PDF(Pubmed)
Abstract：
Mass spectrometry has become the most prominent yet evolving technology in quantitative proteomics. Today, a number of label-free and label-based approaches are available for the relative and absolute quantification of proteins and peptides. However, the label-based methods rely solely on the employment of stable isotopes, which are expensive and often limited in availability. Here we propose a label-based quantification strategy, where the mass difference is identified by the differential alkylation of cysteines using iodoacetamide and acrylamide. The alkylation reactions were performed under identical experimental conditions; therefore, the method can be easily integrated into standard proteomic workflows. Using high-resolution mass spectrometry, the feasibility of this approach was assessed with a set of tryptic peptides of human serum albumin. Several critical questions, such as the efficiency of labeling and the effect of the differential alkylation on the peptide retention and fragmentation, were addressed. The concentration of the quality control samples calculated against the calibration curves were within the ±20% acceptance range. It was also demonstrated that heavy labeled peptides exhibit a similar extraction recovery and matrix effect to light ones. Consequently, the approach presented here may be a viable and cost-effective alternative of stable isotope labeling strategies for the quantification of cysteine-containing proteins.
摘要：
质谱已成为定量蛋白质组学中最突出但不断发展的技术。今天,许多无标记和基于标记的方法可用于蛋白质和肽的相对和绝对定量。然而,基于标签的方法完全依赖于稳定同位素的使用，这是昂贵的，往往是有限的可用性。在这里，我们提出了一种基于标签的量化策略，其中质量差异通过使用碘乙酰胺和丙烯酰胺的半胱氨酸的差异烷基化来鉴定。烷基化反应在相同的实验条件下进行；因此，该方法可以很容易地整合到标准蛋白质组学工作流程中。使用高分辨率质谱，用一组人血清白蛋白的胰蛋白酶肽评估了这种方法的可行性。几个关键问题，如标记效率和差异烷基化对肽保留和片段化的影响，已解决。对照校准曲线计算的质量控制样品的浓度在±20%接受范围内。还证明了重标记肽表现出与轻标记肽相似的提取回收率和基质效应。因此,本文提出的方法可能是定量含半胱氨酸蛋白质的稳定同位素标记策略的可行且具有成本效益的替代方法.
