Chemical cross-linking with a diazirine photoactivatable cross-linker investigated by MALDI- and ESI-MS/MS.
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.
Gomes
Alexandre F
AF
Institute of Chemistry, University of Campinas-UNICAMP and Instituto Nacional de Ciencia e Tecnologia de Bioanalitica, CP 6154, 13083-970, Campinas, Sao Paulo, Brazil.
Gozzo
Fabio C
FC
D000818:Animals; D002853:Chromatography, Liquid; D003432:Cross-Linking Reagents / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D003978:Diazomethane / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D006736:Horses; D013058:Mass Spectrometry / Q000379:methods*; D009211:Myoglobin / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D010777:Photochemistry
D003432:Cross-Linking Reagents; D004220:Disulfides; D009211:Myoglobin; D010455:Peptides; D003978:Diazomethane; D003545:Cysteine