Abstract
By reporting the molar abundance of proteins, absolute quantification determines their stoichiometry in complexes, pathways, or networks. Typically, absolute quantification relies either on protein-specific isotopically labeled peptide standards or on a semiempirical calibration against the average abundance of peptides chosen from arbitrarily selected proteins. In contrast, a generic protein standard FUGIS (fully unlabeled generic internal standard) requires no isotopic labeling, chemical synthesis, or external calibration and is applicable to quantifying proteins of any organismal origin. The median intensity of the peptide peaks produced by the tryptic digestion of FUGIS is used as a single-point calibrant to determine the molar abundance of any codigested protein. Powered by FUGIS, median-based absolute quantification (MBAQ) outperformed other methods of untargeted proteome-wide absolute quantification.
Highlights
Proteomics envelopes multiple workflows for relative and absolute quantification of individual proteins
We developed an untargeted proteome-wide quantification workflow termed median-based absolute quantification (MBAQ) that rely upon a fully unlabeled generic internal standard (FUGIS) based on the common physicochemical properties of proteotypic peptides
The MBAQ workflow relies on a recombinant protein standard consisting of concatenated peptides whose sequences emulate the physicochemical properties shared by typical proteotypic peptides
Summary
Proteomics envelopes multiple workflows for relative and absolute quantification of individual proteins. Peptides quantification relies either on isotopically labeled standards having exactly the same sequence or on a semiempirical calibration against the abundance of selected (or, alternatively, of all detectable) peptides originating from arbitrarily chosen standard proteins.[12,14] Targeted approaches are more accurate, yet they only cover a small selection of proteins that cannot be changed during the experiment. The latter methods work proteomewide; they rely on arbitrary assumptions, and their accuracy is biased by experimental conditions and the properties of individual proteins
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