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PubMed1999

A two-dimensional peptide gel electrophoresis system for phosphopeptide mapping and amino acid sequencing.

A novel two-dimensional electrophoresis system to be carried out on polyacrylamide gels under nondenaturing conditions was developed to efficiently fractionate the peptides resulting from endoproteinase digestion of 32P-labeled proteins. In particular, nondenaturing gel isoelectric focusing was combined with alkaline 40% polyacrylamide gel electrophoresis to generate phosphopeptide maps with high reproducibility, thus allowing both protein fingerprinting and comparative analysis of different samples. The potential application of this method for subsequent amino acid sequencing of the isolated phosphopeptides was further demonstrated by successful manual and automated Edman sequencing. Taken together these data show that such a simple and precise approach is suitable for both analytical and preparative aims.

Amino Acid Sequence↗

PubMed1999

Iron(III)-immobilized metal ion affinity chromatography and mass spectrometry for the purification and characterization of synthetic phosphopeptides.

A method based upon immobilized metal ion affinity chromatography (IMAC) is described for purification of phosphopeptides from the crude preparations of solid-phase peptide synthesis step. Affinity chromatography consists of iron(III) immobilized on iminodiacetate-agarose gel. The method was applied for purification of seven synthetic enkephalin-related phosphorylated peptides. The effectiveness of the method was evaluated by analyzing the IMAC-retained and -nonretained components using reversed-phase (RP) high-performance liquid chromatography (HPLC) and an on-line combination of RP-HPLC and electrospray ionization mass spectrometry. The UV and total ion current chromatograms demonstrated that the phosphopeptides were effectively separated and purified.

Chromatography, Affinity↗

PubMed1999

Compounds capable of generating singlet oxygen represent a source of artifactual data in scintillation proximity assays measuring phosphopeptide binding to SH2 domains.

We developed scintillation proximity assays (SPA) to discover compounds which inhibit phosphopeptide binding to Src homology 2 (SH2) domain proteins Grb2 and Syk. An assay artifact is reported here as a caveat to others. The SPA used an antibody to couple glutathione-S-transferase SH2 domain fusion proteins to scintillant beads coated with protein A. A pyrazoloquinolone and indolocarbazole inhibited [3H]phosphopeptide binding in both assays. Their potency in the SPA increased with prolonged (2 to 24 h) assay exposure to ambient light. They were inactive in absence of light and in an alternate binding assay. Both compounds absorbed visible light and generated singlet oxygen based on 2-methylfuran-trapping experiments. Their inhibitory activity was suppressed by the singlet oxygen scavengers sodium azide and dithiothreitol. The results suggest that compounds, not previously considered photosensitizers, generated enough singlet oxygen to damage oxidant-sensitive SPA components. Therefore, this SPA should be protected from light to minimize occurrence of false positives.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Identification of signalling components in tyrosine kinase cascades using phosphopeptide affinity chromatography.

Various methods are now available to identify the molecular partners of the component of a signal transduction pathway. Some interactions, however, can be technically difficult to detect because they depend upon transient tyrosine phosphorylation. Here, we present a simple affinity chromatography approach based on synthetic phosphopeptides to purify potential partners of phosphotyrosine-containing proteins. With this approach, we confirm the previously characterized interaction between Grb2 and the EGF receptor, and we identify novel partners of the IGF-1 receptor and of the JAK proteins. Methenyltetrahydrofolate synthetase (MTHFS) was identified as a potential mediator of IGF-1R dependent transformation. P85alpha, the regulatory subunit of PI3 kinase, was identified as one of four proteins recruited by a phosphopeptide mimicking a motif conserved in all JAK family members.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Phosphopeptide analysis.

In this chapter we review the various methods available to the experimenter to analyse phosphorylated peptides. The initial steps in such an analysis involve the isolation of the phosphopeptides for analysis, and we outline the various current methods such as immobilised metal affinity chromatography, anti-phosphoamino acid antibodies as well as HPLC (High Pressure Liquid Chromatography) and TLC (Thin Layer Chromatography). The isolated peptides can be analysed by chemical modification followed by Edman degradation or by mass spectrometry (MS). We focus on MS methods and give examples illustrating the selective detection and sequencing of phosphopeptides.

Amino Acid Sequence↗

PubMed2004

An equilibrium thermodynamic model of the sequestration of calcium phosphate by casein phosphopeptides.

Sequestration of calcium phosphate by caseins occurs in the Golgi region of mammary secretory cells during lactation, where it helps to prevent calcification of the gland and to deliver high concentrations of calcium and phosphate to the neonate in the form of milk. Calcium phosphate nanoclusters are formed when a core of amorphous calcium phosphate is sequestered within a shell of casein or casein phosphopeptides. The nanoclusters can form spontaneously from a supersaturated solution or by dispersion of a precipitate of calcium phosphate, demonstrating that they are thermodynamically stable complexes. The average size and chemical composition of the complexes are largely independent of the solution conditions (pH, temperature, peptide concentration, salt composition and rate of reaction) under which they form. Larger, metastable, colloidal particles can form if there is not enough of the phosphopeptide to sequester all the calcium phosphate, or, transiently, if the salt and peptide solutions are mixed together without sufficient care. A thermodynamic model of the sequestration process is presented which makes use of an invariant ion activity product observed in nanocluster-containing solutions. In any given solution that has thermodynamic stability, the extent of the sequestration reaction can be calculated from the empirical formula of the nanoclusters using the criterion that the solution should have the equilibrium value of the invariant ion activity product. Other members of the paralogous group of secretory calcium-binding phosphoproteins to which caseins belong may also be able to sequester calcium phosphate in biological fluids such as saliva and in the extracellular matrix of mineralizing tissues.

Animals↗

PubMed1992

A potential pitfall in protein kinase assay: phosphocellulose paper as an unreliable adsorbent of produced phosphopeptides.

The ability of phosphocellulose paper to retain phosphorylated peptides containing basic amino acid residues was investigated. Some peptide substrates that are commonly used for three different protein kinases were tested. The adsorption onto phosphocellulose paper was strongly dependent on the amino acid composition of the peptides. None of the phosphopeptides studied was adsorbed completely, the amount bound varied from 7 to 93%. Phosphopeptides containing two basic amino acids each differed remarkably in the degree of binding to the phosphocellulose paper (40% RRASVA, 60% FRRLSI, and 80% HRASV was bound). The results presented here indicate that data from phosphorylation experiments obtained so far for different peptides using the phosphocellulose paper method should be judged with caution.

Adsorption↗

PubMed1988

Partially dephosphorylated phosphopeptide AcSer(P)-Ser(P)-Ser(P) is an excellent substrate for casein kinase-2.

The synthetic phosphopeptide AcSer(P)-Ser(P)-Ser(P), reproducing a recurrent feature of casein and other phosphoproteins, once partially dephosphorylated by acid phosphatase, serves as an efficient substrate for casein kinase-2. Previous dephosphorylation beyond 30% hinders subsequent phosphorylation and the entirely dephosphorylated peptide is not a substrate at all. The kinetic constants of the partially dephosphorylated phosphopeptide are much more favourable than those of the synthetic peptides SEEEAA, SSEE and SEE, the latter one being totally inert. Optimal phosphorylation occurs at pH values that ensure complete ionization of the phosphoseryl side chains. These data provide incontrovertible demonstration that phosphoserine can replace carboxylic amino acids as specificity determinant for CK-2, being more effective than glutamic acid itself.

Amino Acid Sequence↗

PubMed1990

Synthetic phosphopeptides are substrates for casein kinase II.

Casein kinase II is a protein serine/threonine kinase that exhibits a preference for acidic substrates. Previous studies have demonstrated that a glutamic acid 3 amino acids C-terminal (+3) to a serine or threonine is required for phosphorylation. To examine the ability of phosphoserine and phosphothreonine residues to serve as specificity determinants for casein kinase II, phosphopeptides containing either of these phosphoamino acids in the +3 position were synthesized and tested as substrates. Phosphopeptides containing phosphoserine in the +3 position were readily phosphorylated. In contrast, corresponding phosphothreonine-containing peptides were very poorly phosphorylated. These results imply that prior phosphorylation of substrate proteins on serine, but not threonine residues, may II.

Amino Acid Sequence↗

PubMed1992

Mapping and sequence-specific identification of phosphopeptides in unfractionated protein digest mixtures by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

We have demonstrated a procedure for the rapid (minutes), sensitive (less than pmol), and sequence-specific identification of phosphopeptides in unfractionated digests of phosphoproteins using matrix-assisted UV laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry. The mass-dependent identification of one specific 13-residue phosphopeptide (S105-K117), observed among the 153 possible trypsin digest fragments of human beta-casein (211 residues), was confirmed by amino acid sequence analysis of the 33P-labeled peptide after isolation by reverse-phase HPLC. MALDI-TOF was also used to monitor the rate and extent to which an 18-residue N-terminal beta-casein peptide (R1-K18) was phosphorylated in vitro. These results demonstrate that MALDI-TOF may be used (i) to facilitate the identification of sequence-specific sites of protein phosphorylation and dephosphorylation, (ii) to monitor protein and peptide phosphorylation and dephosphorylation reaction rates, even in complex unfractionated mixtures, (iii) to determine the minimum primary structure necessary for the phosphorylation of specific protein surface domains, and (iv) to evaluate the effects of intact protein phosphorylation and dephosphorylation on susceptibility to subsequent proteolytic events.

Amino Acid Sequence↗

PubMed1993

SH2 domains recognize specific phosphopeptide sequences.

A phosphopeptide library was used to determine the sequence specificity of the peptide-binding sites of SH2 domains. One group of SH2 domains (Src, Fyn, Lck, Fgr, Abl, Crk, and Nck) preferred sequences with the general motif pTyr-hydrophilic-hydrophilic-Ile/Pro while another group (SH2 domains of p85, phospholipase C-gamma, and SHPTP2) selected the general motif pTyr-hydrophobic-X-hydrophobic. Individual members of these groups selected unique sequences, except the Src subfamily (Src, Fyn, Lck, and Fgr), which all selected the sequence pTyr-Glu-Glu-Ile. The variability in SH2 domain sequences at likely sites of contact provides a structural basis for the phosphopeptide selectivity of these families. Possible in vivo binding sites of the SH2 domains are discussed.

Amino Acid Sequence↗

PubMed1990

Analysis of myosin light chain phosphopeptides in phorbol dibutyrate-contracted artery.

The incorporation of [32P]phosphate into the 20 kDa myosin light chain of phorbol dibutyrate-contracted artery was slightly increased as compared to that of resting muscle. Addition of K+ to the 1-h phorbol dibutyrate-contracted artery immediately doubled the force and greatly increased the light chain phosphorylation. Two-dimensional phosphopeptide mapping of light chain from phorbol dibutyrate-contracted muscle showed distinct peptides phosphorylated on serine residues by myosin light chain kinase and protein kinase C. In addition, the peptide phosphorylated on threonine residue by protein kinase C was revealed for the first time in intact muscle. Upon addition of K+, the distribution of phosphopeptides shifted toward the myosin light chain kinase catalyzed pattern.

Animals↗

PubMed2005

Reduction of non-specific binding in Ga(III) immobilized metal affinity chromatography for phosphopeptides by using endoproteinase glu-C as the digestive enzyme.

The selectivity of immobilized metal affinity chromatography (IMAC) systems for the purification of phosphopeptides is poor. This is particularly a problem with tryptic digests of proteins where a large number of acidic peptides are produced that also bind during IMAC. The hypothesis examined in this work was that the selectivity of IMAC columns for phosphopeptides could be increased by using endoproteinase glu-C (glu-C) for protein digestion. Glu-C cleaves proteins at acidic residues and should reduce the number of acidic residues in peptides. This method was successfully applied to a mixture of model proteins and bovine milk. The percentage of phosphorylated peptides selected from proteolytic digests of the milk sample was increased from 40% with trypsin to 70% with glu-C. Additionally, this method was coupled with stable isotope coding methods to quantitatively compare the concentration of phosphoproteins between samples.

Amino Acid Sequence↗

PubMed2003

Casein-derived bioactive phosphopeptides: role of phosphorylation and primary structure in promoting calcium uptake by HT-29 tumor cells.

Casein phosphopeptides beta-CN(1-25)4P and alpha(s1)-CN(59-79)5P, from beta- and alpha(s1)-casein, respectively, both carrying the characteristic 'acidic motif' Ser(P)-Ser(P)-Ser(P)-Glu-Glu, were chemically synthesized and administered to HT-29 cells differentiated in culture, which are a used model of intestinal epithelium for absorption studies. Both casein phosphopeptides caused an increase of [Ca(2+)](i) due to influx of extracellular Ca(2+). The response was quantitatively higher with beta-CN(1-25)4P than alpha(s1)-CN(59-79)5P. The synthetic peptide corresponding to the 'acidic motif' was ineffective and the dephosphorylated form of beta-CN(1-25)4P almost inactive. The lack of the N-terminally located five amino acids, or sequence modifications within the N-terminal segment of beta-CN(1-25)4P, caused a total loss of activity, whereas the lack of the C-terminal segment preserved activity. In conclusion, the influx of calcium into HT-29 cells caused by beta-CN(1-25)4P appears to depend on the phosphorylated 'acidic motif' and the preceding N-terminal region.

Amino Acid Motifs↗

PubMed1991

Immobilized Fe3+ affinity chromatographic isolation of phosphopeptides.

Immobilized Fe3+ affinity chromatography is suggested as a means of concentrating phosphopeptides that are present in too low a proportion in a complex mixture to be purified by a single-step method. A high-performance liquid chromatographic system and a chelating Superose HR 10/2 column were used. The chromatographic conditions were optimized using a tryptic hydrolysate of whole casein. The unbound fractions did not contain any phosphorylated peptide. All caseinophosphopeptides were retained. Only four other strongly basic peptides were also retained. The quantitative accuracy of the method was evaluated. This method allowed the isolation of phosphopeptides in gastric effluents of calves fed with milk.

Animals↗

PubMed1998

Identification and characterization of receptors for riboflavin carrier protein in the chicken oocyte. Role of the phosphopeptide in mediating receptor interaction.

Riboflavin carrier protein (RCP) is a phosphoglycoprotein found in the egg and the serum of laying birds and other animals. We have investigated the binding of chicken RCP (cRCP) to membranes prepared from the whole chicken oocytes. RCP binding had an absolute requirement for calcium, with an affinity (Kd 10(-8) M) high enough to be physiologically relevant. Ligand blotting experiments using labeled RCP and vitellogenin, with proteins solubilized from oocyte membranes, indicated that RCP and vitellogenin bound specifically to three proteins of Mr 380, 260 and 110 kDa. Vitellogenin also bound to proteins of Mr 515 kDa and 97 kDa, similar in size to those identified by receptor associated protein of RAP. Reduced and carboxyamidated RCP inhibited the binding of 125I-labeled RCP to chicken oocyte membranes, but recombinant RCP expressed in E. coli, and dephosphorylated RCP, failed to interact with the receptors, indicating that post-translational modifications were necessary for ligand-receptor interaction. The purified phosphopeptide, prepared from tryptic digests of egg white RCP, was able to inhibit the binding of RCP to the receptor proteins, with an affinity comparable to native RCP indicating that the phosphopeptide sequence present in RCP serves as the focal point for RCP-receptor interactions.

Amino Acid Sequence↗

PubMed1984

Separation of tryptic phosphopeptides of ribosomal origin by reversed-phase high-performance liquid chromatography.

Phosphorylation sites for cyclic AMP-dependent kinase in ribosomal proteins and their synthetic analogues were converted to tryptic phosphopeptides and analysed by reversed-phase high-performance liquid chromatography (RP-HPLC) using gradients of acetonitrile in water and 0.1% trifluoroacetic acid. Tryptic variants differing by only NH2-terminal basic amino acid residues or phosphoryl groups were not always well resolved under these conditions. The different phospho forms could be resolved by RP-HPLC in phosphate buffers at pH 7.0. A combination of gel permeation chromatography, RP-HPLC and thin-layer cellulose mapping was found to be the most effective strategy for the absolute purification of tryptic phosphopeptides from crude tryptic digests.

Animals↗

PubMed1998

Fragmentation of phosphopeptides in an ion trap mass spectrometer.

A systematic study of the fragmentation pattern of phosphopeptides in an electrospray (ESI) ion trap mass spectrometer is presented. We show that phosphotyrosine- and phosphothreonine-containing peptides show complicated fragmentation patterns. These phosphopeptides were observed to lose the phosphate moiety in the form of H3PO4 and/or HPO3, but were also detected with no loss of the phosphate group. The tendency to lose the phosphate moiety depends strongly on the charge state. Thus, the highest observed charge state tends to retain the phosphate moiety with extensive fragmentation along the peptide backbone. We also show that phosphoserine-containing peptides have relatively simple fragmentation patterns of losing H3PO4. This loss is independent of the charge state. We suggest strategies for the accurate identification of phosphorylation sites using the ion trap mass spectrometer.

Amino Acid Sequence↗