Peptides phosphorylated

This amide, readily formed from an amine and the anhydride or enzymatically using penicillin amidase, is readily cleaved by penicillin acylase (pH 8.1, A -methylpyrrolidone, 65-95% yield). This deprotection procedure works on peptides, phosphorylated peptides, and oligonucleotides, as well as on nonpeptide substrates. The deprotection of racemic phenylacetamides with penicillin acylase can result in enantiomer enrichment of the cleaved amine and the remaining amide. An immobilized form of penicillin G acylase has been developed. ... 

As shown in Fig. 1, the function of E-III is to transfer the phosphoryl group from P-HPr to E-II. P-phosphorylated peptides have been isolated from a number of E-III species and A domains of S. carnosus and S. aureus IIl [27], E. coli II [28],... 

Casein-derived phosphorylated peptides are believed to enhance the bioavailability of calcium from milk and dairy products (Pihlanto and Korhonen, 2003), and a phosphopeptide derived from (3-casein has been shown to increase iron bioavailability (Bouhallab et ah, 2002 Peres, 1999). Other casein-derived peptides have been found to contain antihypertensive activity in rats (Leclerc et ah, 2002 Miguel et ah, 2009). A number of casein fragments demonstrate antibacterial activity (Kilara and Panyam, 2003). 

Ferric ion was immobilized on a Chelating Sepharose Fast Flow column preparatory to the separation of seven enkephalin-related phosphopep-tides.17 Non-phosphorylated peptides flowed through the column, and the bound fraction contained the product. The capacity of the column was found to be 23 pmol/mL by frontal elution analysis. Cupric ion was immobilized on Chelating Superose for the isolation of bovine serum albumin.18 Cupric ion was immobilized on a Pharmacia HiTrap column for the separation of Protein C from prothrombin, a separation that was used to model the subsequent apparently successful separation of Factor IX from prothrombin Factor IX activity of the eluate was, however, not checked.19 Imidazole was used as the displacement agent to recover p-galactosidase from unclarified homogenates injected onto a nickel-loaded IMAC column.20 Pretreatment with nucleases and cleaning in place between injections were required procedures. A sixfold purification factor was observed. 

Waksman G, Kominos D, Robertson SR, Pant N, Baltimore D, Birge RB, Cowbum D, Hanafusa H, Mayer BJ, Overduin M, Resh MD, Rios CB, Silverman L, Kuriyan J. Crystal structure of the phophotyrosine recognition domain SH2 of v-src complexed with tyrosine-phosphorylated peptides. Nature 1992 358 646-653. 

Another means of moving beyond pure protein preparations to high-throughput characterization of proteomes is to enrich for phosphopeptides from complex mixtures by metal affinity chromatography (Andersson and Porath, 1986). Using this method, protein mixtures are proteolyzed to create peptides and phosphorylated peptides are enriched by metal affinity chromatography and subsequently identified by mass spectrometry. This method is limited, however, because in many cases phosphopeptides absorb poorly or nonphosphorylated peptides absorb nonspecifically to the metal affinity resins (Ahn and Resing, 2001). 

A more traditional but still successful method for the detection of a protein phosphorylation is by radioactive labeling with 35P. The labeled protein is digested, the peptides are separated by high-performance liquid chromatography, and the phosphorylated peptides are detected in specific fractions via their radioactivity. The fraction with the phosphorylated peptides can be further analyzed by mass spectrometry (Figeys et al., 1999). 

Fig. 9. Precursor ion scan on an electrospray triple quadrupole mass spectrometer. From all the peptides present of the digested protein only those that are phosphorylated are detected in a precursor ion scan for the phosphate ion (P03, mass 79 Da) in negative ion mode. From the TPX protein three phosphorylated peptides could be detected Ml, AQLTM PSTPTVLK M2, LSETSVNTEQNSK and M3, VQPVQTTPSKDDVSNSATHVC DVK. M, Oxidized methionine C, carbamidomethylated cysteine.

Ding J., Burkhart W., and Kassel D.B. (1994), Identification of phosphorylated peptides from complex mixtures using negative-ion orifice-potential stepping and capillary liquid chromatography/electrospray ionization mass spectrometry, Rapid Commun. Mass Spectrom. 8 94—98. 

IMAP (immobilized metal ion affinity-based fluorescence polarization) Molecular Devices Trivalent metal ion containing beads bind to phosphorylated peptide producing FP signal change... 

POLARIS Chromagen Reactive biotinylated linker covalently binds phosphorylated peptide, producing FP signal change after avidin binding... 

The protein kinase mechanism uses aspartate as a general base, weakening the hydrogen—oxygen bond of the hydroxyl-containing residues. This allows the hydroxyl nucleophile of the substrate to attack the 7-phosphate of ATP, producing a phosphorylated peptide product and ADP (Scheme 2). 

Yu F, Morin X, Cai Y, Yang X, Chia W (2000) Analysis of partner of inscuteable, a novel player of Drosophila asymmetric divisions, reveals two distinct steps in inscuteable apical localization. Cell 100 399 09 Zarling AL, Ficarro SB, White FM, Shabanowitz J, Hunt DF, Engelhard VH (2000) Phosphorylated peptides are naturally processed and presented by major histocompatibihty complex class I molecules in vivo. J Exp Med 192 1755-1762... 

In comparison to the level of cellular serine or threonine phosphorylation, protein tyrosine phosphorylation occurs at quite low levels in normal cells but dramatically increases upon oncogenic transformation or stimulation. Since the first discovery in 1978 that the transforming protein from Rous sarcoma virus (pp60vsrc) exhibited intrinsic kinase activity/5 protein kinase activity has also been shown to be inherent to other growth factor receptors such as epidermal growth factor receptor and the insulin receptor,[6 91 and to involve autophosphorylation processes. The diverse biochemical activity exhibited by protein tyrosine phosphorylation has stimulated the development of chemical methods for the preparation of phosphorylated peptides for use as substrates in elucidating the biochemical and physiological activity of phosphorylated site(s). 

Since the first chemical studies into the preparation of simple phosphoserine-peptides in 1959, several synthetic approaches have been investigated for the solution or solid-phase synthesis of phosphorylated peptides containing phosphoserine Ser(P), phosphothreonine Thr(P), and phosphotyrosine Tyr(P) residues. This section outlines two main methods for the synthesis of phosphorylated peptides which involve (1) the specific incorporation of protected phosphoamino acids in Boc or Fmoc peptide synthesis or (2) the post-assembly phosphorylation of Ser-, Thr- or Tyr-containing peptide-resins. For comprehensive recent reviews on the synthesis of phosphorylated peptides, the reader is directed to refs[141142l... 

Inamori, K., Kyo, M., Nishiya, Y, Inoue, Y., Sonoda, T., Kinoshita, E., Koike, T. and Katayama, Y. (2005) Detection and quantification of on-chip phosphorylated peptides by surface plasmon resonance imaging techniques... 

The crystal structures of the SH2 domains of Src tyrosine kinase and Lck tyrosine kinase in complex with Tyr phosphorylated peptides have enabled important insight to be obtained into recognition of the phosphotyrosine residue and the neighboring amino acids in class lA of SH2 domains. The phosphate residue is boimd in a deep pocket of the SH2 domain, at the end of which an invariant Arg residue (Arg PB5) is located which contacts the negatively charged phosphate by a two-pronged interaction. It can be estimated that a phosphoserine or phosphothreonine residue would be too short to enter into a similar interaction with the Arg residue. 

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