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. 2010 Jul 2;9(7):3384-93.
doi: 10.1021/pr100369y.

Database Search Algorithm for Identification of Intact Cross-Links in Proteins and Peptides Using Tandem Mass Spectrometry

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Free PMC article

Database Search Algorithm for Identification of Intact Cross-Links in Proteins and Peptides Using Tandem Mass Spectrometry

Hua Xu et al. J Proteome Res. .
Free PMC article

Abstract

A new database search algorithm has been developed for identification of intact cross-links in proteins and peptides from tandem mass spectrometric data. Using this algorithm, intact cross-links can be identified and characterized in proteins and peptides with high confidence. The algorithm was tested using BS(3) (bis[sulfosuccinimidyl] suberate) cross-linked Cytochrome C. Five cross-links were identified and verified for spatial plausibility by comparison with its three-dimensional structure at optimized experimental conditions. The distributions of statistical scores for true and false positives and receiver operating characteristic analysis indicate that the algorithm is capable of discriminating true positive cross-linked peptide-spectrum matches from false ones. It has also been demonstrated that the MassMatrix database search engine is capable of searching for intact cross-links in complex Escherichia coli proteome samples cross-linked by BS(3). The new algorithm in MassMatrix offers an additional approach for the discovery of cross-links in proteins and peptides from tandem mass spectrometric data.

Figures

Figure 1
Figure 1
Structure of cross-linking reagent BS3, its reaction with lysine, and the possible dead-end cross-links.
Figure 2
Figure 2
Representative MS/MS spectra obtained for two cross-linked peptides with (a) an intrachain cross-link and (b) an interchain cross-link in the search of tryptic digest of cross-linked Cytochrome C by BS3. Product ions from neutral loss are labeled by * (loss of ammonia) and ′ (loss of water). The lysine residue with a dead-end cross-link is denoted as K*.
Figure 3
Figure 3
(a) Distributions of pptag scores for true and false positive peptide-spectrum matches and (b) ROC curves of the search of a tryptic digest of cross-linked Cytochrome C by BS3. In ROC curve, a value toward the top of the graph indicates higher sensitivity and a value to the left indicates higher specificity.
Figure 4
Figure 4
Cross-links in the BS3 cross-linked Cytochrome C mapped by tandem MS experiment and subsequent database search in MassMatrix. Each cell in the heat map represents a cross-link between two lysine residues. Confidence of the identification of each cross-link is indicated by its score displayed in the heat map. The cross-linking reagent to protein ratio was 25:1 and the final protein concentration was 0.12 mg/mL.
Figure 5
Figure 5
Evaluation of spatial plausibility for Cytochrome C cross-links identified by MassMatrix (PDB code: 1HRC).
Figure 6
Figure 6
Heat maps of identified cross-links for Cytochrome C cross-linked by BS3 at different cross-linking reagent to protein standard ratios of 1:1, 2.5:1, 5:1, 10:1, 25:1, 50:1, and 100:1 with a final protein concentration of 0.12 mg/mL.
Figure 7
Figure 7
(a) Scores of the five nonbackground cross-links, and (b) numbers of all background cross-links (-■-) and the background cross-links at FDR of 5% (-●-) for Cytochrome C cross-linked by BS3 at different cross-linking reagent to protein standard ratios of 1:1, 2.5:1, 5:1, 10:1, 25:1, 50:1, and 100:1 with a final protein concentration of 0.12 mg/mL. The gray area represents the score level of the false positive cross-links.
Figure 8
Figure 8
Heat maps of identified cross-links for Cytochrome C cross-linked by BS3 at a cross-linking reagent to protein ratio of 10:1 and different protein concentrations of 0.06, 0.12, 0.6, and 2.4 mg/mL.
Figure 9
Figure 9
(a) Scores of the five nonbackground cross-links, and (b) numbers of all background cross-links (-■-) and the background cross-links at FDR of 5% (-●-) for Cytochrome C cross-linked by BS3 at a cross-linking reagent to protein ratio of 10:1 and different protein concentrations of 0.06, 0.12, 0.6, and 2.4 mg/mL. The gray area represents the score level of the false positive cross-links.

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