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

Abstract

<jats:p>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 observed by experiments on GroEL and purified bovine heart mitochondria, is the substantial reduction of artificial over-length cross-links when 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 be used to provide new insights into the initial stages of the terminal complement pathway.</jats:p>

Molecular characterization of a complex of Apoptosis Inducing Factor 1 (AIFM1) with cytochrome c oxidase of the mitochondrial respiratory chain

“…We analyzed the organization and interaction landscape of protein complexes in bovine heart mitochondria by combining cross-linking mass spectrometry XL-MS and complexome profiling (Heide et al, 2012;Hevler et al, 2021) thereby adding new information on native state multiprotein complexes of interest, building upon previous work exploring the interactome of mitochondria from different organisms, tissues and cells by XL-MS (Chavez et al, 2020;Linden et al, 2020;Liu et al, 2018;Ryl et al, 2020;Schweppe et al, 2017).…”

“…Mitochondrial membranes from bovine heart were isolated and preserved as described in (Hevler et al, 2021). In order to increase the purity of the preparation and for Tris-buffer removal, frozen crude mitochondria (4 x 15 ml aliquots; 60 mg protein/ml) were thawed on ice, diluted (1:4) with ice-cold SEH buffer (250 mM sucrose, 1 mM EDTA, 20 mM HEPES, pH 7.4 adjusted with NaOH) and centrifuged at 1,000 x g (10 min; 4°C).…”

“…In AIFM1 deficient patients (Ghezzi et al, 2010), ablation of AIFM1 in skeletal and heart muscle affected cytochrome c oxidase (COX) in addition to complex I, whereas in liver a deficiency of complexes I and V was observed (Joza et al, 2005;Pospisilik et al, 2007). In the present study, we explored the molecular interactions of AIFM1 with the multiprotein complexes of the OXPHOS system in heart mitochondria using our recently established complementary experimental approach (Hevler et al, 2021) that combines cross-linking mass-spectrometry (XL-MS) and complexome profiling (Figure 1). Figure 1.…”

Similarities and differences in the structures and proteoform profiles of the complement proteins C6 and C7

“…Unlike the other MAC components, the only known high-resolution structure of C7 is from the fully assembled MAC [9]. The much bettercharacterized C6, the closest homolog of C7, can adopt various conformations in its soluble state [5,7,9,13]. Based on the sequence and domain similarities between C6 and C7, it is tempting to speculate that the soluble C7 could also possess this conformational flexibility.…”

“…This study examines the structure and proteoform profile of C7 by an integrative structural MS-based approach combining native MS, glycopeptide-centric MS, in-gel cross-linking MS (IGX-MS) [13], and structural modelling. Furthermore, we present the proteome profile of C6 and extend on our earlier work, where we showed that C6 adapts a compact conformation in solution [13]. Comparisons to the C6 structure reveals, as expected, an overall high similarity in structure and domain organization.…”