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A large synthetic peptide and phosphopeptide reference library for mass spectrometry–based proteomics

Nature Biotechnology volume 31, pages 557–564 (2013)Cite this article

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Abstract

We present a peptide library and data resource of >100,000 synthetic, unmodified peptides and their phosphorylated counterparts with known sequences and phosphorylation sites. Analysis of the library by mass spectrometry yielded a data set that we used to evaluate the merits of different search engines (Mascot and Andromeda) and fragmentation methods (beam-type collision-induced dissociation (HCD) and electron transfer dissociation (ETD)) for peptide identification. We also compared the sensitivities and accuracies of phosphorylation-site localization tools (Mascot Delta Score, PTM score and phosphoRS), and we characterized the chromatographic behavior of peptides in the library. We found that HCD identified more peptides and phosphopeptides than did ETD, that phosphopeptides generally eluted later from reversed-phase columns and were easier to identify than unmodified peptides and that current computational tools for proteomics can still be substantially improved. These peptides and spectra will facilitate the development, evaluation and improvement of experimental and computational proteomic strategies, such as separation techniques and the prediction of retention times and fragmentation patterns.

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Figure 1: Library design and synthesis.
Figure 2: Peptide library identification rate.
Figure 3: FDR determination for peptide identification.
Figure 4: FLR determination for phosphorylated peptides.
Figure 5: Retention time analysis.

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Acknowledgements

This research was supported in part by the Deutsche Forschungsgemeinschaft International Research Training Group 'Regulation and Evolution of Cellular Systems' (RECESS, GRK 1563) and in part by the PRIME-XS project with the grant agreement number 262067 funded by the European Union 7th Framework Program. H.M. acknowledges the support of the Graduate School at the Technische Universität München, and the authors thank A. Hubauer for expert technical assistance.

Author information

Author notes

  1. Shabaz Mohammed

    Present address: Present address: Departments of Chemistry and Biochemistry, University of Oxford, Physical and Theoretical Chemistry Laboratory, Oxford, UK.,

Authors and Affiliations

  1. Chair for Proteomics and Bioanalytics, Technische Universität München, Freising, Germany

    Harald Marx, Simone Lemeer, Jan Erik Schliep & Bernhard Kuster

  2. Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands

    Lucrece Matheron, Shabaz Mohammed & Albert J R Heck

  3. The Netherlands Proteomics Centre, The Netherlands

    Lucrece Matheron, Shabaz Mohammed & Albert J R Heck

  4. Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Martinsried, Germany

    Jürgen Cox & Matthias Mann

  5. Center for Integrated Protein Science, Munich, Germany

    Bernhard Kuster

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Contributions

H.M., J.E.S. and B.K. designed the study. S.L., J.E.S., L.M. and S.M. performed experiments. H.M., S.L., L.M. and J.C. analyzed data. H.M., S.L., A.J.R.H., M.M. and B.K. wrote manuscript.

Corresponding author

Correspondence to Bernhard Kuster.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–23 (PDF 3226 kb)

Supplementary Table 1

Sequence, site of phosphorylation within the sequence, length and GRAVY score (Hydrophobicity) of the 851 representative sample peptides derived from the consensus of three out of the five publically available human phosphorylation data sets used in this study (XLSX 47 kb)

Supplementary Table 2

Peptide sequence, position of phosphorylation site in the sequence and Gravy score of the seed peptide synthesis of libraries used in this study. For each seed peptide sequence the final number of peptides in the library is given (XLSX 15 kb)

Supplementary Table 3

Search and classification result of HCD data aquired on a Orbitrap Velos. (XLSX 85450 kb)

Supplementary Table 4

Search and classification result of ETD-FT data aquired on a Orbitrap Velos. (XLSX 58136 kb)

Supplementary Table 5

Number of peptide identifications and phosphorylation site localizations at a given global or local false discovery rate (Mascot) (XLSX 526 kb)

Supplementary Table 6

Coefficients for the computation of local and global FDRs and FLRs (XLSX 551 kb)

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Marx, H., Lemeer, S., Schliep, J. et al. A large synthetic peptide and phosphopeptide reference library for mass spectrometry–based proteomics. Nat Biotechnol 31, 557–564 (2013). https://doi.org/10.1038/nbt.2585

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