Phosphorylation is a highly-important modification. Phosphopeptides are low in abundance in the cell and are challenging to observe by mass spectrometry. A good phosphoproteome coverage requires specific enrichment of the phosphopeptides.
Phosphorylation is the most studied, and probably the most important, post-translational protein modification. Due to a low cellular abundance and intrinsic chemical properties, phosphopeptides are notoriously difficult to identify by mass spectrometry.
Experiments that require a high degree of phosphoproteome coverage greatly benefit from adding a selective phosphopeptide enrichment step. The enriched sample can then be analysed as a separate fraction in addition to the unbound, non-phosphorylated fraction. Currently, one of the most frequently-used phosphopeptide enrichment methods utilises titanium dioxide (TiO2) immobilised metal affinity chromatography (IMAC).
Phosphopeptide enrichment with TiO2-IMAC can be performed on SILAC- or isobarically tandem mass tag (TMT)-labelled peptides.