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