phosphorylation on serine, threonine, and tyrosine in the “Differential modification” box.
    • If the experiment was quantitative (i.e. 15N or SILAC), select the appropriate “Metabolic Labeling Search” options. For this example, we selected “yes” and “N15”. If SILAC was used, select “Selected amino acids” and enter the appropriate mass shifts. A full description for this can be found in the QUANTITATION MANUAL.
    • Unique to phosphorylation MS/MS analysis, multistage activation CID fragmentation can be used to improve the fragmentation of peptides after neutral loss of phosphate.[MSA ref] If this method was used, select “Multistage activation mode” option “1” to search for both normal and neutral loss fragment ions and option “2” to search for only neutral loss fragment ions.
      Pic49.png

Filtering of phosphopeptides using DTASelect
The search results from SEQUEST or ProLuCID are filtered by DTASelect. This is performed automatically after the search and can be repeated to adjust the filtering parameters. The primary options for this are shown in the following window and steps. Further options can be found by clicking on the “Additional DTASelect options” link and a general description can be found in a Current Protocols in Bioinformatics chapter.[Cociorva Ref] The following options are generally best for phosphoproteomics.

  1. Make the appropriate “Basic DTASelect 2.0 Parameters” selection for phosphoproteomics.
    • Enter “1” for “Minimum number of peptides per protein (-p)”. Only one phosphorylation site or phosphopeptide may be present or detected for a protein.
    • Select “1” for “Minimum number of tryptic ends per peptide (-y)”. If a “Specificity” of “none” was used in the search, “0” can also be selected to maximize phosphopeptide identifications.
    • Enter a desired “False positive rate (--fp)”. A common FDR is 0.1% at the peptide level.
    • Enter a “Precursor delta mass cutoff (-DM)”. A common value is 10 ppm for Orbitrap data, but can be assessed based on the precision of the instrument used.
  2. Make the appropriate “Advanced DTASelect 2.0 Parameters” selection for phosphoproteomics.
    • Select a “Peptide modification requirement” of “1”.
    • Select “yes” for “Statistics with delta mass (--mass)”.
    • Select “yes” for “Statistics with modifications (--modstat)”.
    • Select “yes” for “Statistics with tryptic status (--trypstat)”.
    • Leave the default of “Both” for “Include heavy search” unless only the unlabeled “Light only” or isotopically-labeled “Heavy only” peptides and proteins need to be identified or quantified.
    • Enter other advanced options as necessary in “Protein ID filter (-e)”, “Peptide sequence filter (-Sic)”, and “Additional DTASelect options”.
    • Select “Overwrite the previous” if the previous DTASelect result is unwanted or “Run as new” if the previous DTASelect result is wanted.
      Pic50.png

Validation of phosphopeptides and phosphorylation sites
DTASelect is used to filter modified and unmodified peptide sequences from SEQUEST and ProLuCID search results. Debunker[Anal Chem. 2007 Feb 15;79(4):1301-10] and Ascore[Nat Biotechnol 24, 1285-1292] can be used to assign statistical confidence in phosphopeptide identification and phosphorylation site localization, respectively, of both unlabeled and isotopically-labeled phosphopeptides. After DTASelect filter run “PhosphoAnalysis” by clicking “Run” as show in the window below.
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The following screen should appear where “Run” should be clicked again.
Note: For phospho peptide quantitation, PhosphoAnalysis MUST be run before Quantitative Analysis.
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When the analysis is complete the “View” and “Rerun” links will appear in the “phosphoanalysis” column.
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Upon clicking “View” in the “phosphoanalysis” column the following screen will appear:
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This above page should appear which lists:

  1. The uniqueness of the phosphopeptide (Unique column).
  2. The most confident phosphorylation site (Corrected phosphopeptide column).
  3. The original identified phosphorylation site (Original phosphopeptide column).
  4. The localization confidence (Localization Score) – if greater than 13 there is 95% confidence in the phosphorylation site and if greater than 19 there is 99% confidence in the phosphorylation site.
  5. The phosphopeptide confidence (Debunker Score) – the value directly corresponds to the confidence on a scale of 1 for phosphoserine and phosphothreonine.
  6. The DTASelect confidence score.
  7. The files which contained the spectra for the identified phosphopeptide (Filename).
  8. The conventional peptide identification scores (XCorr, DeltCN, Conf%, M+H+, CalM+H+, TotalIntensity, SpR, Prob, IonProportion).
  9. The spectral counts of the phosphopeptide in different analyses (Spec Count).
  10. The protein origin of the phosphopeptide (Proteins and Protein Descriptions).

Comparison of phosphopeptides
Pic55.png

The identification©Compare tool can be used to assess the overlap of phosphopeptides between analyses, filter confident phosphopeptides for comparison, and perform label-free comparison by spectral counts. After clicking on the “identification©COMPARE” link, the following screen appears with a link to run a new comparison and lists the previous performed comparisons. Click on “run new identification ©COMPARE”.
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The following screen allows for the selection of different phosphopeptide experiments to compare. To perform a phosphopeptide comparison

  1. Select “phosphopeptide”.
  2. Select the check boxes for the experiments to be compared.
  3. Click “Run”.


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You will return to the comparison page where the results will be listed.
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The results can be viewed either as a Venn diagram or as results page by clicking on “Venn Diagram” or “view”, respectively. A new page will open to display the Venn diagram. The text and circles can be rearranged by left or right clicking and holding down while moving. The image can be printed to a PDF or a printer by clicking “Print”.


Pic59.png

The results page should look as follows which lists:

  1. The uniqueness of the phosphopeptide (Unique column).
  2. The most confident phosphorylation site (Corrected phosphopeptide column).
  3. The original identified phosphorylation site (Original phosphopeptide column).
  4. The localization confidence (Localization Score) – if greater than 13 there is 95% confidence in the phosphorylation site and if greater than 19 there is 99% confidence in the phosphorylation site.
  5. The phosphopeptide confidence (Debunker Score) – the value directly corresponds to the confidence on a scale of 1 for phosphoserine and phosphothreonine.
  6. The files which contained the spectra for the identified phosphopeptide (Filename).
  7. The conventional peptide identification scores (XCorr, DeltCN, Conf%, M+H+, CalM+H+, TotalIntensity, SpR, Prob, IonProportion).
  8. The spectral counts of the phosphopeptide in different analyses (Spec Count).
  9. The protein origin of the phosphopeptide (Proteins and Protein Descriptions).


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In the web-based results, they can be sorted by any column by clicking on the column title. These results can be exported to view in other programs using the different “Export options” at the bottom of the page.
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Alternatively, these results can be copied from within the window that opens when the “view peptide count COMPARE file” link is clicked.
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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 site.

  1. Click “Run now” in the “Quantitative Analysis” column for the associated search and filtering results.
    Pic64.png
  2. Select the appropriate labeling method. For this example we used 15N. For a description of procedures for other labeling methods see the QUANTITATION MANUAL.
    Pic65.png
  3. The default quantitation values for high mass accuracy data (e.g. Orbitrap) and appropriate masses differences for each amino acid will appear in the following table. Click “Submit”.
    Pic66.png
  4. After the Census quantitation from MS1 chromatograms is complete, the following links will be available for both viewing quantitative results for the run (Quantitative Analysis – View data) and validating individual peptide quantitation values (Census - Launch Census).
    Pic67.png


Quantitative comparison and filtering of statistically confident phosphopeptides
The quantitation©COMPARE tool can be used to quantitatively compare phosphopeptides, filter statistically confident phosphopeptides, and perform label-free comparison by spectral counts. To run, click on the “quantitation©COMPARE” link.
Pic68.png

The following screen appears with a link to run a new comparison. Click on “run new quantitative ©COMPARE”.
Pic69.png

The list of experiments which have had PhosphoAnalysis run on them within that project will be listed with relevant experimental information. Start by entering a name for the replicate experiments to be combined for statistical quantitative comparison in the “group name” entry box. Next select the experiment to be combined by checking the boxes next to the experiments as shown below. Then click “Add to group”. The next screen that appears will have the first “Group name” listed of experiments that were combined on the last screen. Repeat the selection of experiment to be grouped for quantitative comparison to another group. Click “Add to group” again and repeat this process until all of the comparisons to be performed are grouped. Then click “Next”.
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The following screen will appear. Name the quantitative comparison in the “Give a name to save” entry box and click “Run it now”.
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The following screen appears with the previously performed comparisons.
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Click on “ANOVA comparison” to view the quantitative comparison results with the following format:
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This page lists:

  1. The uniqueness of the phosphopeptide (Unique column).
  2. The most confident phosphorylation site (Corrected phosphopeptide column).
  3. Sequences before localization analysis
  4. The localization confidence (Localization Score) – if greater than 13 there is 95% confidence in the phosphorylation site and if greater than 19 there is 99% confidence in the phosphorylation site.
  5. The phosphopeptide confidence (Debunker Score) – the value directly corresponds to the confidence on a scale of 1 for phosphoserine and phosphothreonine.
  6. The files which contained the spectra for the identified phosphopeptide (Filename).
  7. The conventional peptide identification scores (XCorr, DeltCN, Conf%, M+H+, CalM+H+, TotalIntensity, SpR, Prob, IonProportion).
  8. The spectral counts of the phosphopeptide in different analyses (Spec Count).
  9. The protein origin of the phosphopeptide (Proteins and Protein Descriptions).

A key feature to this page is the ability to actively filter the phosphopeptides quantified for comparison based on their Localization Score, Debunker Score, or both. To do this, enter the desired cutoff value for either or both in the entry boxes and click “Filter”. To reintegrate from earlier in the manual, if the Ascore is greater than 13 there is 95% confidence in the phosphorylation site and if greater than 19 there is 99% confidence in the phosphorylation site. While the phosphopeptide confidence value (Debunker Score) directly corresponds to the confidence on a scale of 1 for phosphoserine and phosphothreonine.


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After filter as above, the results can be exported by selecting one of the options at the bottom of the page as shown below
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