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. 2008 Apr;5(4):315-8.
doi: 10.1038/nmeth.1192. Epub 2008 Mar 9.

Identification of cross-linked peptides from large sequence databases

Oliver Rinner  1 , Jan Seebacher, Thomas Walzthoeni, Lukas N Mueller, Martin Beck, Alexander Schmidt, Markus Mueller, Ruedi Aebersold
Affiliations

Identification of cross-linked peptides from large sequence databases

Oliver Rinner et al. Nat Methods. 2008 Apr.

Erratum in

  • Nat Methods. 2008 Aug;5(8):748

Abstract

We describe a method to identify cross-linked peptides from complex samples and large protein sequence databases by combining isotopically tagged cross-linkers, chromatographic enrichment, targeted proteomics and a new search engine called xQuest. This software reduces the search space by an upstream candidate-peptide search before the recombination step. We showed that xQuest can identify cross-linked peptides from a total Escherichia coli lysate with an unrestricted database search.

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Figures

Figure 1
Figure 1
Workflow of cross-link search with xQuest in the ion-tag mode. The light and heavy form of isotopic peptide pairs are detected in precursor ion spectra and subjected to separate MS2 sequencing (i). MS2 spectra are compared; fragment ions that are present in both spectra (green) are labeled common-ions; ions with a characteristic isotopic shift (red) are xlink-ions (ii). Common-ions are used to query the ion-index which contains all peptide sequences that can give rise to a specific fragment-ion m/z (iii). Candidate peptides from this ion-index are matched against the union of common- and xlink-ions. Candidate peptides, which match sufficiently well to the spectrum are retained (iv) and recombined to cross-links (v). Only peptide combinations that match the precursor ion mass are retained and scored against the reconstructed MS2 spectrum (vi).
Figure 2
Figure 2
Linear discriminant analysis (LDA) separates true positive MS2 assignments to standard protein cross-links from false positive hits in a large peptide:peptide database. (a) Example spectrum of [M+5H]5+ charged cross-link assigned to β-Lactoglobulin (upper spectrum). The lower spectrum shows the next best random match to unrelated E. coli proteins. Matches (diamonds) are indicated with a mass tolerance of 0.2 Da for common-ions (green) and 0.3 Da for x-link ions (red). (b) Spectral assignments were verified for spatial plausibility with 3D structures or homology models as shown for bovine β-Lactoglobulin. Yellow lines indicate cross-links between the ε-amino groups. The spectrum of the cross-link indicated by the star is shown in a. (c) Sensitivity (TP/(TP+FN)), selectivity (1-FP/(TP+FP)), and specificity (TN/(TN+FP)) of assignments for different k-values (FP: false positive, TP: true positive, FN: false negative, TN: true negative). Error bars indicate s.d. of the bootstrap resampling distributions (n = 1,000). (d) Distribution of discriminant scores with weights derived from the LDA. Inset shows that the distribution of true positive assignment scores is separated from the scores of the large number of false positive assignments.
Figure 3
Figure 3
Inter-protein and intra-protein cross-links were identified from a total E. coli lysate, searched against the total E. coli protein database. (a) Top ranked search hits to cross-links show a highly significant preference for intra-protein cross-links. Search hits ranked at second place show only a slight preference. Almost no intra-protein cross-links were identified with the reversed sequence database. The curves where smoothed with a sliding average window of size 20. (b) An exhaustive search for intra-protein cross-links was performed in enumeration mode against the full E. coli database and a decoy database. True positive hits are clearly separated from false positive hits to cross-links in the decoy database. (c) Several cross-links were found for the RNA polymerase. A homology model was created based on the x-ray structure of Thermus aquaticus (Taq); thin line is the backbone of the Taq structure. Subunits B and B' are color coded for template homology (red: high homology, blue: low homology). A cross-link between B and B' was identified in a low homology region of the complex. The link is shown between the conserved lysine in B and a proline, which corresponds most closely to the lysine in the E. coli sequence. Inset shows a magnification of the linked region. The spectrum of the cross-link is shown and annotated as described above. (d) Examples of cross-links identified within and between complex subunits (colored red-blue and orange-cyan respectively) of Serine hydroxymethyltransferase (GLYA), GroEL (inset shows whole complex), Tryptophanase (TNAA) and the small ribosomal subunit.
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