Silva Jeffrey C, Gorenstein Marc V, Li Guo-Zhong, Vissers Johannes P C, Geromanos Scott J
Waters Corporation, Milford, Massachusetts 01757-3696, USA.
Mol Cell Proteomics. 2006 Jan;5(1):144-56. doi: 10.1074/mcp.M500230-MCP200. Epub 2005 Oct 11.
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 R(2) 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.
相对定量方法在定量蛋白质组学领域占据主导地位。然而，有必要开展绝对定量研究，以准确模拟和理解导致生物样品间蛋白质组变异的复杂分子生物学过程。本文描述了一种蛋白质绝对定量的新方法。该方法基于质谱信号响应与蛋白质浓度之间意外关系的发现：每摩尔蛋白质中三条最强胰蛋白酶肽段的平均质谱信号响应在变异系数小于±10%的范围内保持恒定。给定一个内标，利用这种关系计算出通用信号响应因子。在本研究中，所有测试蛋白质的通用信号响应因子（计数/摩尔）均相同。在有无人血清的情况下，研究了一组浓度不同的六种外源蛋白质。测定标准蛋白质的绝对量，相对误差小于±15%。将每种蛋白质的三条最强肽段的平均质谱信号响应与其计算出的蛋白质浓度作图，得到线性关系，R²值为0.9939。该分析方法用于测定11种常见血清蛋白的绝对浓度，然后将这些浓度与文献中的已知值进行比较。此外，在未分级的大肠杆菌裂解物中，研究了一组已知以功能复合物形式存在的已鉴定蛋白质。计算得到的绝对量用于准确确定它们的化学计量比。
