Using SIM-XL to identify and annotate cross-linked peptides analyzed by mass spectrometry (original) (raw)
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SIM-XL: A powerful and user-friendly tool for peptide cross-linking analysis
Journal of Proteomics, 2015
Chemical cross-linking has emerged as a powerful approach for the structural characterization of proteins and protein complexes. However, the correct identification of covalently linked (cross-linked or XL) peptides analyzed by tandem mass spectrometry is still an open challenge. Here we present SIM-XL, a software tool that can analyze data generated through commonly used cross-linkers (e.g., BS3/DSS). Our software introduces a new paradigm for search-space reduction, which ultimately accounts for its increase in speed and sensitivity.
Detecting Cross-Linked Peptides by Searching against a Database of Cross-Linked Peptide Pairs
Journal of Proteome Research, 2010
Mass spectrometric identification of cross-linked peptides can provide valuable information about the structure of protein complexes. We describe a straightforward database search scheme that identifies and assigns statistical confidence estimates to spectra from cross-linked peptides. The method is well suited to targeted analysis of a single protein complex, without requiring an isotope labeling strategy. Our approach uses a SEQUEST-style search procedure in which the database is comprised of a mixture of single peptides with and without linkers attached and cross-linked products. In contrast to several previous approaches, we generate theoretical spectra that account for all of the expected peaks from a cross-linked product, and we employ an empirical curve-fitting procedure to estimate statistical confidence measures. We show that our fully automated procedure successfully reidentifies spectra from a previous study, and we provide evidence that our statistical confidence estimates are accurate. Figure 1. Various types of molecules produced by a cross-linking protocol. The molecule inside the dotted circle is an interprotein cross-link.
Molecular & Cellular Proteomics, 2011
Knowledge of elaborate structures of protein complexes is fundamental for understanding their functions and regulations. Although cross-linking coupled with mass spectrometry (MS) has been presented as a feasible strategy for structural elucidation of large multisubunit protein complexes, this method has proven challenging because of technical difficulties in unambiguous identification of cross-linked peptides and determination of cross-linked sites by MS analysis. In this work, we developed a novel cross-linking strategy using a newly designed MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). DSSO contains two symmetric collision-induced dissociation (CID)-cleavable sites that allow effective identification of DSSO-cross-linked peptides based on their distinct fragmentation patterns unique to cross-linking types (i.e. interlink, intralink, and dead end). The CID-induced separation of interlinked peptides in MS/MS permits MS 3 analysis of single peptide chain fragment ions with defined modifications (due to DSSO remnants) for easy interpretation and unambiguous identification using existing database searching tools. Integration of data analyses from three generated data sets (MS, MS/MS, and MS 3 ) allows high confidence identification of DSSO cross-linked peptides. The efficacy of the newly developed DSSO-based cross-linking strategy was demonstrated using model peptides and proteins. In addition, this method was successfully used for structural characterization of the yeast 20 S proteasome complex. In total, 13 non-redundant interlinked peptides of the 20 S proteasome were identified, representing the first application of an MS-cleavable cross-linker for the characterization of a multisubunit protein complex. Given its effectiveness and simplicity, this cross-linking strategy can find a broad range of applications in elucidating the structural topology of proteins and protein complexes. Molecular &
CLPM: a cross-linked peptide mapping algorithm for mass spectrometric analysis
2005
Background: Protein-protein, protein-DNA and protein-RNA interactions are of central importance in biological systems. Quadrapole Time-of-flight (Q-TOF) mass spectrometry is a sensitive, promising tool for studying these interactions. Combining this technique with chemical crosslinking, it is possible to identify the sites of interactions within these complexes. Due to the complexities of the mass spectrometric data of crosslinked proteins, new software is required to analyze the resulting products of these studies. Result: We designed a Cross-Linked Peptide Mapping (CLPM) algorithm which takes advantage of all of the information available in the experiment including the amino acid sequence from each protein, the identity of the crosslinker, the identity of the digesting enzyme, the level of missed cleavage, and possible chemical modifications. The algorithm does in silico digestion and crosslinking, calculates all possible mass values and matches the theoretical data to the actual experimental data provided by the mass spectrometry analysis to identify the crosslinked peptides. Conclusion: Identifying peptides by their masses can be an efficient starting point for direct sequence confirmation. The CLPM algorithm provides a powerful tool in identifying these potential interaction sites in combination with chemical crosslinking and mass spectrometry. Through this cost-effective approach, subsequent efforts can quickly focus attention on investigating these specific interaction sites.
Identification of cross-linked peptides from large sequence databases
Nature Methods, 2008
We describe a method to identify cross-linked peptides from complex samples and large protein sequence databases. The advance was achieved by combining isotopically tagged cross-linkers, chromatographic enrichment, targeted proteomics, and a novel search engine called xQuest. This software reduces the search space by an upstream candidatepeptide search before the recombination step; we show that xQuest can identify cross-linked peptides from a total E. coli lysate with an unrestricted database search. Phone number: +41 44 633 31 70 Fax number: +41 44 633 10 51. Author contributions O.R., most of the software design and programming, conducted most of the experiments, and most of the data evaluation; J.S. synthesis of cross-linker, contribution to software design; T.W., scoring, data evaluation, and parts of the experiments; L.N.M., analysis and alignment of MS1 data; M.B., parts of the experiments, data evaluation; A.S., design of mass spectrometric methods for cross-linked samples; M.M, contribution to scoring; R.A., coordination of the project, contribution of ideas and concept.
xComb: A Cross-Linked Peptide Database Approach to Protein−Protein Interaction Analysis
Journal of Proteome Research, 2010
We developed an informatic method to identify tandem mass spectra composed of chemically crosslinked peptides from those of linear peptides and to assign sequence to each of the two unique peptide sequences. For a given set of proteins the key software tool, xComb, combs through all theoretically feasible cross-linked peptides to create a database consisting of a subset of all combinations represented as peptide FASTA files. The xComb library of select theoretical cross-linked peptides may then be used as a database that is examined by a standard proteomic search engine to match tandem mass spectral datasets to identify cross-linked peptides. The database search may be conducted against as many as 50 proteins with a number of common proteomic search engines, e.g. Phenyx, Sequest, OMSSA, Mascot and X!Tandem. By searching against a peptide library of linearized, cross-linked peptides, rather than a linearized protein library, search times are decreased and the process is decoupled from any specific search engine. A further benefit of decoupling from the search engine is that protein cross-linking studies may be conducted with readily available informatics tools for which scoring routines already exist within the proteomic community.
Nonprotein Based Enrichment Method to Analyze Peptide Cross-Linking in Protein Complexes
Analytical Chemistry, 2009
Cross-linking analysis of protein complexes and structures by tandem mass spectrometry (MS/MS) has advantages in speed, sensitivity, specificity, and the capability of handling complicated protein assemblies. However, detection and accurate assignment of the cross-linked peptides are often challenging due to their low abundance and complicated fragmentation behavior in collision-induced dissociation (CID). To simplify the MS analysis and improve the signal-to-noise ratio of the crosslinked peptides, we developed a novel peptide enrichment strategy that utilizes a cross-linker with a cryptic thiol group and using beads modified with a photocleavable cross-linker. The functional cross-linkers were designed to react with the primary amino groups in proteins. Human serum albumin was used as a model protein to detect intra-and intermolecular cross-linkages. Use of this protein-free selective retrieval method eliminates the contamination that can result from avidinbiotin based retrieval systems and simplifies data analysis. These features may make the method suitable to investigate protein-protein interactions in biological samples.