Standard-Free Absolute Quantitation of Antibody Deamidation Degradation and Host Cell Proteins by Coulometric Mass Spectrometry.
Standard-Free Absolute Quantitation of Antibody Deamidation Degradation and Host Cell Proteins by Coulometric Mass Spectrometry.
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DOI：
10.1021/acs.analchem.2c02709
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发表时间：
2022-09-13
影响因子：
7.4
通讯作者：
Chen, Hao
中科院分区：
文献类型：
--
作者：
Ai, Yongling;Gunawardena, Harsha P.;Li, Xuanwen;Kim, Yong-Ick;Dewald, Howard D.;Chen, Hao
蛋白质组的绝对定量策略主要依赖于使用合成的稳定的同位素标记的多肽或蛋白质作为内标，这些合成成本高且耗时长。为了克服这一限制，我们最近开发了一种库仑质谱仪(CMS)方法，用于在不使用标准的情况下对蛋白质进行绝对定量，该方法基于可氧化的替代多肽的电化学氧化，然后用质谱仪测量多肽的氧化产率。在此之前，CMS仅用于单一蛋白质的定量。在这项研究中，首先，我们证明了在不使用任何标准的情况下，用CMS在一次运行中对混合物中的多种蛋白质(例如β-乳球蛋白B、α-乳蛋白和碳酸氢酶)进行绝对定量。用传统的同位素稀释法对CMS定量结果进行了验证。第二，CMS可以用于混合物中低水平目标蛋白的绝对定量；例如，在高度显性的单抗(MAb)存在的情况下，CMS成功地定量了500ppm的有问题的宿主细胞蛋白(HCP)PLBL2，而不使用标准。第三，本研究进一步对单抗重链脱酰胺反应产生的脱酰胺多肽产品进行了前所未有的定量分析。特别是对过去因缺乏标准而未被测定的脱酰胺琥珀酰亚胺中间体，首次用CMS进行了定量。总体而言，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独特的潜在实用价值。
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 spectrometric (CMS) approach for absolute quantitation of proteins without the use of standards, based on the electrochemical oxidation of oxidizable surrogate peptides followed by mass spectrometric 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 traditional isotope dilution method. Second, CMS can be used for absolute quantitation of low-level target protein in mixture; for instance, 500 ppm of PLBL2, a problematic host cell protein (HCP), in the presence of highly dominant monoclonal antibody (mAb) was successfully quantified by CMS with no use of standards. Third, taking one step further, this study conducted the unprecedented quantitative analysis of deamidated peptide products arising from mAb heavy chain deamidation reaction. In particular, the deamidation succinimide intermediate which had not been measured before due to lack of standard was quantified by CMS, for the first time. Overall, our data suggest that CMS has unique potential utilities for quantitative proteomics and for biotherapeutic drug discovery.
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影响因子：
3.8
作者：
Cao, Mingyan;Xu, Weichen;Wang, Jihong
通讯作者：
Wang, Jihong
影响因子：
3.8
作者：
Gupta, Surbhi;Jiskoot, Wim;Rathore, Anurag S.
通讯作者：
Rathore, Anurag S.
影响因子：
4.4
作者：
Hains, Peter G.;Truscott, Roger J. W.
通讯作者：
Truscott, Roger J. W.
影响因子：
2.3
作者：
Liigand, Piia;Kaupmees, Karl;Kruve, Anneli
通讯作者：
Kruve, Anneli
DOI：
10.1021/jasms.1c00121
发表时间：
2021-07-07
影响因子：
3.2
作者：
Ai Y;Zhao P;Fnu PIJ;Chen H
通讯作者：
Chen H