Phosphorylation
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Protein phosphorylation is a ubiquitous and very important post translational modification (PTM), which affects an estimated one third of all proteins. Many critical events involved in cellular response are mediated by phosphorylation and dephosphorylation, such as regulation of enzymatic activity, protein conformational change, protein-protein interaction, and cellular localization.
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Typical quantitative phosphoproteomics workflow
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Phosphorylation events are commonly probed by phosphorylation-specific antibodies. Advances in proteomics technology, including phosphopeptide enrichment, high-accuracy mass spectrometry, and associated bioinformatics now make it possible to analyze entire phosphoproteomes. Despite the sub-stoichiometric nature of phosphorylation it is now possible to identify novel phosphoproteins and, in particular, to localize their phosphorylation sites. Robust quantitative methods can assess the relative change in phosphorylation for several thousand sites in a single experiment. (Macek B. et al, Annu Rev Pharmacol Toxicol, 2009)
To date there have been numerous studies using the MS based phosphoproteomics and our laboratory is one of the pioneers in the field. Our interest focuses on the development and application of technologies used in phosphoproteomics. The adaptation of stable isotope labeling by amino acids in cell culture (SILAC) enables quantitative phosphoproteomic studies in different cellular conditions. Currently, we combine Filter Aided Sample Preparation (FASP), strong-cation ion exchange chromatography (SCX) and titanium dioxide (TiO2) bead chromatography for sample preparation, phosphopeptide separation and enrichment, respectively. High accuracy identification of phosphopeptides with pinpointed phosphorylation sites is routinely performed in the LTQ-Orbitrap Velos mass spectrometer using higher energy collisional dissociation (HCD) which provides high mass accuracy of acquired fragment ions (Nagaraj N et al, J. Proteome Res., 2010).
Recently, we applied high-resolution mass spectrometry–based proteomics to investigate the proteome and phosphoproteome of the human cell cycle on a global scale and quantified 6027 proteins and 20,443 unique phosphorylation sites and their dynamics.(Olsen JV et al, Sci Signal 2010). Further, we developed a generic spike-in SILAC approach to study phosphorylation responses in vivo. Using this method, we identified 15,000 phosphosites and quantitatively compared 10,000 sites in response to insulin treatment in the mouse liver (Monetti M et al, Nat. Methods, 2011).
To enable efficient use of phosphorylation information generated in the lab on various proteomes, we have created a database called the PHOsphorylation SIte Database or PHOSIDA
Recommended protocols
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