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. 2008 Jan;7(1):138-44.
doi: 10.1021/pr070363z. Epub 2007 Dec 12.

Identification and Characterization of Disulfide Bonds in Proteins and Peptides From Tandem MS Data by Use of the MassMatrix MS/MS Search Engine

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Free PMC article

Identification and Characterization of Disulfide Bonds in Proteins and Peptides From Tandem MS Data by Use of the MassMatrix MS/MS Search Engine

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

Abstract

A new database search algorithm has been developed to identify disulfide-linked peptides in tandem MS data sets. The algorithm is included in the newly developed tandem MS database search program, MassMatrix. The algorithm exploits the probabilistic scoring model in MassMatrix to achieve identification of disulfide bonds in proteins and peptides. Proteins and peptides with disulfide bonds can be identified with high confidence without chemical reduction or other derivatization. The approach was tested on peptide and protein standards with known disulfide bonds. All disulfide bonds in the standard set were identified by MassMatrix. The algorithm was further tested on bovine pancreatic ribonuclease A (RNaseA). The 4 native disulfide bonds in RNaseA were detected by MassMatrix with multiple validated peptide matches for each disulfide bond with high statistical scores. Fifteen nonnative disulfide bonds were also observed in the protein digest under basic conditions (pH = 8.0) due to disulfide bond interchange. After minimizing the disulfide bond interchange (pH = 6.0) during digestion, only one nonnative disulfide bond was observed. The MassMatrix algorithm offers an additional approach for the discovery of disulfide bond from tandem mass spectrometry data.

Figures

Figure 1
Figure 1
Classification of disulfide-linked peptides in MassMatrix.
Figure 2
Figure 2
The flow diagram for the search algorithm of disulfide-linked peptides in MassMatrix.
Figure 3
Figure 3
Disulfide-linked peptide matches for (a) type 1A, (b) type 2A, (c) type 2B, and (d) type 3 peptides. Ions with neutral loss of small molecules are labeled by asterisk (*, loss of ammonia) and prime (′, loss of water).
Figure 4
Figure 4
Disulfide bonds mapped by tandem MS experiment and subsequent database search in MassMatrix for digests under (a) basic and (b) acidic conditions. Confidence of the identification for each bond is indicated by the sum of pp values of peptide matches with the bond. Native disulfide bonds are bolded.
Figure 5
Figure 5
Distribution of pp values for peptides without disulfide bonds and disulfide-linked peptides, compared with false positive peptide matches.
Figure 6
Figure 6
ROC curves of the search with decoy sequences. Solid line (−), true positive peptide matches with disulfide bonds vs false positives; dashed line (---), true positive peptides without disulfide bonds vs false positives. False positives include both disulfide-linked false peptides and false peptides without disulfide bonds. In ROC curves, a value to the top indicates higher sensitivity, and a value to the left indicates higher specificity.

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