GPM-MRM

GPM-MRM is functionality that has been added to The GPM to assist researchers in finding signature combinations of peaks in MS/MS spectra. Spectra can be searched by any combination of parent ion mass, accession number or peptide sequence and the results can be pared down by fragment mass presence or uniqueness.

Contents 1 Types of Searches 2 Search Results 3 Example Searches 4 References

Types of Searches

The default values for some of the search parameters are as follows:

• parent ion mass range: 0.1 kDa
• fragment ion mass range: 0.1 kDa
• peak sorting: intensity, decreasing

A search can be performed with any combination of the following parameters:

• parent ion mass
  • returns all spectra within the given range
• sequence
  • returns all spectra applying to that peptide sequence.
  • For finding unique peaks, peaks for that sequence are compared against other spectra within range of all observed weights of that sequence.
• accession number
  • when used in combination with sequence or parent ion mass, specifying the accession number acts as a filter, displaying only those results which both meet the mass or sequence criteria and were found for the given protein.
  • when used alone, it returns a summary page of ten columns. By default, the columns are sorted in order of the number of GPMDB entries for a peptide in z=2 charge state. A dash (—) in the table represents zero.
    1. Peptide Sequence: the peptides sequence identified
    2. Charge: charge states observed for this peptide in this protein
    3. Ret: the calculated HPLC retention time for this peptide
    4. MRMs: the number of spectra used to calculate the information stored in the peakdb tables for this peptide.
    5. Observed z=1: the number of identifications of this peptide stored in GPMDB with charge state 1.
    6. Observed z=2: the number of identifications of this peptide stored in GPMDB with charge state 2.
    7. Observed z=3: the number of identifications of this peptide stored in GPMDB with charge state 3.
    8. log₁₀(ω/ω₀) z=1: the normalized peptide count for this peptide in charge state 1.
    9. log₁₀(ω/ω₀) z=2: the normalized peptide count for this peptide in charge state 2.
    10. log₁₀(ω/ω₀) z=3: the normalized peptide count for this peptide in charge state 3.

The above table also appears when the "(mrm)" link is clicked from the protein.pl page for a specific identification for a protein. When reached from protein.pl, the peptides and associated charge states from that specific identification will be displayed in bold. Please see the Example Searches section for a demonstration of this display mode.

The order of precedence in searching is mass, residue sequence, accession number. If more than one of those parameters is provided, the lower priority parameters will be used to filter results that contain only that parameter.

For any of the above searches, providing the fragment ion mass will filter results that did not include a fragment ion within the given range of the supplied fragment ion mass.

The fragment ion mass range is used to classify fragment masses as unique or not. In a result set, any two masses between different results that differ by less than the fragment ion mass range are considered to be the same, and therefore non-unique. A larger fragment ion mass range will lead to fewer fragment ions being considered unique.

The Pattern parameter can be used to filter out results that do not match a given pattern. For help with the pattern syntax, a short tutorial can be seen at: Regular Expressions Quick Start.

Search Results

The data returned by the search returns in the following order:

• Sequence: the residue sequence of the peptides.
• m/z: the mass-to-charge ratio recorded
• z: the charge
• Accession #'s: the list of identified proteins in which this peptide has been identified
• Peaks: up to 20 mass/intensity peaks from the spectra
  • Beneath the table of mass/intensity peaks, an orange button labeled Spectrum opens a new window that displays the spectrum described by the table above.

Unique fragment ion masses are colour-coded for fast identification, and the rows of results are ordered from fewest to most corresponding accession numbers. The peak/intensity tables can be displayed either by ascending weight or descending intensity. Non-unique peaks can be masked out for a simplified display.

Example Searches

The links below demonstrate the different search and display parameters (changeable parameters show in bold):

• Peptide sequence WIYEDVER: Results
• Parent ion mass 1735 Da, parent mass range of 1.0 Da: Results
• Same as above, but show only results with accession number ENSP00000350063: Results
• Parent ion mass 889 Da, parent mass range of 0.5 Da, fragment ion range of 1.0 Da, filtered by sequence AMGIMNSFVNDIFER and accession number IPI00419833.7, peaks ordered by intensity with only unique peaks shown: Results
• Identification of protein ENSP00000216297 as identified in result file GPM10100150771: Results

References

• Multiple Reaction Monitoring as a Method for Identifying Protein Posttranslational Modifications : Abstract