Phosphopeptides, analysis

R. S. Annan and S. A. Carr. Phosphopeptide Analysis by Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometry. Anal. Chem., 68(1996) 3413-3421. 

Annan R.S. and Carr S. A. (1996), Phosphopeptide analysis by matrix-assisted laser desorption time-of-flight mass spectrometry, Anal. Chem. 68(19), 3413-3421. 

Cao P. and Stults J.T. (1999), Phosphopeptide analysis by on-line immobilized metal-ion affinity chromatography-capillary electrophoresis-electrospray ionization mass spectrometry, J. Chromatogr. 853(1-2), 225-235. 

Dunkley PR, Robinson PJ (1986) Depolarization-dependent protein phosphorylation in synapto-somes mechanisms and significance. Prog Brain Res 69 273-93 Dunkley PR, Baker CM, Robinson PJ (1986) Depolarization-dependent protein phosphorylation in rat cortical synaptosomes characterization of active protein kinases by phosphopeptide analysis of substrates. J Neurochem 46 1692-1703... 

To address some of the issues associated with CID of phos-phopeptides, new strategies for ion activation/dissociation have been introduced recently in the context of phosphoproteomics. In particular, electron transfer dissociation (ETD) is emerging as a promising new strategy for MS/MS-based phosphopeptide analysis (22, 23). 

It should also be noted that besides the conventional production scanning, specialized MS/MS functions have been adopted for phosphopeptide analysis (9). These include precursor ion scanning for monitoring the precursors of the product ion POs" m/z 79) in the negative mode, or precursors of the phosphotyrosine-specihc immonium ion at m/z 216.043 in the positive mode. 

Nnmerons other applications have recently been reported, taking advantages of the technologies here described for phosphopeptide analysis and more general proteomics approaches (Ch. 18). 

It has been shown [18] that when high cone-voltages were used in conjunction with negative-ion electrospray, phosphopeptides produce diagnostic ions at m/z 63 (P02 ) and m/z 79 (P03 ). LC-MS analysis of a trypsin digest of bovine... 

Weng, Q.-P., Kozlowski, M., Belham, C., Zhang, A., Comb, M.J., and Avruch, J. (1998). Regulation of the p70 S6 kinase by phosphorylation in vivo Analysis using site-specific anti-phosphopeptide antibodies. J. Biol. Chem. 273, 16621-16629. 

Identification of associated protein kinases Based on phosphopeptide analyses it became clear that associated kinases modify principally serine and threonine residues. Moreover, the analysis of putative phosphorylation-speciflc consensus sequences of p53, c-Jun, p27, ICSBP and I/cBa revealed that the protein kinase CK2 and a member of the protein kinase C family might be associated with the CSN. 

Schlosser, A., Vanselow, J.T. and Kramer, A. (2005) Mapping of phosphorylation sites by a multi-protease approach with specific phosphopeptide enrichment and NanoLC-MS/MS analysis. Analytical Chemistry, 77, 5243-5250. 

Competition binding analysis of a variety of phosphopeptides derived from different Cdc4 substrates revealed a range of affinities. For example. Sic IP = 24 pM, FarlP = 2.7 pM, Gcn4 = 0.8 pM. Importantly, multi-site phosphorylation dependent interactions with Cdc4 can be recapitulated with synthetic concatamers of low affinity CPD sites based on either the regions T45 or S76 sites in Sicl. 

The last four studies performed by the PSRG have been selected as examples of comparative peptide analysis for this section on the analytics of synthetic peptides. These include techniques for the synthesis and production of cyclic peptides and phosphopeptides and include studies on the potential problems of racemization and deamidation 10 1516191 These studies were selected for the following reasons first, the cyclized and phosphorylated forms of peptides are synthesized and used by many scientists secondly, these studies also address potential problems that can arise using these techniques and thirdly, these studies are provided as references in which readers might find useful methods for each type of analysis. 

Tholey, A., Ionic liquid matrices with phosphoric acid as matrix additive for the facilitated analysis of phosphopeptides by matrix-assisted laser desorption/ ionization mass spectrometry. Rapid Commun. Mass Spec., 20,1761, 2006. 

FIGURE 7.44 MALDI mass spectra from on-CD analysis of the phosphopeptide-con-taining sample, (a) Peptide mass spectrum after concentration/desalting. A database search showed that the sample contained bovine protein disulfide isomerase. (b) Phosphopeptide enrichment by IMAC. Two phosphopeptides at m/z 964 and 2027 were recognized (c) Phosphopeptide enrichment followed by enzymatic on-column dephosphorylation using alkaline phosphatase. Two phosphopeptides at m/z 884 and 1947 were recognized from the mass shifts of 80 Da from (B), which were resulted from dephosphorylation [794]. Reprinted with permission from the American Chemical Society. 

Jost, R. 1993. Functional characteristics of dairy proteins. Trends Food Set Technol. 4, 283-288. Juilleart, M.A., Berrocal, R., Chanton, S., Scherz, J.-C., and Jost, R. 1989. Tryptic phosphopeptides from whole casein. 1. Preparation and analysis by fast protein liquid chromatography. J. Dairy Res. 56, 603-611. 

K20. Kuiper, G. G., and Brinkmann, A. O., Phosphotryptic peptide analysis of the human androgen receptor Detection of a hormone-induced phosphopeptide. Biochemistry 34,1851-1857 (1995). 

Phosphotyrosine phosphatase activity was measured at 30°C in a total volume of 100 /x.L containing 24 mM imidazole (pH 7.2), 1 mM EDTA, 1 mM dithiothreitol, 100 jug of bovine serum albumin, 50 fiM dansyl phosphopeptide, and extracts containing enzyme activity. After 15 minutes, the reaction was terminated by the addition of 20 /x.L of 30% (w/v) trichloroacetic acid. The mixture was centrifuged before injection for HPLC analysis. The reaction was linear with respect to both time and enzyme concentration up to at least 30% substrate conversion. 

A comprehensive analysis of protein phosphorylation includes the identification of the phosphoprotein or phosphopeptide, the localization of the modified amino acid, and if possible, the quantification of phosphorylation. 

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