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Verification of automated peptide identifications from proteomic tandem mass spectra

Nature Protocols volume 1, pages 2213–2222 (2006)Cite this article

Abstract

Shotgun proteomics yields tandem mass spectra of peptides that can be identified by database search algorithms. When only a few observed peptides suggest the presence of a protein, establishing the accuracy of the peptide identifications is necessary for accepting or rejecting the protein identification. In this protocol, we describe the properties of peptide identifications that can differentiate legitimately identified peptides from spurious ones. The chemistry of fragmentation, as embodied in the 'mobile proton' and 'pathways in competition' models, informs the process of confirming or rejecting each spectral match. Examples of ion-trap and tandem time-of-flight (TOF/TOF) mass spectra illustrate these principles of fragmentation.

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Figure 1: Pathways in competition.
Figure 2
Figure 3: These two spectra were both identified as the doubly-charged sequence EIIGVVSQEPVLFATTIAENIR.
Figure 4: This chimeric spectrum is displayed two different ways because it contains fragment ions from two different peptides, DSDYYNMLLK (top) and M*DFSDYDLLK (bottom).
Figure 5: This spectrum was identified as a doubly charged SSLKAGALR (top).
Figure 6: Phosphopeptides can show distinct losses of phosphoric acid both from the precursor and from fragment ions.
Figure 7: This TOF/TOF spectrum was identified as the sequence AVREAAAGLSGPGR by the Mascot algorithm.
Figure 8: Special TOF/TOF fragment ions.
Figure 9: These two peptide identifications are evidence for the presence of calmodulin in a mouse sample.

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Acknowledgements

This work was supported by US National Institutes of Health grant P30 ES000267, by an American Cancer Society Institutional Research Grant (no. IRG-58-009-48) through the Sartain-Lanier Family Foundation and Vanderbilt-Ingram Cancer Center Discovery Grant Programs, and by the Vanderbilt Academic Venture Capital Fund. M. Tyska (Vanderbilt University; supported by a Career Development Award from the Crohn's and Colitis Foundation of America) contributed the spectra of mouse brush border proteins, and R. Peek and A. Franco (Vanderbilt University) contributed H. pylori membrane samples.

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  1. Department of Biochemistry, Mass Spectrometry Research Center, Vanderbilt University Medical Center, Nashville, 37232-8340, Tennessee, USA

    David L Tabb, David B Friedman & Amy-Joan L Ham

  2. Department of Biomedical Informatics, Vanderbilt University Medical Center, Nashville, 37232-8340, Tennessee, USA

    David L Tabb

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Correspondence to David L Tabb.

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Tabb, D., Friedman, D. & Ham, AJ. Verification of automated peptide identifications from proteomic tandem mass spectra. Nat Protoc 1, 2213–2222 (2006). https://doi.org/10.1038/nprot.2006.330

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