%0 Journal Article %A Singh, Pragya %A Shaffer, Scott A. %A Scherl, Alexander %A Holman, Carol %A Pfuetzner, Richard A. %A J. Larson Freeman, Theodore %A Miller, Samuel I. %A Hernandez, Patricia %A Appel, Ron D. %A Goodlett, David R. %D 2008 %T Characterization of Protein Cross-Links via Mass Spectrometry and an Open-Modification Search Strategy %U https://acs.figshare.com/articles/journal_contribution/Characterization_of_Protein_Cross_Links_via_Mass_Spectrometry_and_an_Open_Modification_Search_Strategy/2899954 %R 10.1021/ac801646f.s001 %2 https://acs.figshare.com/ndownloader/files/4598095 %K data sets %K peptide %K 2E %K mass spectrometry %K tandem mass spectra %K mass accuracy %K False positives %K interaction %K database selectivity %K Mass Spectrometry %K pipeline %K LC %K Rcs phosphorelay %K acquisition %K CYP %K mapping disulfide bridges %K membrane lipoprotein %X 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 cross-linked peptides from single LC/MS/MS data sets, without using specialized cross-linkers or custom-written software. To achieve this, each peptide in the pair of cross-linked 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 cross-linked 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 data sets for cross-linked peptides. This open-modification search based pipeline was shown to be useful for characterizing both chemical as well as native cross-links in proteins. The pipeline was validated by characterizing the known interactions in the chemically cross-linked CYP2E1−b5 complex. Utility of this method in identifying native cross-links was demonstrated by mapping disulfide bridges in RcsF, an outer membrane lipoprotein involved in Rcs phosphorelay. %I ACS Publications