Absolute Quantification of Proteins by LCMSE

By Jeffrey C. Silva, M. Gorenstein, Guo‐Zhong Li, J. Vissers, Scott J. Geromanos. Molecular & Cellular Proteomics, 2006

948 citations recorded by the source corpus.

Relative quantification methods have dominated the quantitative proteomics field. There is a need, however, to conduct absolute quantification studies to accurately model and understand the complex molecular biology that results in proteome variability among biological samples. A new method of absolute quantification of proteins is described. This method is based on the discovery of an unexpected relationship between MS signal response and protein concentration: the average MS signal response for the three most intense tryptic peptides per mole of protein is constant within a coefficient of variation of less than ±10%. Given an internal standard, this relationship is used to calculate a universal signal response factor. The universal signal response factor (counts/mol) was shown to be the same for all proteins tested in this study. A controlled set of six exogenous proteins of varying concentrations was studied in the absence and presence of human serum. The absolute quantity of the standard proteins was determined with a relative error of less than ±15%. The average MS signal responses of the three most intense peptides from each protein were plotted against their calculated protein concentrations, and this plot resulted in a linear relationship with an R2 value of 0.9939. The analyses were applied to determine the absolute concentration of 11 common serum proteins, and these concentrations were then compared with known values available in the literature. Additionally within an unfractionated Escherichia coli lysate, a subset of identified proteins known to exist as functional complexes was studied. The calculated absolute quantities were used to accurately determine their stoichiometry.

Subfields: Molecular Biology, Spectroscopy

View Paper: https://www.semanticscholar.org/paper/75f46402603767ef06068749c8fdee35f894941a

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Citations in the corpus

Correlation of mRNA and protein in complex biological samples (2009, positive): “reported an error of ±15% in an absolute quantification study using the average MS signal intensities for the three most abundant peptides per protein as quantitative measure and subsequent normalization to isotopically labeled peptides as internal standards [35].”
Data‐independent acquisition‐based SWATH‐MS for quantitative proteomics: a tutorial (2018, positive): “For this purpose, various strategies have been developed, for example (i) summing or averaging the n-most intense peptides per protein (Silva et al , 2006; Ludwig et al , 2012), (ii) summing up all peptide intensities per sample, regardless of whether they occur across samples or not (iBAQ;…”