通过质谱法测量β3整合素二硫键的氧化还原状态。
Measurement of redox states of the β3 integrin disulfide bonds by Mass Spectrometry.
作者信息
Chiu Joyce
机构信息
Centenary Institute, NHMRC Clinical Trials Centre, Sydney Medical School, the University of Sydney NSW 2006, Sydney, Australia.
出版信息
Bio Protoc. 2019 Feb 5;9(3):e3156. doi: 10.21769/BioProtoc.3156.
Functional disulfide bonds mediate a change in protein function in which they reside when cleaved or formed. To elucidate how a functional disulfide bond controls protein activity, it is critical that the redox state of the bond in the population of protein molecules is known. Measurement of changes in disulfide bond redox state relies on thiol probes and immunoblotting. Such technique only offers a qualitative indication of a change in redox state but not the identity of cysteines involved. A differential cysteine alkylation and mass spectrometry technique is described here that affords precise quantification of protein disulfide bond redox state. The utility of the technique is demonstrated by quantifying the redox state of 24 of the 28 disulfide bonds in human β3 integrin from purified platelets.
功能性二硫键介导其所存在的蛋白质功能的变化，当这些二硫键断裂或形成时。为了阐明功能性二硫键如何控制蛋白质活性，了解蛋白质分子群体中二硫键的氧化还原状态至关重要。二硫键氧化还原状态变化的测量依赖于硫醇探针和免疫印迹。这种技术仅能定性地指示氧化还原状态的变化，而不能确定所涉及的半胱氨酸的身份。本文描述了一种差异半胱氨酸烷基化和质谱技术，该技术能够精确量化蛋白质二硫键的氧化还原状态。通过对纯化血小板中人类β3整合素28个二硫键中的24个的氧化还原状态进行量化，证明了该技术的实用性。
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