Brönstrup Mark
Sanofi-Aventis, Building G878, D-65926 Frankfurt, Germany.
Expert Rev Proteomics. 2004 Dec;1(4):503-12. doi: 10.1586/14789450.1.4.503.
The strong need for quantitative information in proteomics has fueled the development of mass spectrometry-based analytical methods that are able to determine protein abundances. This article reviews mass spectrometry experiments aimed at providing an absolute quantification of proteins. The experiments make use of the isotope-dilution concept by spiking a known amount of synthetic, isotope-labeled reference peptide into the analyte sample. Quantification is achieved by comparing the mass spectrometry signal intensities of the reference with an endogenous peptide that is generated upon proteolytic cleavage of the target protein. In an analogous manner, the level of post-translational modification at a distinct residue within a target protein can be determined. Among the strengths of absolute quantification are low detection limits reaching subfemtomole levels, a high dynamic range spanning approximately five orders of magnitude, low requirements for sample clean-up, and a fast and straightforward method development. Recent studies have demonstrated the compatibility of absolute quantification with various mass spectrometry readout techniques and sample purification steps such as 1D gel electrophoresis, size-exclusion chromatography, isoelectric peptide focusing, strong cation exchange and reversed phase or affinity chromatography. Under ideal conditions, quantification errors and coefficients of variation below 5% have been reported. However, the fact that at the start of the experiment the analyte is a protein and the internal standard is a peptide, severe quantification errors may result due to the selection of unsuitable reference peptides and/or imperfect protein proteolysis. Within the ensemble of mass spectrometry-based quantification methods, absolute quantification is the method of choice in cases where absolute numbers, many repetitive experiments or precise levels of post-translational modifications are required for a few, preselected species of interest. Consequently, prominent application areas include biomarker quantification, the study of post-translational modifications such as phosphorylation or ubiquitination and the comparison of concentrations of interacting proteins.
蛋白质组学中对定量信息的强烈需求推动了基于质谱的分析方法的发展，这些方法能够测定蛋白质丰度。本文综述了旨在对蛋白质进行绝对定量的质谱实验。这些实验利用同位素稀释概念，将已知量的合成同位素标记参考肽加入分析物样品中。通过比较参考肽与目标蛋白经蛋白酶裂解产生的内源性肽的质谱信号强度来实现定量。以类似的方式，可以确定目标蛋白内特定残基的翻译后修饰水平。绝对定量的优点包括低检测限可达亚飞摩尔水平、高动态范围跨越约五个数量级、对样品净化要求低以及方法开发快速直接。最近的研究表明绝对定量与各种质谱读出技术和样品纯化步骤兼容，如一维凝胶电泳、尺寸排阻色谱、等电肽聚焦、强阳离子交换以及反相或亲和色谱。在理想条件下，已报道定量误差和变异系数低于5%。然而，由于实验开始时分析物是蛋白质而内标是肽，选择不合适的参考肽和/或蛋白质不完全水解可能导致严重的定量误差。在基于质谱的定量方法中，对于少数预先选定的感兴趣物种，如果需要绝对数量、多次重复实验或精确的翻译后修饰水平，绝对定量是首选方法。因此，突出的应用领域包括生物标志物定量、磷酸化或泛素化等翻译后修饰的研究以及相互作用蛋白质浓度的比较。
