Advertisement

Analyzing Recombinant Protein Production in Pichia pastoris with Targeted Proteomics

  • Roslyn M. Bill
  • Annegret Ulke-Lemée
  • Stephanie P. Cartwright
  • Rena Far
  • Jay Kim
  • Justin A. MacDonaldEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1923)

Abstract

New mass spectrometry approaches enable antibody-independent tracking of protein production. Herein, we outline an antibody-independent mass spectrometry method for tracking recombinant protein production in the methylotrophic yeast Pichia pastoris system.

Key words

Multiple reaction monitoring Mass spectrometry MRM-MS Yeast Recombinant protein production ZIPK DAPK3 

Notes

Acknowledgments

This work was supported by grants from the Canadian Institutes of Health Research (CIHR, MOP#133543 to JAM), the European Commission Research Executive Agency (Marie Sklodowska-Curie International Incoming Fellowship to JAM and RMB), and the Biotechnology and Biological Sciences Research Council (International Partnering Award BB/P025927/1 to JAM and RMB).

References

  1. 1.
    Gillette MA, Carr SA (2013) Quantitative analysis of peptides and proteins in biomedicine by targeted mass spectrometry. Nat Methods 10(1):28–34.  https://doi.org/10.1038/nmeth.2309 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Ebhardt HA, Root A, Sander C, Aebersold R (2015) Applications of targeted proteomics in systems biology and translational medicine. Proteomics 15(18):3193–3208.  https://doi.org/10.1002/pmic.201500004 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Chen YT, Chen HW, Wu CF et al (2017) Development of a multiplexed liquid chromatrography multiple-reaction monitoring mass spectrometry (LC-MRM/MS) method for evaluaiton of salivary proteins as oral cancer biomarkers. Mol Cell Proteomics 16(5):799–811.  https://doi.org/10.1074/mcp.M116.064758 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Bill RM (ed) (2012) Recombinant protein production in yeast—methods and protocols. In: Methods in molecular biology. New York: Human PressGoogle Scholar
  5. 5.
    MacLean B, Tomazela DM, Shulman N et al (2010) Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 26(7):966–968.  https://doi.org/10.1093/bioinformatics/btq054 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Pino LK, Searle BC, Bollinger JG et al (2017) The Skyline ecosystem: informatics for quantitative mass spectrometry proteomics. Mass Spectrom Rev.  https://doi.org/10.1002/mas.21540
  7. 7.
    Carlson DA, Franke AS, Weitzel DH et al (2013) Fluorescence linked enzyme chemoproteomic strategy for discovery of a potent and selective DAPK1 and ZIPK inhibitor. ACS Chem Biol 8(12):2715–2723.  https://doi.org/10.1021/cb400407c CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Jung S, Danziger SA, Panchaud A, von Haller P, Aitchison JD, Goodlett DR (2015) Systematic analysis of yeast proteome reveals peptide detectability factors for mass spectrometry. J Proteomics Bioinform 8(10):231–239.  https://doi.org/10.4172/jpb.1000374 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Roslyn M. Bill
    • 1
  • Annegret Ulke-Lemée
    • 2
  • Stephanie P. Cartwright
    • 1
  • Rena Far
    • 2
  • Jay Kim
    • 4
  • Justin A. MacDonald
    • 3
    Email author
  1. 1.School of Life and Health SciencesAston UniversityBirminghamUK
  2. 2.Department of Biochemistry and Molecular BiologyCumming School of Medicine, University of CalgaryCalgaryCanada
  3. 3.Department of Biochemistry and Molecular Biology, Cumming School of MedicineUniversity of CalgaryCalgaryCanada
  4. 4.Department of Biochemistry and Molecular BiologyUniversity of CalgaryCalgaryCanada

Personalised recommendations