Median-Based Absolute Quantification of Proteins Using Fully Unlabeled Generic Internal Standard (FUGIS)

• Published in: Journal of proteome research Vol. 21; no. 1; pp. 132 - 141
• Main Authors: Raghuraman, Bharath Kumar, Bogdanova, Aliona, Moon, HongKee, Rzagalinski, Ignacy, Geertsma, Eric R, Hersemann, Lena, Shevchenko, Andrej
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 07.01.2022
• Subjects: Calibration; Isotope Labeling - methods; Peptides - chemistry; Proteome; Reference Standards; absolute quantification of proteins; MS Western workflow; proteome-wide quantification
• ISSN: 1535-3893; 1535-3907
• DOI: 10.1021/acs.jproteome.1c00596

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.