A composite filter for low FDR of protein-protein interactions detected by in vivo cross-linking

In vivo chemical cross-linking combined with LCMSMS of digested extracts (in vivo CX-MS) can reveal stable and dynamic protein-protein interactions at a proteome wide-scale and at the peptide level. In vivo CX-MS requires a membrane permeable and cleavable cross-linker that enables isolation of target peptides and a fast and sensitive search engine to identify the linked peptides. Here we explore the use of the search engine pLink 2 for analysis of a previously obtained LCMSMS dataset from exponentially growing Bacillus subtilis treated in culture with the cross-linker bis(succinimidyl)-3-azidomethyl-glutarate (BAMG). Cross-linked peptide pairs were identified by pLink 2 in very short time at an overall FDR of View larger version (16K): org.highwire.dtl.DTLVardef@974dd1org.highwire.dtl.DTLVardef@1760d10org.highwire.dtl.DTLVardef@1539d5eorg.highwire.dtl.DTLVardef@808e2e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIImproved protocol for identification of PPIs at low FDR by in vivo cross-linking with BAMG C_LIO_LIThe use of all intra-protein cross-linked peptide pairs as true positives C_LIO_LIThe cytosolic aminopeptidase (AMPA_BACSU) interacts with the 50S ribosomal protein L17 C_LIO_LIThe transition state regulator AbrB interacts with elongation factor Tu C_LIO_LIThe essential protein YlaN of unknown function interacts with the iron uptake repressor Fur C_LI SignificanceImportant for reliable identification of PPIs by chemical cross-linking in vivo is a low FDR of non-redundant inter-protein peptide pairs. Here we describe how to recognize the presence of spurious interactions in a dataset of cross-linked peptide pairs enriched by 2D strong cation exchange chromatography and identified by LCMSMS by taking into account chromatographic behavior of cross-linked peptide pairs and protein abundance of corresponding peptides. Based on these criteria we assessed that the FDR of the fraction of non-redundant inter-protein cross-linked peptide pairs was approx. 20-25% by interrogating an entire species specific database at an overall FDR of 5% or 0.1% with a search engine that otherwise scores best in sensitivity among other search engines. We have defined a composite filter to decrease this high FDR of inter-protein cross-linked peptide pairs to only about 2%.