Protein phosphorylation on serine, threonine and tyrosine residues is the prevalent post-translational modification and occurs on more than one-third of all cellular proteins. In the human genome there are 518 protein kinases and about 150 protein phosphatases that regulate phosphorylation networks and control biological processes such as proliferation, differentiation and apoptosis. Phosphorylation takes place mainly on serine residues (86.4%), followed by threonine residues (11.8%) and tyrosine residues (1.8%)[4,5]. The most comprehensive phosphorylation databases such as PhosphoSitePlus, PhosphoELM and Phosida contain together more than a 100,000 phosphorylation sites, half of which have been identified on human proteins. Mass spectrometry has evolved as the major contributing technology to these databases in recent years and will be the key technology in the future as there are only a limited number of phospho-site specific antibodies available.
At the Broad Institute, we study proteome-wide phosphorylation events by means of different quantitative mass spectrometry approaches, such as SILAC and iTRAQ labeling. With less than 5% in abundance, phosphorylated peptides represent only a small fraction of all peptides in tryptic whole proteome samples. Therefore further selective enrichment is required:
- Tyrosine phosphorylated peptides can be enriched with phospho-tyrosine specific antibodies and directly analyzed in a single LC-MS/MS run. Starting with 5 mg of total protein usually yields quantitative information of 50-200 phospho-tyrosine sites (Figure 1-red path)[7,8].
- A global analysis of serine-, threonine- and tyrosine-phosphorylation is achieved by fractionation of tryptic peptides using strong cation exchange (SCX) and subsequent enrichment with immobilized metal affinity chromatography (IMAC). Phosphorylated peptides are separated from non-phosphorylated peptides by SCX at a pH of 2.7 as phosphate groups reduce the solution charge and cause phosphorylated peptides to elute earlier in the gradient. The SCX gradient is optimized for collection of peptides bearing only 1 or 2 positive charges in solution. This allows even partitioning of the phosphopeptides over 12 fractions. Further enrichment with an iron(III)-nitriloacetic acid loaded affinity matrix (commonly called IMAC) allows enrichment of phosphorylated peptides to more than 75% purity[10,11]. Using 5 mg total protein input material more than 10,000 phosphorylation sites can be quantified with 12 LC-MS/MS injections (Figure 1-blue path).
Figure 1: Proteome-wide protein phosphorylation analysis workflow (adapted from reference)
Once the data are acquired on a mass spectrometer we use specialized software packages for phosphopeptide identification, quantitation and phosphosite localization. To extract biologically meaningful data, co-regulated phosphopeptides are then analyzed for linear signature motifs to identify kinase-substrate relationships and mapped to protein interaction networks.
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