Identification of Phosphorylation Sites

Once the identity of phosphopeptides in a digestion mixture is revealed, it is imperative to recognize which amino acid residue (Ser, Thr, or Tyr) is phospho-rylated. This task is accomplished by determining the amino acid sequence of peptide fragments in the protein digest after their fractionation into individual components by RP-HPLC, either off- or online with tandem mass spectrometry [34], The tandem MS methods described below have proved to be practical for this purpose. 

Another feasible proposal to increase the visibility of sequence-specific ions is a pseudo-MS procedure that involves collisional activation of the product ions that are formed by the nentral loss of H3PO4 [60], In this way, the principal neutral-loss product ions are converted into a variety of second-generation sequence-specific ions. A complete MS/MS spectrum is the composite of the product ions that are derived from the original precursor as well as from the neutral-loss products. This pseudo-MS/MS spectrum increases the number and abundance of fragment ions. 

Post-source Decay Tandem Mass Spectrometry The potential of PSD has also been exploited to identify phosphopeptides [61,62]. This technique is used for MALDI-generated ions in a reflectron TOP instrument. PSD can also distinguish any serine-threonine phosphorylation from tyrosine phosphorylation. Phosphotyrosine-containing peptides mostly yield the [MH — HP03J+ (loss of 80 Da) ion, whereas phosphoserine- and threonine-containing peptides produce [MH - H3P041+ (loss of 98 Da) and [M - HP03]+ ions. 

Electron-Capture Dissociation and Electron-Transfer Dissociation As 

Electron-transfer dissociation (ETD) is a variation of ECD in which an ion-ion reaction is conducted in a quadrnpole linear ion trap to transfer an electron to the multiply protonated peptide cation [66]. For example, anthracene anions that are generated in a Cl source have been nsed as electton donors. Analogous to the ECD process, the transfer of an electron induces fragmentation in the peptide backbone to form c- and z -type sequence-specific ions that usually retain the 

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Woods, A., Vertommen, D., Neumann, D., Turk, R., Bayliss, J., Schlattner, U., Wallimann, T., Carling, D., and Rider, M. H. 2003b. Identification of phosphorylation sites in AMP-activated protein kinase (AMPK) for upstream AMPK kinases and study of their roles by site-directed mutagenesis. J Biol Chem 278 28434-28442. 

Trinidad, J.C., Specht, C.G., Thalhammer, A., Schoepfer, R. and Burlingame, A.L. (2006) Comprehensive Identification of Phosphorylation Sites in Postsynaptic Density Preparations. Mol. Cell Proteomics 5, 914-922. 

A.P. Hunter, D.E. Games, Chromatographic and MS methods for the identification of phosphorylation sites in phosphoproteins. Rapid Commun. Mass Spectrom., 8 (1994)... 

P. Stralfors, P. Bjorgell, and P. Belfrage, Hormonal regulation of hormone-sensitive lipase in intact adipocytes identification of phosphorylated sites and effects on the phosphorylation by lipolytic hormones and insulin, Proc. Natl. Acad. 

Differential effects of CC chemokines on CC chemokine receptor 5 (CCR5) phosphorylation and identification of phosphorylation sites on the CCR5 carboxyl terminus. J. Biol. Chem. 274, 8875-8885. 

Fragmentations that form c and z ions are important in the identification of phosphorylation sites. 

Greenberg, R., Groves, M.L., and Dower, H.J. (1984) Human P-casein -amino acid sequence and identification of phosphorylation sites. J. Biol. Chem., 259, 5132-5138. 

Takashiba, M. Chiba, Y. Jigami, Y. Identification of phosphorylation sites in V-linked glycans by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal. Chem. 2006, 78, 5208-5213. 

P. Ferranti, A. Malorni, G. Marino, P. Pucci, G.H. Goodwin, G. Manfioletti and V. Gian-cotti, J. Biol. Chem., 1992, 267, 22486-22489, Mass spectrometric analysis of the HMGY protein from Lewis lung carcinoma. Identification of phosphorylation sites. 

Papac, D.I., Oatis, J.E., Crouch, R.K., and Knapp, D.R., Mass spectrometric identification of phosphorylation sites in bleached bovine rhodopsin, Biochemistry, 32, 5930, 1993. 

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