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Chemical Fractionation, Chromatography/methods, HEK293 Cells, Humans, Ion Mobility Spectrometry/*methods, Peptides/*chemistry
Abstract:
In cross-linking mass spectrometry (XL-MS), the depth and sensitivity of cross-link detection is often limited by the low abundance of cross-links compared to non-cross-linked peptides in the digestion mixture. To improve the identification efficiency of cross-links, here, we present a gas-phase separation strategy using high-field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to the Orbitrap Tribrid mass spectrometers. By enabling an additional peptide separation step in the gas phase using the FAIMS device, we increase the number of cross-link identifications by 22% for a medium complex sample and 59% for strong cation exchange-fractionated HEK293T cell lysate in XL-MS experiments using disuccinimidyl sulfoxide (DSSO) cross-linker. When disuccinimidyl suberate (DSS) cross-linker is in use, we are able to boost cross-link identification by 89% for the medium and 100% for the high complex sample compared to the analyses without FAIMS. Furthermore, we show that, for medium complex samples, FAIMS enables the collection of single-shot XL-MS data with a comparable depth to the corresponding sample fractionated by chromatography-based approaches. Altogether, we demonstrate FAIMS is highly beneficial for XL-MS studies by expanding the proteome coverage of cross-links while improving the efficiency and confidence of cross-link identification.
