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Anal Chem. 2014 Sep 16;86(18):8983-91. doi: 10.1021/ac501526n. Epub 2014 Sep 4.

Cross-linking electrochemical mass spectrometry for probing protein three-dimensional structures.

Author information

1
Center for Intelligent Chemical Instrumentation, Department of Chemistry and Biochemistry and §Edison Biotechnology Institute, Ohio University , Athens, Ohio 45701, United States.

Abstract

Chemical cross-linking combined with mass spectrometry (MS) is powerful to provide protein three-dimensional structure information but difficulties in identifying cross-linked peptides and elucidating their structures limit its usefulness. To tackle these challenges, this study presents a novel cross-linking MS in conjunction with electrochemistry using disulfide-bond-containing dithiobis[succinimidyl propionate] (DSP) as the cross-linker. In our approach, electrolysis of DSP-bridged protein/peptide products, as online monitored by desorption electrospray ionization mass spectrometry is highly informative. First, as disulfide bonds are electrochemically reducible, the cross-links are subject to pronounced intensity decrease upon electrolytic reduction, suggesting a new way to identify cross-links. Also, mass shift before and after electrolysis suggests the linkage pattern of cross-links. Electrochemical reduction removes disulfide bond constraints, possibly increasing sequence coverage for tandem MS analysis and yielding linear peptides whose structures are more easily determined than their cross-linked precursor peptides. Furthermore, this cross-linking electrochemical MS method is rapid, due to the fast nature of electrochemical conversion (much faster than traditional chemical reduction) and no need for chromatographic separation, which would be of high value for structural proteomics research.

PMID:
25141260
PMCID:
PMC4165463
DOI:
10.1021/ac501526n
[Indexed for MEDLINE]
Free PMC Article
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