LabeledProteinQuantification

In a 15N metabolic labeling experiment one sample grows on 15N, is mixed with the 14N sample, and both versions of every peptide are measured in the same run, so the ratio of the light and the heavy peak is a direct comparison of the two samples. How the label enters the search is described in BioFSharp.Mz, and how the ratio is formed from the two peaks in the quantification chapter. LabeledProteinQuantification takes the .quantAndProt rows from JoinQuantPepIonsWithProteins and aggregates them to one light intensity, one heavy intensity and one ratio per protein group and run. It works in up to four steps. The first step is mandatory: every row already holds a light and a heavy intensity, the step divides them into Ratio_LightByHeavy and merges the rows identified from the light and from the heavy form of the same peptide ion. The second step merges charge states and the third merges modified forms of a peptide, both optional. The fourth merges all peptides of a protein group. Every step transforms, filters and aggregates light, heavy and ratio separately.

Inputs and outputs

Logs go to LabeledProteinQuantification_log.txt and LabeledQuantification_log.txt in the output directory.

The tool writes up to five tab separated tables into the output directory. GlobModAggregation.txt is the result of the first step, one row per peptide ion and run with Quant_Light, Quant_Heavy and Ratio_LightByHeavy. ChargeAggregation.txt appears only when the charge step runs and ModificationAggregation.txt only when the modification step runs. ProteinAggregation.txt has one row per protein group and run with the aggregated light, heavy and ratio values, and for each the number of values used, CV, standard deviation and SEM. LabeledQuant.txt is the same table pivoted, one row per protein group and one block of columns per input file, named <run>.Ratio_LightByHeavy, <run>.Quant_Heavy and so on. RatioLFQ reads this file to turn the ratios into comparable intensities per run.

Parameters

An AggregationParams record describes one step. Each of its fields has a Light, a Heavy and a Ratio entry, so the three value series can be treated differently.

Five default files exist. LabeledQuantificationParams.json is the table above. LabeledQuantificationParams_withCorrFilter.json sets Correlation_Light_Heavy_Threshold to Some 0.9. LabeledQuantificationParams_CorrFilter_ChargeAgg.json sets the threshold to Some 0.0 and adds the charge step with Mean. LabeledQuantificationParams_CorrFilter_ChargeAgg_ModAgg.json sets the threshold to Some 0.0 and adds the charge and the modification step, both with Mean. LabeledQuantificationParams_Transform_Filter_Sum.json log2 transforms light, heavy and ratio in the first step, keeps only log2 ratios below 0.0 there, and sums light and heavy in the protein step while the ratio is still averaged.

Writing a parameter file

open System.IO
open ProteomIQon

// Used for the first step and the protein step of the default file: no transform, no filters, mean of light, heavy and ratio.
let meanOfAll : Common.LabeledProteinQuantification.AggregationParams =
    {
        LabeledTransform     = None
        LabeledSingleFilters = None
        LabeledGroupFilters  = None
        LabeledAggregation   =
            {
                Light = NumericAggregation.Mean
                Heavy = NumericAggregation.Mean
                Ratio = NumericAggregation.Mean
            }
    }

let labeledQuantificationParams : Dto.LabeledQuantificationParams =
    {
        Correlation_Light_Heavy_Threshold  = None
        Alignment_QValue                   = None
        ModificationFilter                 = UseModifiedPeptides.All
        AggregateGlobalModificationsParams = meanOfAll
        AggregatePeptideChargeStatesParams = None
        AggregateModifiedPeptidesParams    = None
        AggregateToProteinGroupsParams     = meanOfAll
    }

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

Json.serializeAndWrite outputPath labeledQuantificationParams

Running the tool

The tool installs with dotnet tool install --global ProteomIQon.LabeledProteinQuantification. A single run:

proteomiqon-labeledproteinquantification -i path/to/run.quantAndProt -o path/to/output -p path/to/LabeledQuantificationParams.json

Several runs in one table, from a list or from a directory:

proteomiqon-labeledproteinquantification -i path/to/run1.quantAndProt path/to/run2.quantAndProt -o path/to/output -p path/to/LabeledQuantificationParams.json
proteomiqon-labeledproteinquantification -i path/to/quantAndProt -o path/to/output -p path/to/LabeledQuantificationParams.json

All flags:

proteomiqon-labeledproteinquantification --help