Phosphoproteomics Using iTRAQ
The identification of phosphorylation on proteins has become practicable for many laboratories in recent years, largely due to improvements in mass spectrometry (MS) and the development of methods to selectively enrich for phosphorylated peptides and prot
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1. Introduction A mechanistic understanding of signalling pathways requires the identification of kinases and their targets. Identification of the site(s) of phosphorylation on a protein is only part of the information required to understand their function. The dynamic interplay of phosphorylation and dephosphorylation has important consequences for signalling, as does the stoichiometry of phosphorylation. Traditionally, the majority of phosphorylation events were discovered on a protein-by-protein basis. Global, unbiased studies rely on the selective enrichment and/or detection of phosphoproteins or phosphopeptides, their identification, and quantification. Experimental approaches on the protein level typically use in vivo pulse labelling with 32P (1, 2) or phosphoprotein affinity chromatography (3, 4) followed by 2D-PAGE. Alternatively (or in combination (5)), phospho-specific stains (6) or immunoblotting (7) are
N. Dissmeyer and A. Schnittger (eds.), Plant Kinases: Methods and Protocols, Methods in Molecular Biology, vol. 779, DOI 10.1007/978-1-61779-264-9_17, © Springer Science+Business Media, LLC 2011
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A.M.E. Jones and T.S. Nühse
used for the detection of phosphorylated proteins. Phosphoproteins displaying changes in abundance or staining intensity on 2D-gels still need to be identified. For this reason – and because many proteins, notably membrane proteins, cannot be resolved satisfactorily on 2D-gels – new approaches based on peptide separation and mass spectrometric (MS) identification have been developed. Immobilized metal ion affinity chromatography (IMAC) with chelated Fe3+, Ga3+, and other metals has been used in a range of large-scale studies in yeast (8), plant (9), and human (10) cell cultures. Titanium dioxide (TiO2) has been described as a novel affinity matrix for phosphopeptides by Larsen et al. (11) and is beginning to outstrip IMAC in popularity. Although successful large-scale phosphopeptide sequencing is possible without prefractionation (12), in the vast majority of cases the complex peptide mixture is separated with either cation (13) or anion exchange (9). In an effort to “dig” deeper and deeper into the phosphoproteome, subcellular fractionation has uncovered phosphorylation sites of plant chloroplasts (14), plasma membranes (15), and tonoplasts (16). For quantitative proteomics of animal or bacterial samples, stable isotope labelling with amino acids (SILAC) in cell culture has become the predominant method (17). In plant cells, however, SILAC labelling is relatively inefficient (18). Complete metabolic labelling with 15N via inorganic nitrate and/or ammonia is an alternative that is better suited to plants (19). However, 15N-labelling and SILAC, like most alternative methods, are limited to comparisons of two or three samples at the most. 15N-labelling adds a variable mass increment that depends on the size of the peptide; a fact that complicates analysis. The isobaric tag for relative and absolute quantification (iTRAQ) has four (or recently eight) differential mass tags. T
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