Phosphoprotein analysis: from proteins to proteomes

Abstract

Characterization of protein modification by phosphorylation is one of the major tasks that have to be accomplished in the post-genomic era. Phosphorylation is a key reversible modification occurring mainly on serine, threonine and tyrosine residues that can regulate enzymatic activity, subcellular localization, complex formation and degradation of proteins. The understanding of the regulatory role played by phosphorylation begins with the discovery and identification of phosphoproteins and then by determining how, where and when these phosphorylation events take place. Because phosphorylation is a dynamic process difficult to quantify, we must at first acquire an inventory of phosphoproteins and characterize their phosphorylation sites. Several experimental strategies can be used to explore the phosphorylation status of proteins from individual moieties to phosphoproteomes. In this review, we will examine and catalogue how proteomics techniques can be used to answer specific questions related to protein phosphorylation. Hence, we will discuss the different methods for enrichment of phospho-proteins and -peptides, and then the various technologies for their identification, quantitation and validation.

Figure 1

Schematic representation of phosphoprotein analysis workflow. The most commonly used methods to resolve, to purify or to enrich phosphoproteins are described in the Protein separation section. Peptides resulting from trypsin digestion of phosphoproteins may be analyzed directly by MS or by Edman degradation. Other approaches may be used for the enrichment of phosphopeptides. These methods are depicted in the Peptide separation section. MALDI and ESI are usually used for identification of phosphoproteins. The two predominant techniques for idenditification of the precise sites of phosphorylation are i) Edman degradation and ii) MS analysis (Neutral loss and Precursor ion). These methods are described in the Identification section. 1D/2D GE: On-dimensional/Two-dimensional gel electrophoresis. Chem. Modif.: Chemical Modification. ESI: Electrospray Ionisation. Ib: Immunoblot. IMAC: Immobilized Metal Affinity Chromatography. Ip-α PY: Immunopurification using a phospho-tyrosine antibody. LC: Liquid Chromatography. MALDI: Matrix-Assisted Laser Desorption Ionization. ProQ: Pro-Q Diamond™. AR: Autoradiography. β-Elemin: β-elimination reaction.