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Unrestrictive identification of post-translational modifications through peptide mass spectrometry

Abstract

Proteins are post-translationally modified in vivo as part of cellular regulation and signaling, and undergo further chemical modifications during laboratory processing. Even relatively simple protein samples may carry a wide range of modifications. Peptide tandem mass spectrometry provides a way to study these events. We present a protocol for computational identification of post-translational modifications (PTMs) and the sites where they occur. The protocol performs an unrestrictive search, and requires no prior knowledge of what modifications are present in the sample. We present a largely automated procedure for PTM discovery, and provide a guide for analysis of PTM annotations. This protocol requires you to type out several commands, so you may wish to enlist the help of a colleague familiar with the computer's command-line interface. A typical MS run of up to 25,000 scans can be searched and analyzed in 3 h.

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Figure 1: Partial PTM summary report for search results over a reference collection of mass spectra (see EXAMPLE).
Figure 2: Detailed PTM report for putative modification G+59.

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Acknowledgements

This project was supported by US National Institutes of Health grant NIGMS 1-R01-RR16522. S.T. is supported by US National Science Foundation IGERT training grant DGE0504645.

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Correspondence to Stephen Tanner.

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Tanner, S., Pevzner, P. & Bafna, V. Unrestrictive identification of post-translational modifications through peptide mass spectrometry. Nat Protoc 1, 67–72 (2006). https://doi.org/10.1038/nprot.2006.10

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