Advertisement

Label-Based Mass Spectrometry Approaches for Robust Quantification of the Phosphoproteome and Total Proteome in Toxoplasma gondii

  • Malgorzata BroncelEmail author
  • Moritz TreeckEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2071)

Abstract

Protein phosphorylation plays a key role in regulating biological processes. Over 30% of the proteome is phosphorylated in most organisms and unraveling the function of the kinases that mediate these phosphorylation events requires the technology to reliably measure phosphorylation on proteins under various conditions. Advances in mass-spectrometry instrumentation, sample preparation, and labeling technologies now offer a range of quantification methods, each with their advantages and disadvantages. Here we describe in detail two different quantification methods, that is, stable isotope labeling by amino acids in cell culture and tandem mass tagging, combined with phosphopeptide enrichment strategies to measure the phosphoproteome of Toxoplasma parasites.

Key words

SILAC TMT LC-MS/MS Phosphoproteome Proteome TiO2 IMAC High pH reverse phase fractionation Toxoplasma gondii 

Notes

Acknowledgments

This work was supported by funding to M.T. from the Francis Crick Institute (https://www.crick.ac.uk/), which receives its core funding from Cancer Research UK (FC001189; https://www.cancerresearchuk.org), the UK Medical Research Council (FC001189; https://www.mrc.ac.uk/), and the Wellcome Trust (FC001189; https://wellcome.ac.uk/). M.B. and M.T. are also supported by a grant from the NIH (R01AI123457).

References

  1. 1.
    Treeck M, Sanders JL, Elias JE, Boothroyd JC (2011) The phosphoproteomes of plasmodium falciparum and Toxoplasma gondii reveal unusual adaptations within and beyond the parasites' boundaries. Cell Host Microbe 10:410–419CrossRefGoogle Scholar
  2. 2.
    Treeck M, Sanders JL, Gaji RY et al (2014) The calcium-dependent protein kinase 3 of Toxoplasma influences basal calcium levels and functions beyond egress as revealed by quantitative phosphoproteome analysis. PLoS Pathog 10:e1004197CrossRefGoogle Scholar
  3. 3.
    McCoy JM, Stewart RJ, Uboldi AD et al (2017) A forward genetic screen identifies a negative regulator of rapid Ca2+−dependent cell egress (MS1) in the intracellular parasite Toxoplasma gondii. J Biol Chem 292:7662–7674CrossRefGoogle Scholar
  4. 4.
    Nebl T, Prieto JH, Kapp E et al (2011) Quantitative in vivo analyses reveal calcium-dependent phosphorylation sites and identifies a novel component of the Toxoplasma invasion motor complex. PLoS Pathog 7:e1002222CrossRefGoogle Scholar
  5. 5.
    Jia Y, Marq JB, Bisio H et al (2017) Crosstalk between PKA and PKG controls pH-dependent host cell egress of Toxoplasma gondii. EMBO J 36:3250–3267CrossRefGoogle Scholar
  6. 6.
    Riley NM, Coon JJ (2016) Phosphoproteomics in the age of rapid and deep proteome profiling. Anal Chem 88:74–94CrossRefGoogle Scholar
  7. 7.
    Ong SE, Blagoev B, Kratchmarova I et al (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1:376–386CrossRefGoogle Scholar
  8. 8.
    Thompson A, Schäfer J, Kuhn K et al (2003) Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal Chem 75:1895–1904CrossRefGoogle Scholar
  9. 9.
    McAlister GC, Nusinow DP, Jedrychowski MP et al (2014) MultiNotch MS3 enables accurate, sensitive, and multiplexed detection of differential expression across cancer cell line proteomes. Anal Chem 86:7150–7158CrossRefGoogle Scholar
  10. 10.
    Tyanova S, Temu T, Cox J (2016) The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat Protoc 11:2301–2319CrossRefGoogle Scholar
  11. 11.
    Tyanova S, Temu T, Sinitcyn P et al (2016) The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat Methods 13:731–740CrossRefGoogle Scholar
  12. 12.
  13. 13.
    UniProt C (2015) UniProt: a hub for protein information. Nucleic Acids Res 43:D204–D212CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.The Francis Crick InstituteLondonUK

Personalised recommendations