Peptide Spectrum Matching

Disclaimer this tool needs a peptide database to query against, if you did not create one yet you can do so by using the PeptideDB tool.

An established method to identify acquired MS/MS spectra is the comparison of each spectrum with peptides in a reference database.

Given raw a MS run in the mzLite or mzml format, this tool iterates accross all recorded MS/MS scans and determines the charge state of precursor ions which were selected for fragmentation. With this it is possible to query the peptide data base for every precursor ion mass +/- a tolerance (which defines the so called 'search space') and retrieve peptides that are theoretical candidates for a match. For each of the peptide candidates we create an theoretical spectrum in silico and compare it to the measured MS/MS scan.

To measure similarity we use our own implementations of three established search enginge scores: SEQUEST, Andromeda and XTandem. The search space is extended by so called decoys. Decoys are reversed counterparts of peptides within the search space and allow us to assign a false discovery rate to each scored peptide using the PSMStatistics tool.

Parameters

The following table gives an overview of the parameter set:

Parameter Generation

Parameters are handed to the cli tool as a .json file. you can download the default file here, or use an F# script, which can be downloaded or run in Binder at the top of the page, to write your own parameter file:

#r "nuget: BioFSharp.Mz, 0.1.5-beta"
#r "nuget: Newtonsoft.Json, 12.0.3"
#r "nuget: ProteomIQon, 0.0.5"

open BioFSharp.Mz.SearchDB
open Newtonsoft.Json
open ProteomIQon
open ProteomIQon.Domain
open BioFSharp.Mz

let chargeDetermParams :ChargeState.ChargeDetermParams =
    {
        ExpectedMinimalCharge = 2
        ExpectedMaximumCharge = 5
        Width                 = 1.1
        MinIntensity          = 0.15
        DeltaMinIntensity     = 0.3
        NrOfRndSpectra        = 10000
    }

let andromedaParams: AndromedaParams =
    {
        PMinPMax                = 4,10
        MatchingIonTolerancePPM = 100.
    }

let peptideSpectrumMatchingParams :Dto.PeptideSpectrumMatchingParams =
    {
        ChargeStateDeterminationParams = chargeDetermParams 
        LookUpPPM                      = 30.
        nTerminalSeries                = NTerminalSeries.B
        cTerminalSeries                = CTerminalSeries.Y
        Andromeda                      = andromedaParams
    }


let serialized =
    peptideSpectrumMatchingParams
    |> JsonConvert.SerializeObject

Executing the Tool

Disclaimer this tool needs a peptide database to query against, if you did not create one yet you can do so by using the PeptideDB tool.

To score all MS/MS of an MS run simply call:

proteomiqon-peptidespectrummatching -i "path/to/your/run.mzml" -d "path/to/your/database.sqlite" -o "path/to/your/outDirectory" -p "path/to/your/params.json"

It is also possible to call the tool on a list of MS files. If you have a mulitcore cpu it is possible to score multiple runs in parallel using the -c flag:

proteomiqon-peptidespectrummatching -i "path/to/your/run1.mzml" "path/to/your/run2.mzml" "path/to/your/run3.mzml" -d "path/to/your/database.sqlite" -o "path/to/your/outDirectory" -p "path/to/your/params.json" -c 3

A detailed description of the CLI arguments the tool expects can be obtained by calling the tool:

proteomiqon-peptidespectrummatching --help