Characterization of protein crosslinks via mass spectrometry and an open-modification search strategy

摘要

Protein-protein interactions are key to function and regulation of many biological pathways. To facilitate characterization of protein-protein interactions using mass spectrometry, a new data acquisition/analysis pipeline was designed. The goal for this pipeline was to provide a generic strategy for identifying crosslinked peptides from single LC/MS/MS datasets, without using specialized crosslinkers or custom-written software. To achieve this, each peptide in the pair of crosslinked peptides was considered to be “post-translationally” modified with an unknown mass at an unknown amino acid. This allowed use of an open-modification search engine, Popitam, to interpret the tandem mass spectra of crosslinked peptides. False positives were reduced and database selectivity increased by acquiring precursors and fragments at high mass accuracy. Additionally, a high-charge-state-driven data acquisition scheme was utilized to enrich datasets for crosslinked peptides. This open-modification search based pipeline was shown to be useful for characterizing both chemical as well as native crosslinks in proteins. The pipeline was validated by characterizing the known interactions in chemically crosslinked CYP2E1-b5 complex. Utility of this method in identifying native crosslinks was demonstrated by mapping disulfide bridges in RcsF, an outer membrane lipoprotein involved in Rcs phosphorelay.