Median based absolute quantification of proteins using Fully Unlabelled Generic Internal Standard

By reporting molar abundances of proteins, absolute quantification determines their stoichiometry in complexes, pathways or networks. Typically, absolute quantification relies either on protein-specific isotopically labelled peptide standards or on a semi-empirical calibration against the average abundance of peptides chosen from arbitrary selected proteins. In contrast, a generic protein standard FUGIS (for Fully Unlabelled Generic Internal Standard) requires no isotopic labelling, chemical synthesis or external calibration and is applicable to quantifying proteins of any organismal origin. The median intensity of peptide peaks produced by the digestion of FUGIS is used as a single point calibrant to determine the molar abundance of any co-digested protein. Powered by FUGIS, median based absolute quantification (MBAQ) outperformed other available methods of untargeted proteome-wide absolute quantification.