In situ membrane protein and flagellar enrichment increase the depth of large-scale bacterial interactomics studies using ion mobility and cross-linking mass spectrometry (XL-MS)

Cross-linking mass spectrometry (XL-MS) is an invaluable tool for capturing protein-wide interactomes in an in vivo context, however it remains challenging for complex protein mixtures, particularly those including membrane-embedded and/or membrane-associated proteins. The Gram negative bacterial membrane consists of a variety of sub-structures that include a cytoplasmic inner phospholipid membrane containing hydrophobic transmembrane proteins, a liquid phase periplasm containing soluble proteins, the cell shape and rigidity determinant peptidoglycan (PG) layer and finally, an asymmetric outer membrane (OM) lipid bilayer consisting of phospholipids, OM proteins and external lipopolysaccharide. The membrane facilitates interactions with the environment, including the human host, and is critical for all aspects of virulence, including adherence, host cell invasion, secretion, flagellar motility and antimicrobial resistance. We developed membrane enrichment strategies for in situ cross-linking of membrane proteins and the flagella apparatus from the helical human gastrointestinal pathogen Campylobacter jejuni. We investigated different fractionation approaches, including offline size-exclusion chromatography (SEC) and online gas-phase fractionation with high-field asymmetric waveform ion mobility spectrometry (FAIMS), and then investigated how different protein digestion approaches and gradient lengths affect the success of these strategies in identifying cross-linked peptide species at both the sample preparation and acquisition levels. A hybrid MS2-MS3 fragmentation strategy with or without an internal-stepping compensation voltage scheme showed the benefits of FAIMS for XL-MS increases with sample complexity and gradient length by reducing interference and increasing signal:noise. Significantly, single mg injections were able to yield more cross-links that 8 mg of SEC fractions over the same acquisition time. The use of FAIMS and SEC with different proteases suggests that protease-specific cross-linked peptides respond differently to ion-based separation across several physicochemical properties. In situ membrane protein cross-linking revealed extensive interactions between the major human adherence factor CadF, PG-associated lipoprotein Pal and major OM protein MOMP providing evidence of how C. jejuni maintains membrane integrity. Flagellar apparatus cross-linking revealed flagellin multimerisation and conformational dynamics for the first time, including multimeric interfaces and filament stabilisation. In conclusion, proteome-wide interactome interrogation with XL-MS can be tailored to the needs of the researcher in terms of sample type, preparation, fractionation and acquisition. Optimisation of these parameters can allow, for example, identification of sub-proteome specific interactomes, provide greater depth and coverage of the proteome and interactome, and lead to increased cross-link identification and quality of interactome data.