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Functional Proteomics pp 225–251Cite as

Identification of Unexpected Protein Modifications by Mass Spectrometry-Based Proteomics

Part of the Methods in Molecular Biology book series (MIMB,volume 1871)

Abstract

Peptide identification relies in the majority of mass spectrometry-based proteomics experiments on matching of experimental data against peptide and fragment ion masses derived from in silico digests of protein databases. One of the main drawbacks of this approach is that modifications have to be defined for database searching and therefore no unexpected modifications can be identified in a standard setup. Consequently, in many bottom-up proteomics experiments, unexpected modifications are not identified, even if high-quality fragment ion spectra of the modified peptides were acquired. It is therefore often not straightforward to identify unexpected modifications. In this protocol, we describe a stepwise procedure to identify unexpected modifications at peptides using the database search algorithm Mascot. The workflow includes parallel searches for the identification of known modifications at unexpected amino acids, error tolerant searches for modifications unexpected in the sample but known to the community, and mass tolerant searches for entirely unknown modifications. Furthermore, we suggest a follow-up strategy consisting of (1) verification of identified modifications in the initial dataset and (2) targeted experiments using synthetic peptides.

Key words

  • Mass spectrometry
  • Unexpected modifications
  • Posttranslational modifications
  • Bottom-up proteomics
  • Data analysis
  • Mascot
  • Error tolerant search
  • Mass tolerant search

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Authors and Affiliations

  1. Institute for Biochemistry and Molecular Biology, University of Bonn, Bonn, Germany

    Shiva Ahmadi & Dominic Winter

Authors
  1. Shiva Ahmadi
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  2. Dominic Winter
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Corresponding author

Correspondence to Dominic Winter .

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Editors and Affiliations

  1. Array Bridge Inc., St. Louis, MO, USA

    Xing Wang

  2. Biotech Support Group LLC, Monmouth Junction, NJ, USA

    Matthew Kuruc

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Ahmadi, S., Winter, D. (2019). Identification of Unexpected Protein Modifications by Mass Spectrometry-Based Proteomics. In: Wang, X., Kuruc, M. (eds) Functional Proteomics. Methods in Molecular Biology, vol 1871. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8814-3_15

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  • DOI: https://doi.org/10.1007/978-1-4939-8814-3_15

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