Improving mass spectrometry analysis of protein structures with arginine-selective chemical cross-linkers

Abstract

Chemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS) is widely used to study protein-protein interactions (PPI), protein structures, and even protein dynamics. However, structural information provided by CXMS is still limited, partly because most CXMS experiments use lysine-lysine (K-K) cross-linkers. Although superb in selectivity and reactivity, they are ineffective for lysine deficient regions. Herein, we develop aromatic glyoxal cross-linkers (ArGOs) for arginine-arginine (R-R) cross-linking and the lysine-arginine (K-R) cross-linker KArGO. The R-R or K-R cross-links generated by ArGO or KArGO fit well with protein crystal structures and provide information not attainable by K-K cross-links. KArGO, in particular, is highly valuable for CXMS, with robust performance on a variety of samples including a kinase and two multi-protein complexes. In the case of the CNGP complex, KArGO cross-links covered as much of the PPI interface as R-R and K-K cross-links combined and improved the accuracy of Rosetta docking substantially.

Profiling the Proteome-Wide Selectivity of Diverse Electrophiles

“…A first-in-class method to monitor arginines in the whole proteome. Although arginine has been targeted with glyoxal-based reagents for bioconjugation 61 and crosslinking before, 62 no method is currently available to globally monitor arginines with residue-specific proteomics. We therefore synthesised PhGO-alkyne based on the known reactivity of phenylglyoxals with arginines that eventually produces a stable imidazole derivative (Fig.…”

Interfaces with Structure Dynamics of the Workhorses from Cells Revealed through Cross-Linking Mass Spectrometry (CLMS)

“…Therefore, the ‘n 2 ’ problem creates (n 2 + n)/2 possible cross-links for ‘n’ number of peptides [ 121 ]. There have been many other reports relying on ‘in-solution’ digestion of cross-linked peptides [ 122 , 123 , 124 , 125 , 126 ]. A vital alternative to ‘in-gel’ and ‘in-solution’ digestion protocols, especially when the cross-linked sample contains detergents and contaminants, is ‘Filter Aided Sample Preparation’ (also termed FASP) method.…”

From fuzziness to precision medicine: on the rapidly evolving proteomics with implications in mitochondrial connectivity to rare human disease

“…By examining distance restraints upon considering the most extended conformation of the cross-linker in comparison with various cross-linked residues, protein conformational states and complex topologies can be robustly resolved ( O'Reilly and Rappsilber, 2018 ). This area is also rapidly building upon using classical lysine-lysine into the development of aromatic glyoxal cross-linkers to covalently link arginine, thereby capturing interactions that are lysine deficient ( Jones et al., 2019 ). Also, cross-linking-integrated workflows that couple MS-cleavable cross-linkers and dual fragmentation strategy (i.e., sequential collision-induced dissociation and electron transfer dissociation) to identify cross-links against full proteome databases are being consistently developed and refined ( Liu et al., 2017 ).…”