• Tanveer S Batth and Jesper V Olsen.
• Proteomics Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University ofCopenhagen, Blegdamsvej 3b, 2200, Copenhagen, Denmark.
• Methods Mol. Biol. 2016 Jan 1; 1355: 179-92.

AbstractProtein phosphorylation, a process in which kinases modify serines, threonines, and tyrosines with phosphoryl groups is of major importance in eukaryotic biology. Protein phosphorylation events are key initiators of signaling responses which determine cellular outcomes after environmental and metabolic stimuli, and are thus highly regulated. Therefore, studying the mechanism of regulation by phosphorylation, and pinpointing the exact site of phosphorylation on proteins is of high importance. This protocol describes in detail a phosphoproteomics workflow for ultra-deep coverage by fractionating peptide mixtures based on high pH (basic) reversed-phase chromatography prior to phosphopeptide enrichment and mass spectrometric analysis. Peptides are separated on a C18 reversed-phase column under basic conditions and fractions collected in timed intervals followed by concatenation of the fractions. Each Fraction is subsequently enriched for phosphopeptides using TiO2 followed by LC/MS analysis.

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