Matching Cross-linked Peptide Spectra: Only as Good as the Worse Identification

Abstract

Chemical cross-linking mass spectrometry identifies interacting surfaces within a protein assembly through labeling with bifunctional reagents and identifying the covalently modified peptides. These yield distance constraints that provide a powerful means to model the three-dimensional structure of the assembly. Bioinformatic analysis of crosslinked data resulting from large protein assemblies is challenging because each cross-linked product contains two covalently linked peptides, each of which must be correctly identified from a complex matrix of potential confounders.Protein Prospector addresses these issues through a complementary mass modification strategy in which each peptide is searched and identified separately. We demonstrate this strategy with an analysis of RNA polymerase II. False discovery rates (FDRs) are assessed via comparison of cross-linking data to crystal structure, as well as by using a decoy database strategy. Parameters that are most useful for positive identification of cross-linked spectra are explored. We find that fragmentation spectra generally contain more product ions from one of the two peptides constituting the cross-link. Hence, metrics reflecting the quality of the spectral match to the less confident peptide provide the most discriminatory power between correct and incorrect matches. A support vector machine model was built to further improve classification of cross-linked peptide hits. Furthermore, the frequency with which peptides cross-linked via common acylating reagents fragment to produce diagnostic, cross-linkerspecific ions is assessed.The threshold for successful identification of the cross-linked peptide product depends upon the complexity of the sample under investigation. Protein Prospector, by focusing the reliability assessment on the least confident peptide, is better able to control the FDR for results as larger complexes and databases are ana- Most proteins are organized into stable assemblies that communicate among themselves through transient proteinprotein interaction networks to catalyze cellular phenomena. Chemical cross-linking mass spectrometry directly measures protein-protein interactions by using bifunctional cross-linking reagents to covalently link surfaces of interacting partners (1-3). Following proteolysis, mass spectrometry is used to identify the covalently linked peptides and modified residues. This information, taken together with the geometry of the cross-linking reagent, provides distance constraints that are reflective of the three-dimensional structure of the protein complex. Cross-linking-derived distance constraints provide a powerful means by which to integrate atomic resolution structures of individual protein subunits or subassemblies with low-resolution electron-microscopy-derived structures, as well as to clarify molecular details that are unresolved in electron density maps. For instance, this approach has recently been applied to modeling the RNA Pol II preinitiation complex (4), several chromatin remodeling complexes (5, 6), th...

MS-Viewer: A Web-based Spectral Viewer for Proteomics Results

“…Each cross-linked peptide in the complex is reported in a separate column in the uploaded tabular file, where the mass of the second peptide with cross-linker is reported as a mass modification on the relevant residue in each peptide (13). Fig.…”

“…delimited text files), an author could potentially use the same file for submission to the journal and MSViewer (13). It is also possible to create hyperlinks from a table outside of MS-Viewer directly to spectra (e.g.…”

Human Liver Cytochrome P450 3A4 Ubiquitination

“…gp78, CYP3A4, etc., such TPAL cross-links could include those of protein homodimers as well. Subsequent tryptic/Lys-C digestion of the TPAL cross-linked proteins, subfractionation of the cross-linked peptide digests by size-exclusion chromatography, followed by LC-MS/MS analyses of the TPAL crosslinked peptide digests wherein quadruply charged and higher precursors were selected and dissociated by ETD or sometimes by HCD ("Experimental Procedures"), yielded MS data from which peak lists were generated as described previously (56) and further analyzed as detailed under "Experimental Procedures." Precursor ion annotations were also inspected manually.…”

“…Peak lists were then searched against this database using Protein Prospector to assign cross-linked peptides (56). Carbamidomethylation of cysteine was considered as a fixed modification.…”

“…Interpretation of MS Data-Peak lists from all MS runs were generated as described previously (56). Peak lists were first searched against the SwissProt Database with no organism restrictions to identify proteins present in the samples.…”