Quantification

Label-Free quantification

• MetaMorpheus uses FlashLFQ to extract MS1 intensity and quantify peptides identified by MS2 identifications below a 0.01 Q-value.
• 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.
• Peptides are quantified by apex intensity, meaning that the most intense peak that meets the quantification criteria will be reported as the intensity.
• Because modified peptides can ionize at different efficiencies than unmodified peptides, and can thus affect the intensity of the detected ion, peptide intensity should only be compared to the same peptide with the same mods across runs. Make sure you're comparing apples to apples.

Tandem-Mass-Tag

TMT for HCD data is possible for up to 10-plex samples. Intensity is reported as a list of 10 intensities, one for each plex. The ion intensity is reported in order of each plex's diagnostic ion: 126.127725, 127.124760, 127.131079, 128.128114, 128.134433, 129.131468, 129.137787, 130.134822, 130.141141, 131.138176 with the "|" as the delimiter between each plex.

Proteins

• Protein quantification is based on unique+razor peptide intensity. If your favorite protein has no unique or razor peptides, you may need to manually sum peptide intensities.