Mass spectrometry‐based approaches have enabled important breakthroughs in quantitative proteomics in the last decades. This development is reflected in the better quantitative assessment of protein levels as well as to understand post‐translational modifications and protein complexes and networks. Nowadays, the focus of quantitative proteomics shifted from the relative determination of proteins (ie, differential expression between two or more cellular states) to absolute quantity determination, required for a more‐thorough characterization of biological models and comprehension of the proteome dynamism, as well as for the search and validation of novel protein biomarkers. However, the physico‐chemical environment of the analyte species affects strongly the ionization efficiency in most mass spectrometry (MS) types, which thereby require the use of specially designed standardization approaches to provide absolute quantifications. Most common of such approaches nowadays include (i) the use of stable isotope‐labeled peptide standards, isotopologues to the target proteotypic peptides expected after tryptic digestion of the target protein; (ii) use of stable isotope‐labeled protein standards to compensate for sample preparation, sample loss, and proteolysis steps; (iii) isobaric reagents, which after fragmentation in the MS/MS analysis provide a final detectable mass shift, can be used to tag both analyte and standard samples; (iv) label‐free approaches in which the absolute quantitative data are not obtained through the use of any kind of labeling, but from computational normalization of the raw data and adequate standards; (v) elemental mass spectrometry‐based workflows able to provide directly absolute quantification of peptides/proteins that contain an ICP‐detectable element. A critical insight from the Analytical Chemistry perspective of the different standardization approaches and their combinations used so far for absolute quantitative MS‐based (molecular and elemental) proteomics is provided in this review.

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质谱绝对定量蛋白质组学的标准化方法

在过去的几十年中，基于质谱的方法在定量蛋白质组学上取得了重要突破。这种进步反映在对蛋白质水平的更好定量评估以及对翻译后修饰和蛋白质复合物与网络的了解上。如今，定量蛋白质组学的重点已从蛋白质的相对测定（即两个或多个细胞状态之间的差异表达）转移到绝对量测定，这对于生物学模型的更全面表征和蛋白质组动力学的理解也是必需的用于寻找和验证新型蛋白质生物标志物。但是，在大多数质谱（MS）类型中，分析物种类的物理化学环境会严重影响电离效率，因此，需要使用专门设计的标准化方法来提供绝对定量。如今，此类方法中最常见的方法包括（i）使用稳定的同位素标记的肽标准品，胰蛋白酶消化目标蛋白后预期的目标蛋白型肽的同位素同源物；（ii）使用稳定的同位素标记的蛋白质标准品来补偿样品制备，样品损失和蛋白水解步骤；（iii）等压试剂，其在MS / MS分析中裂解后可提供最终可检测的质量偏移，可用于标记分析物和标准样品；（iv）无标签方法，其中绝对定量数据不是通过使用任何种类的标签获得的，而是通过原始数据和适当标准品的计算归一化获得的；（v）基于元素质谱的工作流程能够直接对包含ICP可检测元素的肽/蛋白质进行绝对定量。这篇综述提供了从分析化学的角度对迄今为止用于绝对定量质谱（分子和元素）蛋白质组学的不同标准化方法及其组合的重要见解。