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在线电化学还原二硫键:提高了促肾上腺皮质激素和铁调素的 FTICR-CID 和 -ETD 覆盖度。

On-line electrochemical reduction of disulfide bonds: improved FTICR-CID and -ETD coverage of oxytocin and hepcidin.

机构信息

Leiden University Medical Center (LUMC), Center for Proteomics and Metabolomics, 2300 RC, Leiden, The Netherlands.

出版信息

J Am Soc Mass Spectrom. 2013 Dec;24(12):1980-7. doi: 10.1007/s13361-013-0725-7. Epub 2013 Sep 10.

Abstract

Particularly in the field of middle- and top-down peptide and protein analysis, disulfide bridges can severely hinder fragmentation and thus impede sequence analysis (coverage). Here we present an on-line/electrochemistry/ESI-FTICR-MS approach, which was applied to the analysis of the primary structure of oxytocin, containing one disulfide bridge, and of hepcidin, containing four disulfide bridges. The presented workflow provided up to 80% (on-line) conversion of disulfide bonds in both peptides. With minimal sample preparation, such reduction resulted in a higher number of peptide backbone cleavages upon CID or ETD fragmentation, and thus yielded improved sequence coverage. The cycle times, including electrode recovery, were rapid and, therefore, might very well be coupled with liquid chromatography for protein or peptide separation, which has great potential for high-throughput analysis.

摘要

特别是在中、自上而下的肽和蛋白质分析领域,二硫键会严重阻碍片段化,从而阻碍序列分析(覆盖率)。在这里,我们提出了一种在线/电化学/ESI-FTICR-MS 方法,该方法应用于含有一个二硫键的催产素和含有四个二硫键的铁调素的一级结构分析。所提出的工作流程提供了两个肽中二硫键高达 80%(在线)的转化率。通过最小化样品制备,这种还原导致 CID 或 ETD 片段化时产生更多的肽骨架断裂,从而提高了序列覆盖率。包括电极恢复在内的循环时间非常快,因此很可能与用于蛋白质或肽分离的液相色谱法耦合,这对高通量分析具有巨大的潜力。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d30d/3837188/db56e37d05fb/13361_2013_725_Fig1_HTML.jpg

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