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Conserved peptide fragmentation as a benchmarking tool for mass spectrometers and a discriminating feature for targeted proteomics.

Toprak UH, Gillet LC, Maiolica A, Navarro P, Leitner A, Aebersold R - Mol. Cell Proteomics (2014)

Bottom Line: In both cases, confidence in peptide identification is directly related to the quality of spectral matches.Altogether, this study validates the use of the normalized spectral contrast angle as a sensitive spectral similarity measure for targeted proteomics, and more generally provides a methodology to assess the performance of spectral comparisons and to support the rational selection of the most appropriate similarity measure.The algorithms used in this study are made publicly available as an open source toolset with a graphical user interface.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, 8093 Zurich, Switzerland;

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Target-decoy separation characteristics of (A) dot product, (B) spectral correlation, (C) normalized spectral contrast angle. The blue curves denote the target assay similarities whereas the red curves denote the decoy assay similarities in a TripleTOF shotgun/SWATH mode comparison. The lognormal/gamma fit parameters as well as the fit errors (norm of residuals) are reported for each fit.
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Figure 5: Target-decoy separation characteristics of (A) dot product, (B) spectral correlation, (C) normalized spectral contrast angle. The blue curves denote the target assay similarities whereas the red curves denote the decoy assay similarities in a TripleTOF shotgun/SWATH mode comparison. The lognormal/gamma fit parameters as well as the fit errors (norm of residuals) are reported for each fit.

Mentions: The results are shown in Fig. 5 and supplemental Fig. S12. As expected, for all scores, the target shotgun assays displayed relatively good levels of fragmentation similarity (equivalent to 10–28% perturbation depending on the score used) compared with that of the SWATH MS integrated fragment signals. In contrast, the relative fragment ion intensities of the decoy assays displayed a quasi-random match (equivalent to 100% perturbation) compared with that of the corresponding SWATH MS integrated signals. Overall, all similarity measures achieved good discrimination between the target and decoy score distributions except spectral correlation, which attributed quite early relatively high scores to decoy spectra (Fig. 5B). Interestingly and despite the compressed response anticipated from Fig. 1, the dot product achieved equivalent performance in separating targets and decoys compared with the normalized spectral contrast angle, after remapping the raw score values onto the corresponding perturbation benchmark spectra set. Using one of those two similarity measures, very few decoy assays displayed high similarity scores compared with their corresponding target assays. Therefore, in principle, any instrument offering overall intrinsic fragmentation reproducibility and assay transferability performance above this threshold (35% in the case of the normalized spectral contrast angle) should support effective peptide identification in SWATH MS targeted extraction via relative fragment intensity. Higher fragmentation reproducibility, of course, would be expected to further increase the discrimination between targets and decoys and would thus strengthen again the power of such fragmentation similarity scores for peptide identifications.


Conserved peptide fragmentation as a benchmarking tool for mass spectrometers and a discriminating feature for targeted proteomics.

Toprak UH, Gillet LC, Maiolica A, Navarro P, Leitner A, Aebersold R - Mol. Cell Proteomics (2014)

Target-decoy separation characteristics of (A) dot product, (B) spectral correlation, (C) normalized spectral contrast angle. The blue curves denote the target assay similarities whereas the red curves denote the decoy assay similarities in a TripleTOF shotgun/SWATH mode comparison. The lognormal/gamma fit parameters as well as the fit errors (norm of residuals) are reported for each fit.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4125737&req=5

Figure 5: Target-decoy separation characteristics of (A) dot product, (B) spectral correlation, (C) normalized spectral contrast angle. The blue curves denote the target assay similarities whereas the red curves denote the decoy assay similarities in a TripleTOF shotgun/SWATH mode comparison. The lognormal/gamma fit parameters as well as the fit errors (norm of residuals) are reported for each fit.
Mentions: The results are shown in Fig. 5 and supplemental Fig. S12. As expected, for all scores, the target shotgun assays displayed relatively good levels of fragmentation similarity (equivalent to 10–28% perturbation depending on the score used) compared with that of the SWATH MS integrated fragment signals. In contrast, the relative fragment ion intensities of the decoy assays displayed a quasi-random match (equivalent to 100% perturbation) compared with that of the corresponding SWATH MS integrated signals. Overall, all similarity measures achieved good discrimination between the target and decoy score distributions except spectral correlation, which attributed quite early relatively high scores to decoy spectra (Fig. 5B). Interestingly and despite the compressed response anticipated from Fig. 1, the dot product achieved equivalent performance in separating targets and decoys compared with the normalized spectral contrast angle, after remapping the raw score values onto the corresponding perturbation benchmark spectra set. Using one of those two similarity measures, very few decoy assays displayed high similarity scores compared with their corresponding target assays. Therefore, in principle, any instrument offering overall intrinsic fragmentation reproducibility and assay transferability performance above this threshold (35% in the case of the normalized spectral contrast angle) should support effective peptide identification in SWATH MS targeted extraction via relative fragment intensity. Higher fragmentation reproducibility, of course, would be expected to further increase the discrimination between targets and decoys and would thus strengthen again the power of such fragmentation similarity scores for peptide identifications.

Bottom Line: In both cases, confidence in peptide identification is directly related to the quality of spectral matches.Altogether, this study validates the use of the normalized spectral contrast angle as a sensitive spectral similarity measure for targeted proteomics, and more generally provides a methodology to assess the performance of spectral comparisons and to support the rational selection of the most appropriate similarity measure.The algorithms used in this study are made publicly available as an open source toolset with a graphical user interface.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, 8093 Zurich, Switzerland;

Show MeSH
Related in: MedlinePlus