Absolute quantification of proteins by LCMSE -: A virtue of parallel MS acquisition
Absolute quantification of proteins by LCMSE -: A virtue of parallel MS acquisition
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DOI：
10.1074/mcp.m500230-mcp200
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发表时间：
2006-01-01
影响因子：
7
通讯作者：
Geromanos, SJ
中科院分区：
文献类型：
--
作者：
Silva, JC;Gorenstein, MV;Geromanos, SJ
相对定量方法主导了定量蛋白质组学领域。然而，需要进行绝对定量研究，以准确建模和理解导致生物样品之间蛋白质组变异的复杂分子生物学。描述了一种蛋白质绝对定量的新方法。该方法基于 MS 信号响应与蛋白质浓度之间意外关系的发现：每摩尔蛋白质中三种最强烈的胰蛋白酶肽的平均 MS 信号响应在小于 +/- 10% 的变异系数内恒定。给定内部标准，该关系用于计算通用信号响应因子。本研究中测试的所有蛋白质的通用信号响应因子（计数/摩尔）均相同。在不存在和存在人血清的情况下研究了一组不同浓度的六种外源蛋白的对照。标准蛋白的绝对量测定的相对误差小于+/-15%。将每种蛋白质中三种最强肽的平均 MS 信号响应与计算出的蛋白质浓度作图，该图得出 R-2 值为 0.9939 的线性关系。应用分析确定 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，还研究了已知作为功能复合物存在的已鉴定蛋白质的子集。计算出的绝对量用于准确确定其化学计量。
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.