Phosphopeptides identification

The study that serves to illustrate a possible bioanalytical stat-egy for characterization of a human phosphoproteome concerns characterization of protein phosphorylation in a human pituitary tissue (25). The analytical methodology involves in-gel IFF for protein separation, IMAC for enrichment of phospho-peptides after digestion, LC-MS/MS and database searches for phosphopeptide identification and localization of the phoshory-lated amino acid residues. 

J.W. Flora, D.C. Muddiman, Selective, sensitive, and rapid phosphopeptide identification of enzymatic digests using ESI-FT-ICR-MS with IRMPD, Anal. Chem., 73 (2002)2305. 

Figure 4 Phosphopeptide identification by MALDI-TOF-MS mapping combined with alkaline phosphatase treatment. (A) The MALDI-TOF-MS spectrum of a proteolytic digest. Phospho-peptides are indicated by peaks shifted by multiples of 80 Da (HPO3 = 80Da) relative to predicted unphosphorylated peptide masses. (B) The disappearance of such peaks upon treatment with a phosphatase confirms their identity as phosphopeptides. (Reprinted with permission from McLachlin DT and Chart BT (2001) Analysis of posphorylated proteins and peptides by mass spectrometry (review). Current Opinion in Chemicai Bioiogy5 5) 591-602 Elsevier.)...

Figure 2.7. Identification ofphosphoproteins by site-specific chemical modification. A. Method of Zhou et al. (2001) involves trypsin digest of complex protein mixture followed by addition of sulfhydryl groups specifically to phosphopeptides. The sulfhydryl group allows capture of the peptide on a bead. Elution of the peptides restores the phosphate and the resulting phosphopeptide is analyzed by tandem mass spectrometry. B. Method of creates a biotin tag in place of the phosphate group. The biotin tag is used for subsequent affinity purification. The purified proteins are proteolyzed and identified by mass spectrometry.

Busman M., Schey K.L., Oatis Jr. J.E., and Knapp D.R.J. (1996), Identification of phosphorylation sites in phosphopeptides by positive and negative mode electrospray ionization tandem mass spectrometry, J. Am. Soc. Mass Spectrom. 7, 243-249. 

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. 

Ellegard, K.H., Gammelgard-Larsen, C., Sorensen, E.S., and Fedosov, S. 1999. Process scale chromatographic isolation, characterization and identification of tryptic bioactive casein phosphopeptides. Int. Dairy J. 9, 639-652. 

Hata, I., Higasiyama, S., and Otani, H. 1998. Identification of a phosphopeptide in bovine asl-casein digest as a factor influencing proliferation and immunoglobulin production in lymphocyte cultures. J. Dairy Res. 65, 569—578. 

Gram FI, Schmitz R, Zuber JF, Baumann G, Identification of phosphopeptide ligands for the Src-homology 2 (SH2) domain of Grb2 by phage display, Eur. J. Biochem., 246(3) 633-637, 1997. 

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. 

Fractionation of proteins by strong cation exchange (SCX) chromatography, followed by IMAC enrichment of phosphopeptides from SCX fractions, led to a comprehensive identification of phosphoproteins of PSD isolated from mouse brain using LC-MS/MS (Trinidad et al. 2006). In this study, phosphorylation site(s) were mapped to 287 proteins from a total of 1,264 unique proteins identified. This translates into a 23% phosphorylation rate, comparable to an expected 33% rate in the general proteome (Johnson et al. 2005). The 287 phosphoproteins were derived from a total of 998 unique phosphorylated peptides, and the phosphorylations were mapped to 723 unique sites. Most of these occurred on serines, to a lesser extent on threonines, and only minimally on tyrosines (Figure 5A). 

While all these strategies rely on specific properties of phosphopeptides in MS analysis, a more global approach involving shotgun protein identification strategies and SEQUEST database searching (Ch. 18.3.2) is demonstrated by the group of Yates for protein complexes and lens tissue proteins [29]. 

M. Adamczyk, J.C. Gebler, J. Wu, Identification of phosphopeptides by chemical modification with an isotopic tag and ion trap MS, Rapid Commun. Mass Spectrom., 16(2002)999. 

A general protocol for mass spectrometric analysis of phosphoproteins is illustrated in Figure 15 various steps of this protocol are cleavage of purified phosphoproteins, isolation and preferential enrichment of phosphopeptides, selective detection of phosphopeptides in the digest, identification of the phosphorylation sites using tandem mass spectrometry, and identification of phosphopeptides/proteins through a database search. 

Several versions of the precursor-ion scan can be found in the literature for selective identification of phosphopeptides. In one version, the precursor-ion scan of m/z 79 is performed in the negative-ion mode.100 In another version, termed phosphotyrosine-specific immonium-ion (PSI) scanning, only phosphotyrosine-containing peptides are detected by monitoring the CID-produced immonium ion of m/z 216.043 in the positive-ion mode.101,102... 

Another ion activation method that is well suited for identification and sequence analysis of phosphopep-tides in the positive and negative polarity modes is infrared multiphoton dissociation (IRMPD).105,112 In this technique, phosphopeptides are irradiated with 10.6 pm photons emitted from a C02 laser. The phosphate group behaves like a chromophore for these photons, allowing evenly distributed cleavages in the peptide chain and more sequence coverage than the CID technique.105... 

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