PSMBasedQuantification

PSMBasedQuantification turns the identifications from PSMStatistics into peptide ion intensities. For every identified peptide ion (sequence, modification state and charge) it takes the scan times of its MS/MS identifications, extracts an ion chromatogram of the monoisotopic m/z in a window around them, detects the peaks in that trace and fits the peak closest to the identification. The fitted area is the reported quantity. For metabolically labeled samples the tool computes the m/z of the partner ion (the heavy form of a light identification, or the other way round) and quantifies that trace as well, so every row can carry a light and a heavy value.

Inputs and outputs

The input must be .mzlite. A directory given to -i is also scanned for .mzML, but the tool opens every file with the mzlite reader and fails on anything else, so convert first. With one file per flag the pair is used as given. With lists, the n-th mzlite file is paired with the n-th qpsm file unless you pass -mf, which pairs files by their base name (run1.mzlite with run1.qpsm) and skips mzlite files without a partner.

For every run the tool writes run.quant, tab separated with a header and one row per quantified peptide ion. Next to the identifiers and the best q-value and PEP value of the ion it reports the fitted quantity (Quant_Light, Quant_Heavy), the apex intensity, the fit parameters, the difference between identification and fitted scan time, the extracted traces and the isotope pattern for both channels. QuantBasedAlignment, AlignmentBasedQuantification, AlignmentBasedQuantStatistics and JoinQuantPepIonsWithProteins read .quant files.

With -dc the tool writes one chart per peptide ion into run_plots, plus mzErrorAndCorrection.html and precMzCorrected.html for the m/z calibration it derives from the identifications. -z packs that directory into run_plots.zip and deletes it. Delete an existing run.quant in the output directory before a rerun, the tool appends to it. Logs go to PSMBasedQuantification_log.txt and run_log.txt in the output directory.

Parameters

The wavelet parameters:

The default file is QuantificationParams.json. To run without baseline correction set BaseLineCorrection = None in the script below.

Writing a parameter file

open ProteomIQon
open ProteomIQon.Domain
open FSharp.Stats.Signal

let waveletParams : WaveletParameters =
    {
        Borderpadding          = None
        BorderPadMethod        = Padding.BorderPaddingMethod.Random
        InternalPaddingMethod  = Padding.InternalPaddingMethod.LinearInterpolation
        HugeGapPaddingMethod   = Padding.HugeGapPaddingMethod.Zero
        HugeGapPaddingDistance = 100.
        MinPeakDistance        = None
        MinPeakLength          = Some 0.1
        MaxPeakLength          = 1.5
        NoiseQuantile          = 0.01
        MinSNR                 = 0.01
    }

let quantificationParams : Dto.QuantificationParams =
    {
        PerformLabeledQuantification = Labeling.N15Labeling
        FragPipe                     = false
        XicExtraction =
            {
                ScanTimeWindow = 2.
                MzWindow_Da    = Window.Estimate
                XicProcessing  = XicProcessing.Wavelet waveletParams
                TopKPSMs       = None
            }
        BaseLineCorrection = Some { MaxIterations = 10; Lambda = 6; P = 0.05 }
    }

// Replace the temp folder with your project folder.
let outputPath = System.IO.Path.Combine(System.IO.Path.GetTempPath(), "QuantificationParams.json")

Json.serializeAndWrite outputPath quantificationParams

Running the tool

Install with dotnet tool install --global ProteomIQon.PSMBasedQuantification, then quantify one run:

proteomiqon-psmbasedquantification -i path/to/run.mzlite -ii path/to/run.qpsm -d path/to/database.db -o path/to/output -p path/to/QuantificationParams.json

Several runs, paired by position in the two lists, three in parallel:

proteomiqon-psmbasedquantification -i path/to/run1.mzlite path/to/run2.mzlite path/to/run3.mzlite -ii path/to/run1.qpsm path/to/run2.qpsm path/to/run3.qpsm -d path/to/database.db -o path/to/output -p path/to/QuantificationParams.json -c 3

Two directories, paired by file name:

proteomiqon-psmbasedquantification -i path/to/mzliteFolder -ii path/to/qpsmFolder -d path/to/database.db -o path/to/output -p path/to/QuantificationParams.json -c 3 -mf

All flags:

proteomiqon-psmbasedquantification --help