Relative Quantification of Phosphorylated and Glycosylated Peptides from the Same Sample Using Isobaric Chemical Labelling with a Two-Step Enrichment Strategy

Post-translational modifications (PTMs) are essential for the regulation of all cellular processes. The interplay of various PTMs on a single protein or different proteins comprises a complexity that we are far from understanding in its entirety. Reliable strategies for the enrichment and accurate quantification of PTMs are needed to study as many PTMs on proteins as possible. In this protocol we present a liquid chromatography-tandem mass spectrometry (LC/MS/MS)-based workflow that enables the enrichment and quantification of phosphorylated and N-glycosylated peptides from the same sample. After extraction and digestion of proteins, we label the peptides with stable isotope-coded tandem mass tags (TMTs) and enrich N-glycopeptides and phosphopeptides by using zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) and titanium dioxide (TiO₂) beads, respectively. Labelled and enriched N-glycopeptides and phosphopeptides are further separated by high pH (basic) reversed-phase chromatography and analyzed by LC/MS/MS. The enrichment strategies, together with quantification of two different PTM types from the same sample, allow investigation of the interplay of those two PTMs, which are important for signal transduction inside the cell (phosphorylation), as well as for messaging between cells through decoration of the cellular surface (glycosylation).