Advertisement

Online LC-FAIMS-MS/MS for the Analysis of Phosphorylation in Proteins

  • Hongyan Zhao
  • Andrew J. Creese
  • Helen J. Cooper
Part of the Methods in Molecular Biology book series (MIMB, volume 1355)

Abstract

High-field asymmetric waveform ion mobility spectrometry (FAIMS) is a gas-phase separation technique which, when coupled with liquid chromatography tandem mass spectrometry, offers benefits for analysis of complex proteomics samples such as those encountered in phosphoproteomics experiments. Results from LC-FAIMS-MS/MS are typically complementary, in terms of proteome coverage and isomer identification, to those obtained by use of solution-phase separation methods, such as prefractionation with strong cation-exchange chromatography. Here, we describe the protocol for large-scale phosphorylation analysis by LC-FAIMS-MS/MS.

Key words

Phosphorylation Ion mobility spectrometry FAIMS 

Notes

Acknowledgments

The Advion Triversa Nanomate, Dionex LC and Thermo Fisher Velos Orbitrap mass spectrometer used in this research were funded through the Birmingham Science City Translational Medicine: Experimental Medicine Network of Excellence Project, with support from Advantage West Midlands (AWM). The Chinese Scholarship Council is gratefully acknowledged for funding. HJC is an EPSRC Established Career Fellow (EP/L023490/1).

References

  1. 1.
    Swearingen KE, Moritz RL (2012) High-field asymmetric waveform ion mobility spectrometry for mass spectrometry-based proteomics. Expert Rev Proteomics 9:505–517CrossRefPubMedGoogle Scholar
  2. 2.
    Guevremont R (2004) High-field asymmetric waveform ion mobility spectrometry: a new tool for mass spectrometry. J Chromatogr A 1058:3–19CrossRefPubMedGoogle Scholar
  3. 3.
    Venne K, Bonneil E, Eng K et al (2005) Improvement in peptide detection for proteomics analyses using nanoLC-MS and high-field asymmetry waveform ion mobility mass spectrometry. Anal Chem 77:2176–2186CrossRefPubMedGoogle Scholar
  4. 4.
    Saba J, Bonneil E, Pomies C et al (2009) Enhanced sensitivity in proteomics experiments using FAIMS coupled with a hybrid linear Ion trap/orbitrap mass spectrometer. J Proteome Res 8:3355–3366CrossRefPubMedGoogle Scholar
  5. 5.
    Bridon GL, Bonneil E, Muratore-Schroeder T et al (2011) Improvement of phosphoproteome analyses using FAIMS and decision tree fragmentation. Application to the insulin signaling pathway in Drosophila melanogaster S2 cells. J Proteome Res 11:927–940CrossRefPubMedGoogle Scholar
  6. 6.
    Creese AJ, Smart J, Cooper HJ (2013) Large-scale analysis of peptide sequence variants: the case for high field asymmetric waveform ion mobility spectrometry. Anal Chem 85:4836–4843PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Villén J, Gygi SP (2008) The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry. Nat Protoc 3:1630–1638PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Creese AJ, Shimwell NJ, Larkins KP et al (2013) Probing the complementarity of FAIMS and strong cation exchange chromatography in shotgun proteomics. J Am Soc Mass Spectrom 24:431–443PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Swearingen KE, Hoopmann MR, Johnson RS et al. (2012) Nanospray FAIMS fractionation provides significant increases in proteome coverage of unfractionated complex protein digests. Mol Cell Proteomics 11 doi:10.1074/m111.014985Google Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Hongyan Zhao
    • 1
  • Andrew J. Creese
    • 1
  • Helen J. Cooper
    • 1
  1. 1.School of BiosciencesUniversity of BirminghamEdgbaston, BirminghamUK

Personalised recommendations