Phosphoproteomics: Detection, Identification and Importance of Protein Phosphorylation

IntroductionReversible protein phosphorylation is one of the most important and well explored posttranslational modifications.It is estimated that 30-50% of the proteins are phosphorylated at some time point (Kalume, Molina, & Pandey, 2003).Phosphorylation is a major regulatory mechanism that controls many basic cellular processes.It may mediate a signal from the plasma membrane to the nucleus using a cascade of proteins, by which to regulate physiological and pathological processes such as cell growth, proliferation, differentiation and apoptosis (Blume-Jensen & Hunter, 2001;Hunter, 2000).Protein phosphorylation may result in alteration in protein-protein interactions, protein intracellular localization, and its activity (Blume-Jensen & Hunter, 2001;Kalume et al., 2003).Approximately 30% of drug discovery programs and R&D investment by the pharmaceutical industry target protein kinases.Knowledge of exactly when and where phosphorylation occurs and the consequences of this modification for the protein of interest can lead to an understanding of the detailed mechanism of the protein action, and ultimately to the discovery of new drug targets.Protein phosphorylation is a fast and reversible process.It is catalyzed by kinases by attaching phosphate groups onto specific amino acids.Opposed to phosphorylation, dephosphorylation removes the phosphate groups from proteins by phosphatases.Dephosphorylation plays important role in balancing the protein phosphorylation status in signaling proteins.About 2-3% of the human genome encodes 518 distinct protein kinases (Manning et al., 2002).Four types of phosphorylation have been described based on the phosphorylation sites: (a) O-phosphorylation (serine, threonine and tyrosine), (b) Nphosphorylation (arginine, histidine and lysine), (c) S-phosphorylation (cysteine) and (d) acylphosphorylation (aspartic acid and glutamic acid) (Reinders & Sickmann, 2005).Currently, analytical methods have mainly been developed for O-phosphorylation, which is due to chemical stability of O-phosphorylation in acidic and in neutral milieu.Therefore, Ophosphorylation is the best studied among various types of phosphorylation (Reinders, 2002).In eukaryotic cells, phosphorylation occurs primarily on serine (pSer), threonine (pThr), and tyrosine (pTyr) residues, that is estimated to be in the ratio of 1800:200:1/pSer: pThr: pTyr (Kersten et al, 2006).As aforementioned, phosphorylation is of importance for cell signaling and drug development.The lack of technologies to study all types of phosphorylation, differences in abundance and high dynamics make it difficult to have a comprehensive cover of all www.intechopen.comIntegrative Proteomics 216 phosphorylation events in cells.This chapter summarizes strategies that have been developed to characterize the phosphoproteome.These strategies include identification of phosphoproteins and phosphopeptides, localization of the exact phosphorylation sites and quantitation of phosphorylation.In addition, the applications of phosphoproteomics in life science are discussed. Phosphorylation Detection of phosphoproteins 2.1.1 Radioactive labeling of proteins with 32 P isotopeRadioactive labeling of proteins with 32 P or 33 P is the oldest, but still one of the most sensitive approaches for detection of phosphorylation.Under the appropriate condition, the phosphoryl groups of 32 P or 33 P are enzymatic added to the proteins.The phosphorylated proteins are then detected by autoradiography.Therefore, radioactive labeling detects all types of phosphorylation, and is not specific to only one type of phosphorylation.The proteins can be labeled with 32 P/ 33 P isotopes in vitro and in vivo.For in vitro labeling, [ -32 P/ 33 P]-ATP is used.It is fast and convenient process, that requires (semi)purified kinase and substrate (Springer, 1991).A kinase phosphorylates its substrate in a defined mixture of the kinase, substrate, buffer, ions, ATP and [ 32 P/ 33 P]-ATP.However, since the enzymatic reaction takes place in vitro, the major disadvantage is that it may not reflect the kinase activity under physiological conditions.This problem was overcome by introduction of the in vivo metabolic labeling (Wyttenbach & Tolkovsky, 2006).[ 32 P/ 33 P]Orthophosphate is used in in vivo labeling as a source of the isotope.The radioactive orthophosphates are incorporated during metabolic processes by kinases in cells.The significant advantage of in vivo labeling is that it provides a more accurate scenario of physiological enzymatic events, and reflects cellular responses as a consequence of treatments.The drawback of in vivo labeling also exists, e.g. it has been reported that in vivo labeling with doses of radioactivity may induce DNA fragmentation, DNA repair processes, subsequently may result in the cell cycle arrest and apoptosis.Another concern is that for in vivo labeling is usually used phosphate-free medium to culture cells.This medium may differ from the medium cells are cultured.Therefore, in vivo labeling experiments are often limited in time to 4-8 hours.The third concern of radioactive labeling (in vitro and in vivo) is that only very small amount of radioactivity will be incorporated in proteins.This requires protocols for thorough removal of non-incorporated radioactivity from phosphorpoteins.The fourth concern of radioactive labeling is safety requirements.As the assays use radioactivity, corresponding safety rules have to be applied.Thus, it is very important to control quantity of the isotope and duration of labeling, take care of safety issues, and to minimize artificially-induced changes in phosphorylation. Phospho-specific antibodiesIn 1981, the first documented phospho-antibody was produced in rabbits immunized with benzonyl phosphonate conjugated to keyhole limpet hemocyanin (KLH) (Ignatoski, 2001).This antibody broadly recognized proteins containing phosphotyrosine.After that, there has been a rapid development in production of the phospho-antibodies.Nowadays, a large amount of phospho specific antibodies targeted to different amino acids (Ser, Thr, Tyr) at distinct sites in proteins have been produced, and widely used in the basic and clinic research (Ignatoski, 2001;Izaguirre, Aguirre, Ji, Aneskievich, & Haimovich, 1999).The www.intechopen.com