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Quantification
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MetaMorpheus uses FlashLFQ to extract MS1 intensity and quantify peptides identified by MS2 identifications below a 0.01 Q-value. Instructions for performing peptide and protein quantification can be found by following this link.
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Quantification occurs after identification. All MS1 peaks around the identifying MS2 spectrum within the retention time window are checked for an expected isotopic distribution. FlashLFQ's peakfinding algorithm is used to find MS1 peaks that meet the expected isotopic distribution.
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Peptides are quantified by apex intensity, meaning that the most intense isotopic envelope that meets the quantification criteria will be reported as the intensity (rather than integrated peak intensity).
- Protein label-free quantification is also handled by FlashLFQ. See the FlashLFQ page for a description of how protein quantification is handled.
FlashLFQ outputs several text files, described here. The .tsv files are convenient to view with Microsoft Excel.
AllQuantifiedPeaks.tsv - Each chromatographic peak is shown here, even peaks that were not quantifiable (peak intensity = 0). Details about each peak, such as number of PSMs mapped, start/apex/end retention times, ppm error, etc are contained in this file. A peptide can have multiple peaks over the course of a run (e.g., oxidized peptidoforms elute at different times, etc). Ambiguous peaks are displayed with a | (pipe) delimiter to indicate more than one peptide mapped to that peak.
AllQuantifiedPeptides.tsv - Peptide intensities are summed by full sequence; this makes it convenient to compare modified peptidoform intensities across runs. For each identified peptide, FlashLFQ finds the apex intensities for all relevant charge states within a narrow retention time window around the identification. FlashLFQ finds the highest apex intensity and then sums all the isotopic forms for that charge state. This single value is reported in the QuantifiedPeptides.tsv file. Note, this intensity is not a peak area or integration across multiple charge states and the full retention time. It is a charge state at a single time.
AllQuantifiedProteins.tsv - Lists protein accession and in the future will include gene and organism if the TSV contains it. The intensity is either a) the sum of the 3 most intense peptides or b) (Advanced protein quant) a weighted-average of the intensities of the peptides assigned to the protein. The weights are determined by how well the peptide co-varies with the other peptides assigned to that protein. See Diffacto.