Evaluation of phosphopeptide enrichment strategies for quantitative TMT analysis of complex network dynamics in cancer-associated cell signalling

• An overview is presented to highlight critical steps of quantitative phosphoproteomics.
• Mass spectrometry limitations in phosphpo-peptides identification.
• A comparison of two experimental workflows (SCX and antibody based) to enrich phosphopeptides shows that the two methods detect different phosphorylation sites.

Defining alterations in signalling pathways in normal and malignant cells is becoming a major field in proteomics. A number of different approaches have been established to isolate, identify and quantify phosphorylated proteins and peptides. In the current report, a comparison between SCX prefractionation versus an antibody based approach, both coupled to TiO2 enrichment and applied to TMT labelled cellular lysates, is described. The antibody strategy was more complete for enriching phosphopeptides and allowed the identification of a large set of proteins known to be phosphorylated (715 protein groups) with a minimum number of not previously known phosphorylated proteins (2).

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