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

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.

Automated summary

Cross-linking mass spectrometry has become an important method for studying protein structures and interactions, with the combination of blue native PAGE and in-gel cross-linking mass spectrometry (IGX-MS) offering a more efficient workflow that avoids certain time-consuming steps. IGX-MS not only reduces the unwanted over-length cross-links compared to traditional in-solution cross-linking, but also allows for the parallel analysis of co-occurring protein complexes using minimal sample amounts. Additionally, IGX-MS has been shown to provide new insights into the initial stages of the terminal complement pathway through experiments on complement components C5, C6, and C5b6 complex.