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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).
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To normalize intensity data, you must specify an Experimental Design.
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.
AllProteins.tsv - Lists protein accessions, peptides, and protein intensities. The median polish algorithm (example) is used on the peptide log2-transformed intensities, where the biological replicates are the columns and the peptides that are digestion products of the protein being quantified are the rows. After the median polish algorithm is complete, the column effects correspond to a relative protein quantity in the various biological replicates. These sample quantities are then un-logged and multiplied by a constant value, which is the top 3 peptide intensities in the first condition where there is at least 1 valid intensity value. The C# code is here. The output of protein intensities depends on the Experimental Design configuration. Please review the Experimental Design page so that you get the output that you desire.
Stable isotope labels can be searched and quantified using FlashLFQ in MetaMorpheus. In the "Search Task" window, under the "Quantification" header, select "SILAC/SILAM". If there are no unlabeled peptides in your sample(s), uncheck "Quantify unlabeled peptides/proteins". To specify your label(s), click "Add Isotope Label", which will bring up a new window. This window allows you to specify which amino acid is labeled, as well as which atoms on said amino acid are isotopically labeled. Start by typing in the single character abbreviation for your amino acid (Example: 'K' for lysine). This will automatically populate the other fields. After specifying the amino acid, specify how many of each atom is isotopically labeled. The fields will automatically update to accommodate your edits. If one of your conditions uses multiple labels (i.e. both K and R are labeled), input the information for a single label and then select "Add Additional Labels to This Condition". This will bring up a new window that allows for additional labels to be added to that condition. To close all windows, click "Save Label(s)".
Multiplexed SILAC in MetaMorpheus assumes 100% incorporation of the labeled amino acids. For turnover experiments, where an organism is fed one label and then switched to another, proteins frequently contain "recycled" amino acids, such as both a heavy and a light label on the same molecule at two different sites. These partially labeled peptides are useful in the analysis to determine the rate of recycling within the sample. MetaMorpheus automatically consolidates this data and outputs it as "Original" and "NewlySynthesized" protein. Turnover experiments can be used by specifying the start and end conditions, where the start is the food the organism was originally on and the end is the food the organism was on during sample harvest.