Selective cross-linking of coinciding protein assemblies by in-gel cross-linking mass spectrometry.
EMBO J
; 40(4): e106174, 2021 02 15.
Article
em En
| MEDLINE
| ID: mdl-33459420
ABSTRACT
Cross-linking mass spectrometry has developed into an important method to study protein structures and interactions. The in-solution cross-linking workflows involve time and sample consuming steps and do not provide sensible solutions for differentiating cross-links obtained from co-occurring protein oligomers, complexes, or conformers. Here we developed a cross-linking workflow combining blue native PAGE with in-gel cross-linking mass spectrometry (IGX-MS). This workflow circumvents steps, such as buffer exchange and cross-linker concentration optimization. Additionally, IGX-MS enables the parallel analysis of co-occurring protein complexes using only small amounts of sample. Another benefit of IGX-MS, demonstrated by experiments on GroEL and purified bovine heart mitochondria, is the substantial reduction of undesired over-length cross-links compared to in-solution cross-linking. We next used IGX-MS to investigate the complement components C5, C6, and their hetero-dimeric C5b6 complex. The obtained cross-links were used to generate a refined structural model of the complement component C6, resembling C6 in its inactivated state. This finding shows that IGX-MS can provide new insights into the initial stages of the terminal complement pathway.
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MEDLINE
Assunto principal:
Espectrometria de Massas
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Proteínas do Sistema Complemento
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Complemento C5
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Complemento C6
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Reagentes de Ligações Cruzadas
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Mitocôndrias Cardíacas
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article