October 11, 2005 | Jeffrey C. Silva, Marc V. Gorenstein, Guo-Zhong Li, Johannes P. C. Vissers, and Scott J. Geromanos
The article describes a novel method for absolute quantification of proteins using LCMS (liquid chromatography-tandem mass spectrometry). The method leverages an unexpected relationship between MS signal response and protein concentration, where 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%. This relationship is used to calculate a universal signal response factor, which can be applied to any protein in a mixture to determine its absolute concentration. The method was validated using a controlled set of six exogenous proteins of varying concentrations in the absence and presence of human serum, achieving an absolute error of less than ±15%. The technique was also applied to determine the absolute concentrations of 11 common serum proteins and a subset of identified proteins in an unfractionated Escherichia coli lysate, demonstrating its utility in understanding protein stoichiometry and complex biological networks. The method provides a valuable tool for studying protein dynamics and interactions in various biological systems.The article describes a novel method for absolute quantification of proteins using LCMS (liquid chromatography-tandem mass spectrometry). The method leverages an unexpected relationship between MS signal response and protein concentration, where 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%. This relationship is used to calculate a universal signal response factor, which can be applied to any protein in a mixture to determine its absolute concentration. The method was validated using a controlled set of six exogenous proteins of varying concentrations in the absence and presence of human serum, achieving an absolute error of less than ±15%. The technique was also applied to determine the absolute concentrations of 11 common serum proteins and a subset of identified proteins in an unfractionated Escherichia coli lysate, demonstrating its utility in understanding protein stoichiometry and complex biological networks. The method provides a valuable tool for studying protein dynamics and interactions in various biological systems.