Phosphorylation is one of the quickest post-translational modifications that controls downstream signaling pathways regulating processes like cell proliferation, survival, and differentiation. Nowadays, mass spectrometry-based phosphoproteomics is a well-established method providing unprecedented characterization and quantification of phosphorylated proteins and peptides in complex samples. A comprehensive phosphoproteomics workflow consists of protein digestion, phosphopeptide enrichment, sample fractionation, chromatographic separation, and final detection by mass spectrometry. Each of these stages provides its own contribution to overall data variability and should be optimized thoroughly. This review aims to provide an overview of current developments in individual steps of phosphoproteomics workflow with a special focus on applied analytical methods. Recent efforts in all experimental steps are discussed. Finally, possible future development in the field of (phospho)proteomics is proposed.

Department of Chemistry Faculty of Science Masaryk University Kamenice 5 625 00 Brno Czech Republic

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