Identification of Unexpected Protein Modifications by Mass Spectrometry-Based Proteomics

  • Shiva Ahmadi
  • Dominic WinterEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1871)


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 


  1. 1.
    Aebersold R, Mann M (2016) Mass-spectrometric exploration of proteome structure and function. Nature 537:347–355. Scholar
  2. 2.
    Kalli A, Smith GT, Sweredoski MJ et al (2013) Evaluation and optimization of mass spectrometric settings during data-dependent acquisition mode: focus on LTQ-orbitrap mass analyzers. J Proteome Res 12:3071–3086. Scholar
  3. 3.
    Eng JK, McCormack AL, Yates JR (1994) An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J Am Soc Mass Spectrom 5:976–989. Scholar
  4. 4.
    Griss J, Perez-Riverol Y, Lewis S et al (2016) Recognizing millions of consistently unidentified spectra across hundreds of shotgun proteomics datasets. Nat Methods 13:651–656. Scholar
  5. 5.
    Nielsen ML, Savitski MM, Ra Z (2006) Extent of modifications in human proteome samples and their effect on dynamic range of analysis in shotgun proteomics. Mol Cell Proteomics 5:2384–2391. Scholar
  6. 6.
    Nesvizhskii AI, Roos FF, Grossmann J et al (2006) Dynamic spectrum quality assessment and iterative computational analysis of shotgun proteomic data: toward more efficient identification of post-translational modifications, sequence polymorphisms, and novel peptides. Mol Cell Proteomics 5:652–670. Scholar
  7. 7.
    Chick JM, Kolippakkam D, Nusinow DP et al (2015) A mass-tolerant database search identifies a large proportion of unassigned spectra in shotgun proteomics as modified peptides. Nat Biotechnol 33:743–749. Scholar
  8. 8.
    Tanner S, Shu H, Frank A et al (2005) InsPecT: identification of posttranslationally modified peptides from tandem mass spectra. Anal Chem 77:4626–4639. Scholar
  9. 9.
    Jensen ON (2004) Modification-specific proteomics: characterization of post-translational modifications by mass spectrometry. Curr Opin Chem Biol 8:33–41. Scholar
  10. 10.
    Zhao Y, Jensen ON (2009) Modification-specific proteomics: strategies for characterization of post-translational modifications using enrichment techniques. Proteomics 9:4632–4641. Scholar
  11. 11.
    Müller T, Winter D (2017) Systematic evaluation of protein reduction and alkylation reveals massive unspecific side effects by iodine-containing reagents. Mol Cell Proteomics 16:1173–1187. Scholar
  12. 12.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. Scholar
  13. 13.
    Boersema PJ, Raijmakers R, Lemeer S et al (2009) Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat Protoc 4:484–494. Scholar
  14. 14.
    Shevchenko A, Wilm M, Vorm O et al (1996) Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68:850–858. Scholar
  15. 15.
    Chin Y, Aiken GR, O’Loughlin E (1994) Molecular weight, polydispersity, and spectroscopic properties of aquatic humic substances. Environ Sci 28:1853–1858. Scholar
  16. 16.
    Williams A, Frasca V (2001) Ion-exchange chromatography. Curr Protoc Protein Sci 15:8.2.1–8.2.30Google Scholar
  17. 17.
    Chen J, Lee CS, Shen Y et al (2002) Integration of capillary isoelectric focusing with capillary reversed-phase liquid chromatography for two-dimensional proteomics separation. Electrophoresis 23:3143–3148.<3143::AID-ELPS3143>3.0.CO;2-7CrossRefPubMedGoogle Scholar
  18. 18.
    Nühse TS, Stensballe A, Jensen ON et al (2003) Large-scale analysis of in vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry. Mol Cell Proteomics 2:1234–1243. Scholar
  19. 19.
    Beausoleil SA, Jedrychowski M, Schwartz D et al (2004) Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc Natl Acad Sci 101:12130–12135. Scholar
  20. 20.
    Michel PE, Reymond F, Arnaud IL et al (2003) Protein fractionation in a multicompartment device using Off-GelTM isoelectric focusing. Electrophoresis 24:3–11. Scholar
  21. 21.
    Huber LA, Pfaller K, Vietor I (2003) Organelle proteomics: implications for subcellular fractionation in proteomics. Circ Res 92:962–968. Scholar
  22. 22.
    Rappsilber J, Ishihama Y, Mann M (2003) Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal Chem 75:663–670. Scholar
  23. 23.
    Verheggen K, Raeder H, Berven FS et al (2017) Anatomy and evolution of database search engines-a central component of mass spectrometry based proteomic workflows. Mass Spectrom Rev.
  24. 24.
    Brosch M, Yu L, Hubbard T et al (2009) Accurate and sensitive peptide identification with mascot percolator. J Proteome Res 8:3176–3181. Scholar
  25. 25.
    Bantscheff M, Schirle M, Sweetman G et al (2007) Quantitative mass spectrometry in proteomics: a critical review. Anal Bioanal Chem 389:1017–1031. Scholar
  26. 26.
    Creasy DM, Cottrell JS (2002) Error tolerant searching of uninterpreted tandem mass spectrometry data. Proteomics 2:1426–1434.<1426::AID-PROT1426>3.0.CO;2-5CrossRefPubMedGoogle Scholar
  27. 27.
    Seidler J, Zinn N, Boehm ME et al (2010) De novo sequencing of peptides by MS/MS. Proteomics 10:634–649. Scholar
  28. 28.
    Winter D, Steen H (2011) Optimization of cell lysis and protein digestion protocols for the analysis of HeLa S3 cells by LC-MS/MS. Proteomics 11:4726–4730. Scholar
  29. 29.
    Yu YQ, Gilar M, Lee PJ et al (2003) Enzyme-friendly, mass spectrometry compatible surfactant for in-solution enzymatic digestion of proteins. Anal Chem 75:6023–6028. Scholar
  30. 30.
    Kollipara L, Zahedi RP (2013) Protein carbamylation: in vivo modification or in vitro artefact? Proteomics 13:941–944. Scholar
  31. 31.
    Deutsch EW, Mendoza L, Shteynberg D et al (2015) Trans-Proteomic Pipeline, a standardized data processing pipeline for large-scale reproducible proteomics informatics. Proteomics Clin Appl 9:745–754. Scholar
  32. 32.
    Holman JD, Tabb DL, Mallick P (2014) Employing ProteoWizard to convert raw mass spectrometry data. Curr Protoc Bioinformatics 46:13.24.1–13.24.9.

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute for Biochemistry and Molecular BiologyUniversity of BonnBonnGermany

Personalised recommendations