Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation.
Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation.
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DOI：
10.3791/62671
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发表时间：
2021-06-21
期刊：
影响因子：
--
通讯作者：
Qian WJ
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文献类型：
--
作者：
Gaffrey MJ;Day NJ;Li X;Qian WJ
蛋白质巯基的可逆氧化修饰最近成为细胞功能的重要介质。在这里，我们描述了一个定量的氧化还原蛋白质组学方法，利用树脂辅助捕获（RAC）结合串联质量标签（TMT）的同量异位素标记和液相色谱-串联质谱（LC-MS/MS）的详细程序，允许在蛋白质组水平的氧化蛋白硫醇的多路化学计量定量。氧化半胱氨酸残基的位点特异性定量信息为此类修饰的功能影响提供了额外的见解。该工作流程可适用于许多样品类型，包括培养的细胞（例如，哺乳动物、原核生物）和整个组织（例如，心脏、肺、肌肉），其最初被裂解/均质化，并且游离硫醇被烷基化以防止人工氧化。氧化的蛋白质硫醇然后被硫醇亲和树脂还原和捕获，这通过允许进行消化、标记和洗涤程序而无需额外转移蛋白质/肽来简化和简化工作流程步骤。最后，洗脱标记的肽并通过LC-MS/MS分析，以揭示与整个蛋白质组中的硫醇氧化相关的全面化学计量变化。该方法大大提高了对与蛋白质巯基氧化相关的生理和病理生理状态下的氧化还原依赖性调节的作用的理解。
Reversible oxidative modifications on protein thiols have recently emerged as important mediators of cellular function. Herein we describe the detailed procedure of a quantitative redox proteomics method that utilizes resin-assisted capture (RAC) in combination with tandem mass tag (TMT) isobaric labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to allow multiplexed stochiometric quantification of oxidized protein thiols at the proteome level. The site-specific quantitative information on oxidized cysteine residues provides additional insight into the functional impacts of such modifications. The workflow is adaptable across many sample types, including cultured cells (e.g., mammalian, prokaryotic) and whole tissues (e.g., heart, lung, muscle), which are initially lysed/homogenized and with free thiols being alkylated to prevent artificial oxidation. The oxidized protein thiols are then reduced and captured by a thiol-affinity resin, which streamlines and simplifies the workflow steps by allowing the proceeding digestion, labeling, and washing procedures to be performed without additional transfer of proteins/peptides. Finally, the labeled peptides are eluted and analyzed by LC-MS/MS to reveal comprehensive stoichiometric changes related to thiol oxidation across the entire proteome. This method greatly improves the understanding of the role of redox-dependent regulation under physiological and pathophysiological states related to protein thiol oxidation.
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DOI：
10.1042/bcj20180556
发表时间：
2018-10-31
期刊：
The Biochemical journal
影响因子：
--
作者：
Mitchell AR;Yuan M;Morgan HP;McNae IW;Blackburn EA;Le Bihan T;Homem RA;Yu M;Loake GJ;Michels PA;Wear MA;Walkinshaw MD
通讯作者：
Walkinshaw MD
影响因子：
5.5
作者：
Pace NJ;Weerapana E
通讯作者：
Weerapana E
影响因子：
14.8
作者：
通讯作者：
--
影响因子：
7
作者：
Guo, Jia;Nguyen, Amelia Y.;Qian, Wei-Jun
通讯作者：
Qian, Wei-Jun
影响因子：
17.1
作者：
Duan J;Kodali VK;Gaffrey MJ;Guo J;Chu RK;Camp DG;Smith RD;Thrall BD;Qian WJ
通讯作者：
Qian WJ