期刊
JOURNAL OF MASS SPECTROMETRY
卷 45, 期 8, 页码 892-899出版社
WILEY
DOI: 10.1002/jms.1776
关键词
cross-linking; mass spectrometry, diazirine; fragmentation; photoactivatable
资金
- Sao Paulo Proteomics Network [FAPESP 2004/14846-0, FINEP 01 07 0290.00]
- Instituto Nacional de Ciencia e Tecnologia de Bioanalitica [FAPESP 08/57805-2, CNPq 573672/2008-3]
- CNPq
Crystallography and nuclear magnetic resonance are well-established methods to study protein tertiary structure and interactions. Despite their usefulness, such methods are not applicable to many protein systems. Chemical cross-linking of proteins coupled with mass spectrometry allows low-resolution characterization of proteins and protein complexes based on measuring distance constraints from cross-links. In this work, we have investigated cross-linking by means of a heterobifunctional cross-linker containing a traditional N-hydroxysuccinimide (NHS) ester and a UV photoactivatable diazirine group. Activation of the diazirine group yields a highly reactive carbene species, with potential to increase the number of cross-links compared with homobifunctional, NHS-based cross-linkers. Cross-linking reactions were performed on model systems such as synthetic peptides and equine myoglobin. After reduction of the disulfide bond, the formation of intra- and intermolecular cross-links was identified and the peptides modified with both NHS and diazirine moieties characterized. Fragmentation of these modified peptides reveals the presence of a marker ion for intramolecular cross-links, which facilitates identification. Copyright (c) 2010 John Wiley & Sons, Ltd.
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