NHS-Ester Tandem Labeling in One Pot Enables 48-Plex Quantitative Proteomics,Analytical Chemistry

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NHS-Ester Tandem Labeling in One Pot Enables 48-Plex Quantitative Proteomics
Analytical Chemistry ( IF 6.7 ) Pub Date : 2021-09-16 , DOI: 10.1021/acs.analchem.1c01314
Sansi Xing ₁ , Akshat Pai ₁ , Ruilin Wu ₁ , Yu Lu ₁

Affiliation

Stable-isotope labeling strategies are extensively used for multiplex quantitative proteomics. Hybrid-isotope labeling strategies that combine the use of isotopic mass difference labeling and isobaric tags can greatly increase sample multiplexity. In this work, we present a novel hybrid-isotope labeling approach that we termed NHS-ester tandem labeling in one pot (NETLOP). We first optimized 16-plex isobaric TMTpro labeling of lysine residues followed by 2-plex or 3-plex isotopic mTRAQ labeling of peptide N-termini, both of which with commercially available NHS-ester reactive reagents. We then demonstrated the utility of the NETLOP approach by labeling HeLa cell samples and performing proof-of-principle quantitative 32-plex and 48-plex proteomic analyses, each in a single LC-MS/MS experiment. Compared to current hybrid-isotope labeling methods, our NETLOP approach requires no sample cleanup between different labeling steps to minimize sample loss, induces no retention time shifts that compromise quantification accuracy, can be adapted to other NHS-ester isotopic labeling reagents to further increase multiplexity, and is compatible with samples from any origin in a wide array of biological and clinical proteomics applications.

中文翻译：

NHS-酯串联标记在一锅中实现 48-Plex 定量蛋白质组学

稳定同位素标记策略广泛用于多重定量蛋白质组学。结合使用同位素质量差异标记和同量异位标记的混合同位素标记策略可以大大提高样品的多重性。在这项工作中，我们提出了一种新颖的混合同位素标记方法，我们将其称为 NHS-酯串联标记一锅法 (NETLOP)。我们首先优化了赖氨酸残基的 16 重同量异位 TMTpro 标记，然后是肽 N 末端的 2 重或 3 重同位素 mTRAQ 标记，两者均使用市售 NHS 酯反应试剂。然后，我们通过标记 HeLa 细胞样本并进行原理验证定量 32 重和 48 重蛋白质组学分析来证明 NETLOP 方法的实用性，每个分析都在单个 LC-MS/MS 实验中。与目前的混合同位素标记方法相比，

更新日期：2021-09-28

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