Selective cross‐linking of coinciding protein assemblies by in‐gel cross‐linking mass spectrometry

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. image Cross‐linking mass spectrometry experiments in‐solution are complicated by co‐occurring protein complexes of different stoichiometries, hampering confident identification of whether cross‐link originates e.g. from monomeric or dimeric species. Here, an alternative strategy combining blue‐native PAGE with in‐gel cross‐linking mass spectrometry (IGX‐MS) enables selective analysis of coinciding protein assemblies. IGX‐MS makes cross‐linker optimization and buffer exchange steps nearly obsolete. IGX‐MS is efficient and thus applicable to very low sample amounts. IGX‐MS enables the generation of assembly specific cross‐link restraints, also of highly complex samples such as mitochondrial respiratory (super‐)complexes. IGX‐MS comes with a substantial reduction of over‐length crosslinks compared to in‐solution XL‐MS. As the native structure is maintained in the gel, IGX‐MS provides distance restraints useful for structural modelling.