Label-Based Mass Spectrometry Approaches for Robust Quantification of the Phosphoproteome and Total Proteome in Toxoplasma gondii
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 wordsSILAC TMT LC-MS/MS Phosphoproteome Proteome TiO2 IMAC High pH reverse phase fractionation Toxoplasma gondii
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).