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PubMed2004

Phosphopeptide-selective column-switching RP-HPLC with a titania precolumn.

A methodology of phosphopeptide-selective analysis coupled with column-switching HPLC utilizing titania as precolumn media is presented. Phosphopeptides were selectively enriched on titania packing within a protein/peptide mixture without any additional procedure, and analyzed by column-switching high-performance liquid chromatography. First, phospho-compounds were separated from complex mixtures by trapping them under acidic conditions on a titania packing, where non-phosphorylated compounds were effused out of the precolumn. Subsequently, phospho-compounds were desorbed from the titania column under a specific condition and analyzed. The behavior of phospho-compounds on a titania surface, especially adsorption/desorption, was precisely examined and optimized. A phosphoric buffer was successively employed for the elution of phosphopeptides on a titania surface by competition with the free phosphate group. From the successes of a selective concentration/analysis of phosphopeptides with column-switching HPLC with a titania precolumn, a novel phosphopeptide-selective RP-HPLC analysis has been shown to have an application possibility as a tool for phosphoproteomics.

Chromatography, High Pressure Liquid↗

PubMed2004

Chemo-affinity of titania for the column-switching HPLC analysis of phosphopeptides.

A column-switching high-performance liquid chromatography (HPLC) system for the determination of phosphopeptides has been developed. The method is based on the selective adsorption of phosphopeptides on a titania (TiO2) precolumn and successive HPLC separation of the phosphopeptides on an anion-exchange column with a UV detector (215 nm). The recoveries of phosphopeptides were tested using authentic phosphopeptides [Gln-Ile-Ser(p)-Val-Arg, Ile-Ser(p)-Val-Arg and Lys-Gln-Ile-Ser(p)-Val-Arg] at an injection amount of 1 microg. The recoveries were 74.3, 79.6, and 82.6%, respectively, while the corresponding dephosphopeptides were not retained on the titania precolumn.

Chromatography, Affinity↗

PubMed1980

Phosphopeptide substrates of a phosphoprotein phosphatase from rat liver.

The substrate specificity of a preparation of phosphoprotein phosphatase (Mr = 32 000) from rat liver was investigated. Phosphopeptides based on the structure Leu-Arg-Arg-Ala-Ser(P)-Val-Ala-Glx-Leu and Ala-Arg-Thr-Lys-Arg-Ser-Gly-Ser(P)-Val-Tyr-Glu-Pro-Leu-Lys were used. These phosphopeptides correspond to the phosphorylation sites of rat liver pyruvate kinase (type L) and the beta subunit of rabbit muscle phosphorylase b kinase, respectively. A decrease in the apparent Km values and a concomitant increase in Vmax values was observed when the number of amino acyl residues after the phosphoseryl residue in the respective phosphopeptides were increased from 2 to 4, 5, or 6. Most of the phosphopeptides investigated generally showed apparent Km values higher than the values obtained with phosphopyruvate kinase. Ala-Ser(P)-Val-Ala and Gly-Ser(P)-Val-Tyr appeared to be the shortest phosphopeptides that could be dephosphorylated rapidly. These findings support the hypothesis that a small part of the phosphoprotein may be sufficient to fulfill the minimal requirements for its dephosphorylation.

Amino Acid Sequence↗

PubMed2003

Phosphopeptide binding specificities of BRCA1 COOH-terminal (BRCT) domains.

Protein phosphorylation by protein kinases may generate docking sites for other proteins. It thus allows the assembly of signaling complexes in response to kinase activation. Several protein domains that bind phosphoserine or phosphothreonine residues have been identified, including the 14-3-3, PIN1, FHA, KIX, WD-40 domain, and polo box (Yaffe, M. B., and Elia, A. E. (2001) Curr. Opin. Cell Biol. 13, 131-138; Elia, A. E., Cantley, L. C., and Yaffe, M. B. (2003) Science 299, 1228-1231). The BRCA1 COOH-terminal (BRCT) domains are protein modules found in many proteins that regulate DNA damage responses (Koonin, E. V., Altschul, S. F., and Bork, P. (1996) Nat. Genet. 13, 266-268). Whether BRCT domains can mediate phosphorylation-dependent interactions has not been systematically investigated. We report here that the BRCT domains also recognize phosphopeptides. Oriented peptide library analysis indicated that the BRCT domains from BRCA1, MDC1, BARD1, and DNA Ligase IV preferred distinct phosphoserine-containing peptides. In addition, the interaction between BRCA1 and the BRCT binding motif of BACH1 was required for BACH1 checkpoint activity. Furthermore, BRCT domains of the yeast DNA repair protein Rad9 could bind phosphopeptides, suggesting that the BRCT domains represent a class of ancient phosphopeptide-binding modules. Potential targets of BRCT domains were identified through data base search. Structural analysis of BRCA1 BRCT repeats also predicted conserved residues that may form the phosphopeptide-binding pocket. Thus, the BRCT repeats are a new family of phosphopeptide-binding domains in DNA damage responses.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Physicochemical characterization of casein phosphopeptide-amorphous calcium phosphate nanocomplexes.

Milk caseins stabilize calcium and phosphate ions and make them available to the neonate. Tryptic digestion of the caseins yields phosphopeptides from their polar N-terminal regions that contain clusters of phosphorylated seryl residues. These phosphoseryl clusters have been hypothesized to be responsible for the interaction between the caseins and calcium phosphate that lead to the formation of casein micelles. The casein phosphopeptides stabilize calcium and phosphate ions through the formation of complexes. The calcium phosphate in these complexes is biologically available for intestinal absorption and remineralization of subsurface lesions in tooth enamel. We have studied the structure of the complexes formed by the casein phosphopeptides with calcium phosphate using a range of physicochemical techniques including x-ray powder diffraction, scanning electron microscopy, transmission electron microscopy, and equilibrium binding analyses. The amorphous nature of the calcium phosphate phase was confirmed by two independent methods: x-ray powder diffraction and selected area diffraction. In solution, the ion activity product of a basic amorphous calcium phosphate phase was the only ion product that was a function of bound phosphate independent of pH, consistent with basic amorphous calcium phosphate being the phase stabilized by the casein phosphopeptides. Detailed investigations of calcium and calcium phosphate binding using a library of synthetic homologues and analogues of the casein phosphopeptides have revealed that although the fully phosphorylated seryl-cluster motif is pivotal for the interaction with calcium and phosphate, other factors are also important. In particular, calcium binding and calcium phosphate stabilization by the peptides was influenced by peptide net charge, length, and sequence.

Animals↗

PubMed1997

Immunogenicity of casein phosphopeptides derived from tryptic hydrolysis of beta-casein.

The immunogenicity of phosphopeptides derived from tryptic hydrolysis of beta-casein (CN) was investigated in a rat model system. The titers of specific immunoglobulin (Ig)G and IgE antibodies made in response to intraperitoneal sensitization to beta-CN, casein phosphopeptides, and skim milk proteins were examined using indirect and amplified indirect ELISA, respectively. Serum IgG antibodies from rats injected with beta-CN were significantly more reactive to beta-CN, casein phosphopeptides, and skim milk proteins coated on microtiter plate wells than were the IgG antibodies generated in rats that had been subjected to other treatments. A significant difference in titers because of the time of sampling (14 or 21 d postinjection) was noted for IgE but not for IgG. Rats that were injected with casein phosphopeptides did not produce IgG antibodies that crossreacted with either skim milk proteins or beta-CN. Specific antibody levels for the IgE class rarely exceeded those of unimmunized controls. The findings suggest that immunogenicity of the phosphopeptides was reduced compared with that of native beta-CN and skim milk proteins.

Animals↗

PubMed2000

Cell cycle-dependent phosphorylation of centrosomes: localization of phosphopeptide specific antibodies to the centrosome.

The microtubule nucleation capacity of the centrosome increases dramatically as cells progress from interphase into mitosis. The increase in nucleation capacity of the centrosome correlates with the cell cycle-dependent localization of the mitotic protein monoclonal-2 (MPM-2) phosphoepitope-specific antibody to the mitotic centrosome. Therefore, the phosphorylation state of centrosomal components may regulate the microtubule nucleation capacity of this organelle during mitosis. Neither the identity of the MPM-2 kinase(s) nor all of the MPM-2-reactive phosphoproteins associated with the centrosome have been fully elucidated. Only recently have the characteristics of the MPM-2 epitope site been defined, and we used this information to prepare polyclonal antibodies against synthetic phosphopeptides containing potential MPM-2 epitopes derived from the sequences of two MPM-2-reactive proteins, topoisomerase II, and microtubule associated protein 1B (MAP1B). We demonstrate that these phosphopeptide-specific antibodies also localize to the centrosome in a cell cycle-dependent fashion. Thus, polyclonal antibodies have been generated against defined phosphopeptides that reiterate many of the immunofluorescence staining properties exhibited by the MPM-2 antibody. These new phosphopeptide-specific antibodies will provide additional probes to examine the phosphorylation of centrosomal components and the functional consequences of their phosphorylation during mitosis.

Amino Acid Sequence↗

PubMedDate not supplied

Phosphopeptide mapping and phosphoamino acid analysis by electrophoresis and chromatography on thin-layer cellulose plates.

Identification of protein phosphorylation sites is essential in order to evaluate the contribution of individual sites to the regulation of a particular protein by phosphorylation. Here we review a method we have developed for the identification of phosphorylation sites based on digestion of 32P-labeled proteins with site-specific proteases and separation of the digestion products in two dimensions on thin-layer cellulose plates using electrophoresis in the first dimension followed by chromatography. This method is very sensitive, requiring only a few hundred 32P-disintegrations per minute to obtain reproducible phosphopeptide maps. We also report methods for the analysis of the phosphoamino acid content of both intact phosphoproteins and individual phosphopeptides recovered from two-dimensional separations, in which the material is subjected to partial acid hydrolysis, and the hydrolysis products are separated on thin-layer cellulose plates by electrophoresis in one or two dimensions. Finally, we describe methods for analyzing the structure of isolated phosphopeptides by secondary digestion with site-specific proteases, by manual Edman degradation, and by immunoprecipitation, and indicate how this information can be used in conjunction with the two-dimensional mobility of the peptide to deduce the identity of a phosphopeptide from the known sequence of a protein.

Amino Acid Sequence↗

PubMed2006

Ionic liquid matrices with phosphoric acid as matrix additive for the facilitated analysis of phosphopeptides by matrix-assisted laser desorption/ionization mass spectrometry.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful tool for the analysis and characterization of protein phosphorylation on the peptide level. In this study, the applicability of ionic liquid matrices (ILM) formed by combination of the crystalline MALDI matrix 2,5-dihydroxybenzoic acid (DHB) with pyridine or n-butylamine was tested for the analysis of phosphopeptides. Low ionization efficiency in both positive and negative ion mode was observed in acid-free sample preparations. Upon addition of 0.1% trifluoroacetic acid (TFA), ion formation was increased, but analogously to the situation described earlier for pure DHB, best results were obtained upon use of 1% phosphoric acid as matrix additive. The samples prepared in this way were significantly more homogeneous than preparations with pure DHB, thus avoiding the need for time-consuming search for hot spots. Other characteristics like metastable fragmentation of phosphopeptides did not differ from that observed in classical preparations. The limits of detection for synthetic phosphopeptides and singly or multiply phosphorylated peptides from tryptic digests of alpha- and beta-casein were comparable with those obtained when using pure DHB; in some cases even higher signal intensities could be observed in the ILM. The use of ILM in combination with 1% phosphoric acid as matrix additive significantly facilitates analysis of phosphopeptides by MALDI-MS.

Caseins↗

PubMed2001

Characterization of phosphopeptides from protein digests using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nanoelectrospray quadrupole time-of-flight mass spectrometry.

A two-step mass spectrometric method for characterization of phosphopeptides from peptide mixtures is presented. In the first step, phosphopeptide candidates were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) based on their higher relative intensities in negative ion MALDI spectra than in positive ion MALDI spectra. The detection limit for this step was found to be 18 femtomoles or lower in the case of unfractionated in-solution digests of a model phosphoprotein, beta-casein. In the second step, nanoelectrospray tandem mass (nES-MS/MS) spectra of doubly or triply charged precursor ions of these candidate phosphopeptides were obtained using a quadrupole time-of-flight (Q-TOF) mass spectrometer. This step provided information about the phosphorylated residues, and ruled out nonphosphorylated candidates, for these peptides. After [(32)P] labeling and reverse-phase high-performance liquid chromatography (RP-HPLC) to simplify the mixtures and to monitor the efficiency of phosphopeptide identification, we used this method to identify multiple autophosphorylation sites on the PKR-like endoplasmic reticulum kinase (PERK), a recently discovered mammalian stress-response protein.

Amino Acid Sequence↗

PubMed2000

Detection of phosphopeptides by fluorescence polarization in the presence of cationic polyamino acids: application to kinase assays.

We have studied the interaction of several phosphopeptides with cationic polyamino acids such as polyarginine and polylysine by fluorescence polarization. The phosphopeptides used were labeled with fluorescein, and their net charges at the experimental pH of 7. 5 were 0, -1, -2, and -3. These phosphopeptides represent the products of enzymatic phosphorylation reactions of the corresponding nonphosphorylated precursors by the protein kinase A, Akt1 (protein kinase Balpha), and protein kinase C. We found that these phosphopeptides bind more strongly to the cationic polyamino acids studied than their nonphosphorylated analogs. This preferential binding of the phosphorylated peptides could be conveniently detected by an increase in the fluorescence polarization signal of the attached fluorescein residue. We have exploited this observation to develop a new approach for the detection of kinase activity that does not require radioactivity or separation of substrate from product. We have successfully used this method to perform K(m) determinations of the kinase enzymes for their substrates and K(i) determinations of one of their inhibitors. This method for measuring kinase activity might be particularly useful for high-throughput screening applications.

Amino Acids↗

PubMed2006

Optimization of the cellular import of functionally active SH2-domain-interacting phosphopeptides.

Phosphopeptides interacting with src homology 2 (SH2) domains can activate essential signaling enzymes in vitro. When delivered to cells, they may disrupt protein-protein interactions, thereby influencing intracellular signaling. We showed earlier that phosphopeptides corresponding to the inhibitory motif of Fcgamma receptor IIb and a motif of the Grb2-associated binder 1 adaptor protein activate SH2-containing tyrosine phosphatase 2 in vitro. To study the ex vivo effects of these peptides, we have now compared different methods for peptide delivery: (i) permeabilization of the target cells and (ii) the use of cell-permeable vectors, which are potentially able to transport biologically active compounds into B cells. We found octanoyl-Arg(8) to be an optimal carrier for the delivery of phosphopeptides to the cells. With this strategy, the function of cell-permeable SHP-2-binding phosphopeptides was analyzed. These peptides modulated the protein phosphorylation in B cells in a dose- and time-dependent manner.

Adaptor Proteins, Signal Transducing↗

PubMed2004

ATP-phosphopeptide conjugates as inhibitors of Src tyrosine kinases.

A number of Src SH2 domain inhibitors enhance the kinase catalytic activity by switching the closed inactive to the open active conformation. ATP-phosphopeptide conjugates were designed and synthesized as Src tyrosine kinase inhibitors based on a tetrapeptide sequence pTyr-Glu-Glu-Ile (pYEEI) and ATP to block the SH2 domain signaling and substrate phosphorylation by ATP, respectively. In general, ATP-phosphopeptide conjugates with optimal linkers such as compounds 5 and 7 (K(i) = 1.7-2.6 microM) showed higher binding affinities to the ATP-binding site relative to the other ATP-phosphopeptide conjugates having short or long linkers, 1-4 and 6, (K(i) = 10.1-16.1 microM) and ATP (K(m) = 74 microM). These ATP-phosphopeptide conjugates may serve as novel templates for designing protein tyrosine kinase inhibitors to block SH2 mediated protein-protein interactions and to counter the activation of enzyme that resulted from the SH2 inhibition.

Adenosine Triphosphate↗

PubMed2003

Profiling substrate phosphorylation at the phosphopeptide level.

The identification of substrates is a key aspect in the study of the biological function of protein kinases. The procedure here described is aimed at profiling substrate phosphorylation at the phosphopeptide level by sequentially involving (i). the assessment of the in vitro activity of individual protein kinases on a complex mix of immobilized proteins, (ii). the fractionation of the phosphopeptides being released upon proteolysis of substrates, and (iii). the final identification of the targeted sequences. In particular, the protein sample is spotted onto nitrocellulose membrane and then subjected to a solid-phase kinase assay in the presence of [32P]ATP, prior to solid-phase proteolytic digestion and two-dimensional phosphopeptide mapping. Radiolabeled phosphopeptides are subsequently isolated and sequenced to identify the substrates being targeted by the examined protein kinase. Using the gamma-isotype of p21-activated protein kinase (gamma-PAK) and its known in vitro substrates, I verified that both the specificity of substrate phosphorylation and its efficiency are similar upon solid- and liquid-phase conditions. To demonstrate the feasibility of the overall experimental system, I then employed a fairly crude cell extract as a source of candidate substrates and successfully identified the sequence of a putative substrate of gamma-PAK.

Peptide Mapping↗

PubMed1997

Calcium-dependent phosphorylation of glial fibrillary acidic protein (GFAP) in the rat hippocampus: a comparison of the kinase/phosphatase balance in immature and mature slices using tryptic phosphopeptide mapping.

In previous work we showed that phosphorylation of the astrocyte marker glial fibrillary acidic protein (GFAP) in hippocampal slices from adult rats is dependent on external Ca2+, whereas in slices from immature rats aged 12-16 days postnatal 32P incorporation into GFAP is inhibited by external Ca2+. The nature of this late developmental change in Ca2+ sensitivity for GFAP phosphorylation was investigated in the present work by comparing in immature and adult animals phosphorylation of GFAP by endogenous protein kinase activity in cytoskeletal fractions and tryptic phosphopeptide maps prepared from cytoskeletal fractions labelled with [gamma-32P]ATP and from slices labelled with [32P]phosphate. Cytoskeletal fractions prepared from immature and adult hippocampus both contained endogenous protein kinase activity towards GFAP and other proteins stimulated by Ca2+/calmodulin and by cyclic AMP. The maps of GFAP isolated from the cytoskeletal fractions labelled in the presence of Ca2+/calmodulin were very similar and exhibited two major and several minor phosphopeptides. Comparison with maps derived from these fractions labelled in the presence of cyclic AMP showed that one of the major phosphopeptides was either directly or indirectly phosphorylated by Ca2+/calmodulin-stimulated kinase activity. Maps derived from GFAP isolated from adult slices labelled in the presence of Ca2+ and immature slices labelled in the absence of Ca2+ were qualitatively identical, with minor differences from the cytoskeletal maps. At both ages the slice maps displayed the phosphopeptide phosphorylated through the activity of a Ca2+/calmodulin kinase in the cytoskeletal fractions. By its migration properties this peptide appears to correspond to a sequence containing a site shown by other workers to be phosphorylated in vitro by CaM kinase II, suggesting that even in the absence of external Ca2+, kinase activity directly or indirectly dependent on Ca2+ was occurring in the immature slices. The near identity of the phosphorylation sites at the two ages suggest that the change in Ca2+ sensitivity of GFAP phosphorylation during development is not due to a change in the balance of kinase and phosphatase activities, but rather to a change in the mechanism(s) whereby Ca2+ controls the relative activity of these enzymes.

Animals↗

PubMed2000

Detection and sequencing of phosphopeptides affinity bound to immobilized metal ion beads by matrix-assisted laser desorption/ionization mass spectrometry.

Consecutive enzymatic reactions of analytes which are affinity bound to immobilized metal ion beads with subsequent direct analysis of the products by matrix-assisted laser desorption/ionization mass spectrometry have been used for detecting phosphorylation sites. The usefulness of this method was demonstrated by analyzing two commercially available phosphoproteins, beta-casein and alpha-casein, as well as one phosphopeptide from a kinase reaction mixture. Agarose loaded with either Fe3+ or Ga3+ was used to isolate phosphopeptides from the protein digest. Results from using either metal ion were complementary. Less overall suppression effect was achieved when Ga3+-loaded agarose was used to isolate phosphopeptides. The selectivity for monophosphorylated peptides, however, was better with Fe3+-loaded agarose. This technique is easy to use and has the ability to analyze extremely complicated phosphopeptide mixtures. Moreover, it eliminates the need for prior high-performance liquid chromatography separation or radiolabeling, thus greatly simplifying the sample preparation.

Amino Acid Sequence↗

PubMed1999

Enhanced detection of phosphopeptides in matrix-assisted laser desorption/ionization mass spectrometry using ammonium salts.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has been used successfully to detect phosphorylation sites in proteins. Applications may be limited by the low response of phosphopeptides compared to nonphosphorylated peptides in MALDI MS. The addition of ammonium salts to the matrix/analyte solution substantially enhances the signal for phosphopeptides. In examples shown for equimolar mixtures, the phosphorylated peptide peaks become the largest peaks in the spectrum upon ammonium ion addition. This can allow for the identification of phosphopeptides in an unfractionated proteolytic digestion mixture. Sufficient numbers of protonated phosphopeptides can be generated such that they can be subjected to postsource decay analysis, in order to confirm the number of phosphate groups present. The approach works well with the common MALDI matrices such as alpha-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid, and with ammonium salts such as diammonium citrate and ammonium acetate.

Amino Acid Sequence↗

PubMed2000

Effects of bioactive substances in milk on mineral and trace element metabolism with special reference to casein phosphopeptides.

Bioactivity of phosphopeptides yielded after tryptic hydrolysis of casein (CPP) was reported more than 50 years ago when CPP were found to improve calcium balance in rachitic newborns. Several investigations have been carried out to study the effects of CPP mainly on calcium metabolism but also on other minerals like iron and zinc. Most of the experiments were in vitro studies or short-term experiments like the effects of CPP after single meals or their effect on mineral disappearance from intestinal everted sac or ligated loop. Investigations on calcium balance were also mainly short term, i.e. 3-4 weeks, and mainly done in rats. A few experiments have been carried out in minipigs, an animal model that is closer to the human than the rat. Studies in human were rare and short term. To date a variety of other peptides have been isolated after enzymatic hydrolysis, and some have been investigated for bioactivity, with equivocal findings. Bioactivity of phosphopeptides seemed to be more obvious when investigations were done in vitro or short term. Results were less clear in metabolic balance studies, especially under physiological conditions. The composition of the basal diet, i.e. content of calcium and phytate, or the protein source had a significant impact on the effect of phosphopeptides. It was concluded that phosphopeptides revealed positive effects on mineral solubility and absorbability, and bone mineralisation under certain experimental conditions. Accordingly they could have a beneficial effect on bone health for some groups of the population.

Animals↗