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A more recent version of this article appeared on January 1, 2006.
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Submitted on July 26, 2005
Revised on September 23, 2005
Accepted on October 11, 2005

Absolute quantification of proteins by LCMSE: A virtue of parallel MS acquisition

Jeffrey C. Silva, Marc V. Gorenstein, Guo-Zhong Li, Johannes P. C. Vissers, and Scott J. Geromanos

Core Technology/Proteomics, Waters Corporation, Milford, MA 01757-3696

Corresponding Author: jeff_silva{at}waters.com

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 which 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 employed to calculate a universal signal response factor. The universal signal response factor (counts/mole) 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 response 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 R^2 value 0.9939. The analyses were extended to determine the absolute concentration of eleven common serum proteins and these concentrations were then compared to known values available in the literature. Additionally, within an unfractionated E. 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.


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