PDF(Pubmed)
Abstract：
Proteomic absolute quantitation strategies mainly rely on the use of synthetic stable isotope-labeled peptides or proteins as internal standards, which are highly costly and time-consuming to synthesize. To circumvent this limitation, we recently developed a coulometric mass spectrometry (CMS) approach for absolute quantitation of proteins without the use of standards, based on the electrochemical oxidation of oxidizable surrogate peptides, followed by mass spectrometry measurement of the peptide oxidation yield. Previously, CMS was only applied for single-protein quantitation. In this study, first, we demonstrated absolute quantitation of multiple proteins in a mixture (e.g., β-lactoglobulin B, α-lactalbumin, and carbonic anhydrase) by CMS in one run, without using any standards. The CMS quantitation result was validated with a traditional isotope dilution method. Second, CMS can be used for absolute quantitation of a low-level target protein in a mixture; for instance, 500 ppm of PLBL2, a problematic host cell protein (HCP), in the presence of a highly abundant monoclonal antibody (mAb) was successfully quantified by CMS with no use of standards. Third, taking one step further, this study demonstrated the unprecedented quantitative analysis of deamidated peptide products arising from the mAb heavy chain deamidation reaction. In particular, absolute quantitation of the deamidation succinimide intermediate which had not been performed before due to the lack of standard was conducted by CMS, for the first time. Overall, our data suggest that CMS has potential utilities for quantitative proteomics and biotherapeutic drug discovery.
摘要：
蛋白质组学绝对定量策略主要依靠使用合成的稳定同位素标记的肽或蛋白质作为内标，合成成本高且耗时。为了规避这种限制，我们最近开发了一种库仑质谱（CMS）方法，用于在不使用标准品的情况下对蛋白质进行绝对定量，基于可氧化替代肽的电化学氧化，随后是肽氧化产率的质谱测量。以前,CMS仅应用于单蛋白定量。在这项研究中,首先,我们证明了混合物中多种蛋白质的绝对定量(例如，β-乳球蛋白B,α-乳清蛋白,和碳酸酐酶)通过CMS在一次运行中，不使用任何标准。采用传统的同位素稀释法对CMS定量结果进行了验证。第二,CMS可用于混合物中低水平靶蛋白的绝对定量；例如，500ppm的PLBL2，一种有问题的宿主细胞蛋白（HCP），在高度丰富的单克隆抗体（mAb）存在下，通过CMS成功定量，无需使用标准品。第三，更进一步，这项研究证明了对mAb重链脱酰胺化反应产生的脱酰胺化肽产物的前所未有的定量分析。特别是,绝对定量的脱酰胺琥珀酰亚胺中间体之前没有进行过，由于缺乏标准由CMS进行，第一次。总的来说,我们的数据表明,CMS在定量蛋白质组学和生物治疗药物发现方面具有潜在的应用价值.
