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PubMed1995

Mass spectrometric analysis of 21 phosphorylation sites in the internal repeat of rat profilaggrin, precursor of an intermediate filament associated protein.

Profilaggrin, a highly phosphorylated protein synthesized in mammalian cornified epithelia, is the precursor of filaggrin, a protein that is involved in aggregation of keratin during terminal differentiation. Possible functions for the phosphorylation include preventing premature aggregation of keratin, packing profilaggrin into a storage granule, association of other proteins with the granule, and/or regulating proteolytic processing of profilaggrin. As a first step in characterizing the phosphorylation of rat profilaggrin, tryptic peptides of filaggrin and profilaggrin were fractionated by reverse-phase HPLC and analyzed by ionspray mass spectrometry. Nine putative phosphopeptides were identified as those with masses 80 Da (or multiples of 80 Da) greater than the predicted unphosphorylated masses. The six that were phosphorylated to a high stoichiometry were analyzed further. Several multiply phosphorylated peptides underwent neutral loss of H3PO4 during collisional activation, complicating interpretation of the MS/MS spectra. In order to circumvent this problem, an alternative strategy was applied in which peptide mixtures were treated with Ba(OH)2, resulting in beta-elimination of H3PO4 and generation of dehydrated serine or threonine at the site of phosphorylation. Peptides containing dehydrated serine or threonine fragmented well, providing unequivocal identification of multiple phosphorylation sites in peptides as long as 39 amino acids. The phosphopeptides (with phosphorylated residues underlined) were GQQHSGHPQVYYYGVEETEDESDAQQGHHQQQQQQR, GGQAGSHSESEASGGQAGR, HTSRPEQSPDTAGR, GESPAGQQSPDR, EASASQSSDSEGHSGAHAGIGQGQTSTTHR, and GSSESQASDSEGHSDYSEAHTQGAHGGIQTSSQR.

Amino Acid Sequence↗

PubMed1993

Synthesis of phosphotyrosine-containing peptides and their use as substrates for protein tyrosine phosphatases.

Prior methods for the chemical synthesis of phosphotyrosine-containing peptides involved the incorporation of fully protected phosphoamino acids into the peptide chain or phosphorylation of free phenol side chains after peptide assembly is complete. The present work describes a novel and general methodology for the solid-phase synthesis of phosphopeptides, featuring direct incorporation of N alpha-(9-fluorenylmethyloxycarbonyl)-O-phospho-L-tyrosine (unprotected side chain). This technique obviated the formation of peptide byproducts containing tyrosine H-phosphonate, a previously unrecognized side reaction from literature phosphorylation/oxidation approaches. Phosphopeptides corresponding to the tyrosine phosphorylation site of adipocyte lipid binding protein were synthesized by the newer, preferred method. These peptides were purified and characterized by high-performance liquid chromatography (HPLC), capillary zone electrophoresis (CZE), amino acid analysis (AAA), fast atom bombardment mass spectrometry (FABMS), and 31P nuclear magnetic resonance (31P NMR). The synthetic peptides were tested as substrates for two distinct protein tyrosine phosphatases, rat brain protein tyrosine phosphatase (PTPase) and human acid phosphatase. Substrate specificity was measured at pH 6.0 and 37 degrees C, using a colorimetric assay for released inorganic phosphate. Kinetic analysis revealed that both the rat brain PTPase and the human adipocyte acid phosphatase catalyzed peptide dephosphorylation but with different rates and affinities. The rat brain PTPase displayed classical Michaelis-Menten kinetics, with Km's of 68 +/- 9 microM and 42 +/- 11 microM and kcat/Km values of 4.9 x 10(5) s-1 M-1 and 6.9 x 10(5) s-1 M-1 determined for phosphorylated peptides of lengths 4 and 10 residues, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Nitrophenylphosphatase↗

PubMed2000

Role of electrostatic interactions in SH2 domain recognition: salt-dependence of tyrosyl-phosphorylated peptide binding to the tandem SH2 domain of the Syk kinase and the single SH2 domain of the Src kinase.

SH2 domains are small protein domains that bind specifically to tyrosyl-phosphorylated sequences. Because phosphorylation contributes a large part of the binding free energy, it has been postulated that electrostatic interactions may play an important role in SH2 domain recognition. To test this hypothesis, we have examined the salt dependence of the interaction between tyrosyl-phosphorylated peptides and SH2 domains. The dependence of the binding constant, K(obs), on [NaCl] was shown to be strong for binding of the tandem SH2 domain of the Syk kinase (Syk-tSH2) to doubly phosphorylated peptides derived from immune-receptor tyrosine activation motifs (dpITAMs): the slopes of plots of log(K(obs)) versus log [NaCl], designated SK(obs), ranged from -2.6 +/- 0.1 to -3.1 +/- 0.2. Binding of the single SH2 domain of the Src kinase to its consensus singly phosphorylated peptide (sequence pYEEI where pY indicates a phosphotyrosine) was also highly dependent on [NaCl] with a SK(obs) value of -2.4 +/- 0.1. The ability of salt to disrupt the interactions between Syk-tSH2 and dpITAM peptides was shown to be anion-dependent with the inhibitory effect following the order: phosphate > Cl(-) > F(-). For the Syk-tSH2 system, interactions in the pY-binding pockets were shown to be responsible for a large portion of the total salt dependence: removal of either phosphate from the dpITAM peptide reduced the magnitude of SK(obs) by 40-60% and weakened binding by 2-3 orders of magnitude. Consistent with this finding, binding of the single amino acid Ac-pY-NH(2) was characterized by a large salt dependence of binding and was also dependent on the identity of the perturbing anion. The role of peptide residues C-terminal to the pY, which are implicated in determining the specificity of the phosphopeptide-SH2 domain interaction, was next probed by comparing the binding of the Src SH2 domain to a peptide containing the pYEEI sequence with that of a lower affinity variant pYAAI peptide: the magnitude of SK(obs) for the variant peptide was reduced to -1.3 +/- 0.1 as compared to -2.4 +/- 0.1 for the pYEEI peptide, indicating that in addition to pY, residues conferring peptide binding specificity contribute significantly to the salt dependence of SH2 domain binding. This study shows that electrostatic interactions play important roles not only in mediating pY recognition and binding but also in contributing to the specificity of the interactions between tyrosyl phosphopeptides and SH2 domains.

Amino Acid Sequence↗

PubMed2000

Dimer formation through domain swapping in the crystal structure of the Grb2-SH2-Ac-pYVNV complex.

Src homology 2 (SH2) domains are key modules in intracellular signal transduction. They link activated cell surface receptors to downstream targets by binding to phosphotyrosine-containing sequence motifs. The crystal structure of a Grb2-SH2 domain-phosphopeptide complex was determined at 2.4 A resolution. The asymmetric unit contains four polypeptide chains. There is an unexpected domain swap so that individual chains do not adopt a closed SH2 fold. Instead, reorganization of the EF loop leads to an open, nonglobular fold, which associates with an equivalent partner to generate an intertwined dimer. As in previously reported crystal structures of canonical Grb2-SH2 domain-peptide complexes, each of the four hybrid SH2 domains in the two domain-swapped dimers binds the phosphopeptide in a type I beta-turn conformation. This report is the first to describe domain swapping for an SH2 domain. While in vivo evidence of dimerization of Grb2 exists, our SH2 dimer is metastable and a physiological role of this new form of dimer formation remains to be demonstrated.

Adaptor Proteins, Signal Transducing↗

PubMed1991

Control of insulin receptor autophosphorylation by polypeptide substrates: inhibition and stimulation by interaction with the catalytic subunit.

Autophosphorylation of purified insulin receptor, in the absence of insulin, was stimulated by selected polypeptide substrates. In the presence of 1 microM insulin these peptides inhibited autophosphorylation. Stimulation was observed with reduced [S-(carboxamidomethyl)cysteinyl]lysozyme (RCAM-lysozyme) and three peptides generated by CNBr cleavage, V8 proteinase digestion and/or chemical modification. We also generated two peptide substrates from RCAM-lysozyme which did not stimulate receptor autophosphorylation and were very weak inhibitors. As a control peptide, the simple substrate angiotensin inhibited receptor autophosphorylation in the absence or presence of insulin. However, stimulatory peptide, but not insulin, significantly shifted the concentration dependence for inhibition by angiotensin. The stimulatory peptides also increased autophosphorylation of the cloned cytoplasmic domain of the kinase [R-BIRK; Villalba, M., Wente, S. R., Russell, D. S., Ahn, J., Reichelderfer, C. F., & Rosen, O. M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7848]. Therefore, stimulation occurs by interaction with the cytoplasmic process of the beta-subunit and not through interaction with the insulin binding alpha-subunit of the native receptor. Autophosphorylation was analyzed by mapping 32P-labeled tryptic phosphopeptides from the beta-subunit and from R-BIRK. Nearly identical phosphopeptide maps were found, comparing first, basal R-BIRK and basal native receptor, second, peptide- and insulin-stimulated native receptor, and third, peptide-stimulated R-BIRK and insulin-stimulated native receptor. Therefore, R-BIRK functions as a basal-state enzyme and can be stimulated in an insulin-like manner. On the basis of these observations, stimulation by insulin and by peptides yields similar functional results, but by apparently different mechanisms.

Adenosine Triphosphate↗

PubMed1984

Highly phosphorylated region of chicken riboflavin-binding protein: chemical characterization and 31P NMR studies.

Phosphate residues on chicken riboflavin-binding protein (RBP) have been implicated in the recognition and deposition of the protein-vitamin complex in egg yolk by the ovary. We demonstrate that all of the phosphate of RBP is linked to serine and that most if not all of the phosphoserine residues are contained in a single tryptic peptide having a composition SerP8, Glx6, His2, Leu2, Ala, Met, Lys. Despite differences in the tissue of synthesis, RBPs isolated from egg yolk and egg white yield phosphopeptides virtually identical in their amino acid composition and 101-MHz 31P NMR spectra. This implies that posttranslational phosphorylation of RBP is the same in liver and oviduct. The 31P NMR spectrum of the phosphopeptide is quite different from that of the phosphoprotein. The 31P NMR spectra of egg yolk and egg white RBP are quite similar but not identical and are unaffected by the binding of riboflavin. Optimal resolution of seven phosphorus resonances was obtained near pH 7.0. The titration behavior of all the phosphoserine residues is similar. We propose that this highly anionic peptide, exposed at the surface of RBP, is the principal determinant for the uptake of RBP by the vitelline membrane of the ovarian follicle.

Amino Acids↗

PubMed1986

Phosphorylation-dephosphorylation of pyruvate dehydrogenase from bakers' yeast.

The pyruvate dehydrogenase complex was purified to homogeneity from bakers' yeast (Saccharomyces cerevisiae). No pyruvate dehydrogenase kinase activity was detected at any stage of the purification. However, the purified pyruvate dehydrogenase complex was phosphorylated and inactivated with purified pyruvate dehydrogenase kinase from bovine kidney. The protein-bound radioactivity was localized in the pyruvate dehydrogenase alpha subunit. The phosphorylated, inactive pyruvate dehydrogenase complex was dephosphorylated and reactivated with purified pyruvate dehydrogenase phosphatase from bovine heart. Tryptic digestion of the 32P-labeled complex yielded a single phosphopeptide, which was purified to homogeneity. The sequence of the phosphopeptide was established to be Tyr-Gly-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Thr-Thr-Tyr-Arg. This sequence is very similar to the sequence of a tryptic phosphotetradecapeptide derived from the alpha subunit of bovine kidney and heart pyruvate dehydrogenase: Tyr-His-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Val-Ser-Tyr-Arg.

Adenosine Triphosphate↗

PubMed1987

Separate domains of the insulin receptor contain sites of autophosphorylation and tyrosine kinase activity.

We have studied the structure and function of the solubilized insulin receptor before and after partial proteolytic digestion to define domains in the beta-subunit that undergo autophosphorylation and contain the tyrosine kinase activity. Wheat germ agglutinin purified insulin receptor from Fao cells was digested briefly at 22 degrees C with low concentrations (5-10 micrograms/mL, pH 7.4) of trypsin, staphylococcal V8 protease, or elastase. Autophosphorylation of the beta-subunit was carried out before and after digestion, and the [32P]phosphoproteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, detected by autoradiography, and analyzed by tryptic peptide mapping by use of reverse-phase high-performance liquid chromatography. Mild trypsin digestion reduced the apparent molecular mass of the beta-subunit from 95 to 85 kDa, and then to 70 kDa. The 85-kDa fragment was not immunoprecipitated by an antibody directed against the C-terminal domain of the beta-subunit (alpha Pep-1), indicating that this region of the receptor was lost. The 85-kDa fragment contained about half of the [32P]phosphate originally found in the beta-subunit, and tryptic peptide mapping showed that two major tryptic phosphopeptides (previously called pY2 and pY3) were removed. Three other tryptic phosphopeptides (pY1, pY1a, and pY4) were found in the 85- and 70-kDa fragments. Treatment of the intact receptor with staphylococcal V8 protease also converted the beta-subunit to an 85-kDa fragment that did not bind to alpha Pep-1, contained about 50% of the initial radioactivity, and lacked pY2 and pY3. Elastase rapidly degraded the receptor to inactive fragments between 37 and 50 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1978

Sites of in vivo phosphorylation of histone H5.

Previous studies have suggested that the phosphorylation and dephosphorylation of histone H5 play an important role in controlling the condensation of avian erythrocyte chromatin. The present work locates in the polypeptide chain the major sites at which H5 is phosphorylated in vivo. The majority of the radioactivity in 32P-labeled H5 is clustered in two regions of the molecule. Nearly 50% of the 32P is found in the amino-terminal N-bromosuccinimide (NBS) peptide (residues 1-28); the remainder is confined to three phosphopeptides arising from the C-terminal half of the molecule (residues 100-200). All phosphopeptides are found in a tryptic digest of monophosphorylated H5, indicating the phosphorylation of a given site is a random event. Automatic Edman degradation of the amino-terminal fragment shows that the radioactivity is equally divided between serines at positions 3 and 7. The C-terminal phosphorylated tryptic peptides share some features with the C-terminal phosphorylation sites in H1. If, as has been postulated, the sites of phosphorylation are in or near DNA combining regions, then H5 may have two DNA combining sites. The location of the phosphorylation sites is discussed in relation to a possible mechanism for controlling chromatin condensation.

Amino Acid Sequence↗

PubMed1978

Sites of phosphorylation on pyruvate dehydrogenase from bovine kidney and heart.

The highly purfied pyruvate dehydrogenase complex (EC 1.2.4.1) and uncomplexed pyruvate dehydrogenase from bovine kidney and heart mitochondria were phosphorylated and inactivated with pyruvate dehydrogenase kinase and [gamma-32P]ATP. Tryptic digestion of the phosphorylated pyruvate dehydrogenase yielded three phosphopeptides, a mono- (site 1) and a di- (sites 1 and 2) phosphorylated tetradecapeptide and a monophosphorylated nonapeptide (site 3). The amino acid sequences of the three phosphopeptides were established to be Tyr-His-Gly-His-Ser(P)-Met-Ser-Asn-Pro-Gly-Val-Ser-Tyr-Arg, Tyr-His-Gly-His-Ser(P)-Met-Ser-Asn-Pro-Gly-Val-Ser(P)-Tyr-Arg, and Tyr-Gly-Met-Gly-Thr-Ser(P)-Val-Glu-Arg. Phosphorylation proceeded markedly faster at site 1 than at sites 2 and 3, and phosphorylation at site 1 correlated closely with inactivation of pyruvate dehydrogenase. Complete inactivation of pyruvate dehydrogenase was associated with incorporation at site 1 of 1.0--1.6 mol of phosphoryl groups per mol of enzyme. Since pyruvate dehydrogenase is a tetramer (alpha2beta2) and since phosphorylation occurs only on the alpha subunit, the possibility of half-site reactivity is considered.

Adenosine Triphosphate↗

PubMed2002

Phosphorylation of p47phox sites by PKC alpha, beta II, delta, and zeta: effect on binding to p22phox and on NADPH oxidase activation.

Production of superoxide anions by the multicomponent enzyme of human neutrophil NADPH oxidase is accompanied by extensive phosphorylation of p47(phox), one of its cytosolic components. p47(phox) is an excellent substrate for protein kinase C (PKC), but the respective contribution of each PKC isoform to this process is not clearly defined. In this study, we found that PKC isoforms known to be present in human neutrophils (PKC alpha, beta, delta, and zeta) phosphorylate p47(phox) in a time- and concentration-dependent manner, with apparent K(m) values of 10.33, 3.37, 2.37, and 2.13 microM for PKC alpha, beta II, delta, and zeta, respectively. Phosphopeptide mapping of p47(phox) showed that, as opposed to PKC zeta, PKC alpha, beta II, and delta are able to phosphorylate all the major PKC sites. The use of p47(phox) mutants identified serines 303, 304, 315, 320, 328, 359, 370, and 379 as targets of PKC alpha, beta II, and delta. Comparison of the intensity of phosphopeptides suggests that Ser 328 is the most phosphorylated serine. The ability of each PKC isoform to induce p47(phox) to associate with p22(phox) was tested by using an overlay technique; the results showed that all the PKC isoforms that were studied induce p47(phox) binding to the cytosolic fragment of p22(phox). In addition, PKC alpha, beta II, delta, and zeta were able to induce production of superoxide anions in a cell-free system using recombinant cytosolic proteins. Surprisingly, PKC zeta, which phosphorylates a subset of selective p47(phox) sites, induced stronger activation of the NADPH oxidase. Taken together, these results suggest that PKC alpha, beta II, delta, and zeta expressed in human neutrophils can individually phosphorylate p47(phox) and induce both its translocation and NADPH oxidase activation. In addition, phosphorylation of some serines could have an inhibitory effect on oxidase activation.

Alanine↗

PubMed2003

Nuclear magnetic resonance structure of the P395S mutant of the N-SH2 domain of the p85 subunit of PI3 kinase: an SH2 domain with altered specificity.

Understanding the specificity of Src homology 2 (SH2) domains is important because of their critical role in cell signaling. Previous genetic analysis has characterized mutants of the N-terminal src homology 2 (SH2) domain of the p85 subunit of phosphoinositide 3-kinase (PI3K). The P395S mutant exhibits a specificity for phosphopeptide binding different from that of the wild-type SH2. The P395S mutant has an increased affinity for the platelet-derived growth factor receptor (PDGFr) compared to polyomavirus middle T antigen (MT). Solution structures of the P395S mutant of the p85 N-SH2 alone and complexed to a PDGFr phosphopeptide were determined to explain the change in specificity. Chemical shift perturbations caused by different peptides were compared for mutant and wild-type structures. The results show that the single P395S mutation has broad effects on the structure. Furthermore, they provide a rationale for the observed changes in binding preference.

Amino Acid Sequence↗

PubMed1996

Purification of bovine thymus cytosolic C-terminal Src kinase (CSK) and demonstration of differential efficiencies of phosphorylation and inactivation of p56lyn and pp60c-src by CSK.

The C-terminal src kinase (CSK) is a ubiquitously expressed, cytosolic enzyme capable of phosphorylating and inactivating several plasma membrane-bound src-family protein tyrosine kinases in vitro [Nada, S., Okada, M., MacAuley, A., Cooper, J.A., & Nakagawa, H. (1990) Nature 351, 69-72; Bergman, M., Mustelin, T., Oetken, C., Partanen, J., Flint, N.A., Amrein, K.E., Autero, M., Burn, P., & Alitalo, K. (1992) EMBO J. 11, 2919-2924]. We purified CSK to apparent homogeneity from bovine thymus cytosol to study in vitro how the purified enzyme recognizes the various src-family kinases as its substrates. A novel assay method was developed for assaying the ability of CSK to inactivate src-family tyrosine kinases. With this assay method, we demonstrated that CSK inactivated p56lyn with a significantly higher efficiency than pp60c-src. Phosphopeptide mapping of CSK-phosphorylated p56lyn and pp60c-src shows that the consensus tyrosine residue (also termed tail tyrosine) in the C-terminal regulatory domain of p56lyn was phosphorylated by CSK with an efficiency much higher than that of pp60c-src. Thus, the higher efficiency of inactivation of p56lyn by CSK is a result of the ability of p56lyn to serve as a better substrate of CSK. The synthetic peptides derived from the C-terminal portion of p56lyn and pp60c-src were much poorer substrates than the intact src-family kinases for CSK, indicating that the local structure around the tail tyrosine is not sufficient to direct efficient phosphorylation of p56lyn by CSK. Nevertheless, the slightly higher efficiency displayed by CSK in phosphorylating the peptide derived from the C-terminal portion of p56lyn than that from pp60c-src suggests that the structural differences between the C-terminal portions of p56lyn and pp60c-src contribute to the differential efficiencies displayed by CSK in phosphorylating the two kinases. Determination of the CSK-phosphorylation site in the src-C-terminal peptide by phosphopeptide mapping reveals that the whole C-terminal regulatory domain and an adjacent part of the protein kinase domain contain some of the structural determinants directing CSK to phosphorylate the consensus tail tyrosine of the src-family kinases.

Amino Acid Sequence↗

PubMed1997

Phosphorylation of the acidic ribosomal P proteins in Saccharomyces cerevisiae: a reappraisal.

Previous reports had pointed to serines 62 and 71/79 as possible phosphorylation sites in the yeast acidic ribosomal proteins YP1 alpha and YP2 alpha, respectively. However, it has been found that mutation of these serine residues did not affect the phosphorylation level of the proteins. A detailed examination of the YP2 alpha tryptic digest from the in vivo labeled protein demonstrates the existence of a totally trypsin-insensitive site at lysine 88 that led to a misinterpretation of previous results. The unique YP2 alpha tryptic phosphopeptide obtained contains, in addition to serines 71 and 79, a serine at position 96 near the carboxyl end, which automatic Edman degradation confirmed as the phosphorylated residue. In addition, by using Staphyloccocus protease V8, it was possible to obtain phosphopeptides containing only serine 96, whose phosphorylation has likewise been confirmed by radioactive labeling as well as by chemical methods. A similar analysis of the other 12 kDa acidic proteins, YP1 alpha, YP1 beta, and YP2 beta, has shown the presence of equivalent phosphorylation sites in the four P proteins, which correspond to position 96 in proteins YP1 alpha, YP1 beta, and YP2 alpha and position 100 in YP2 beta. This conclusion has been confirmed by the fact that mutation of serine 96 in proteins YP1 alpha and YP2 alpha abolishes their capacity to be phosphorylated in vivo. The mutation of the phosphorylation site of the individual acidic proteins seems not to alter their interaction with the ribosome. However, it has been found that the level of phosphorylation of the stalk proteins has an effect on the response of the cells to some specific metabolic conditions, indicating that it may modulate the translation of specific proteins.

Amino Acid Sequence↗

PubMed1999

Identification of the sites phosphorylated by cyclic AMP-dependent protein kinase on the beta 2 subunit of L-type voltage-dependent calcium channels.

Voltage-dependent L-type calcium (Ca) channels are heteromultimeric proteins that are regulated through phosphorylation by cAMP-dependent protein kinase (PKA). We demonstrated that the beta 2 subunit was a substrate for PKA in intact cardiac myocytes through back-phosphorylation experiments. In addition, a heterologously expressed rat beta 2a subunit was phosphorylated at two sites in vitro by purified PKA. This beta 2a subunit contains two potential consensus sites for PKA-mediated phosphorylation at Thr164 and Ser591. However, upon mutation of both of these residues to alanines, the beta 2a subunit remained a good substrate for PKA. The actual sites of phosphorylation on the beta 2a subunit were identified by phosphopeptide mapping and microsequencing. Phosphopeptide maps of a bacterially expressed beta 2a subunit demonstrated that this subunit was phosphorylated similarly to the beta 2 subunit isolated from heart tissue and that the phosphorylation sites were contained in the unique C-terminal region. Microsequencing identified three serine residues, each of which conformed to loose consensus sites for PKA-mediated phosphorylation. Mutation of these residues to alanines resulted in the loss of the PKA-mediated phosphorylation of the beta 2a subunit. The results suggest that phosphorylation of the beta 2a subunit by PKA occurs at three loose consensus sites for PKA in the C-terminus and not at either of the two strong consensus sites for PKA. The results also highlight the danger of assuming that consensus sites represent actual sites of phosphorylation. The actual sites of PKA-mediated phosphorylation are conserved in most beta 2 subunit isoforms and thus represent potential sites for regulation of channel activity. The sites phosphorylated by PKA are not substrates for protein kinase C (PKC), as the mutated beta 2 subunits lacking PKA sites remained good substrates for PKC.

Amino Acid Sequence↗

PubMed2000

Preparation of novel functional oligophosphopeptides from hen egg yolk phosvitin.

Novel hen egg phosvitin phosphopeptides (PPP) with molecular masses of 1-3 kDa were prepared from tryptic hydrolysis following partial alkaline dephosphorylation. The phosvitin treated with various NaOH concentrations (0.05-0.4 N) resulted in different dephosphorylated proteins ranging from 17.5 to 96.3% of phosphate retention. The protein was digested into 10-20 amino acid peptides with trypsin. Calcium-binding properties of PPP were compared with those of commercial casein phosphopeptides in vitro. The PPP with 35% phosphate retention is shown to be effective for enhancing calcium binding capacity and inhibiting the formation of insoluble calcium phosphate. The results provide potential novel functional oilgophosphopeptides as nutraceuticals.

Amino Acids↗

PubMed2006

Derivatized nanoparticle coated capillaries for purification and micro-extraction of proteins and peptides.

Various methods are used to enrich or purify a protein of interest from other proteins and components in a crude cell lysate or other sample. One of the most powerful methods is affinity purification, also called affinity chromatography, whereby the proteins of interest are purified by virtue of their specific binding properties to an immobilized ligand. Affinity purification is becoming more widely used for exploring post-translation modifications and protein-protein interactions, especially with a view toward developing new general tag systems and strategies of chemical derivatization on peptides for affinity selection. Our work was aimed to immobilize proteins or ligands for affinity purification of antibodies, fusion-tagged proteins and other proteins and peptides. Selected proteins or peptides are efficiently extracted and enriched using chemically derivatized walls of a fused silica capillary column. In this paper, we present an open tubular capillary, where the inner wall of a fused silica capillary was derivatized by covalent binding of modified polystyrene latex particles. The capillaries were derivatized with iminodiacetic acid and loaded with Fe3+ or Ni2+ for the purification and enrichment of phosphopeptides or His-tagged proteins, respectively. The latex coated capillaries have been successfully applied to enrich phosphopeptides from beta-casein tryptic digest and ovalbumin tryptic digest at a micro volume scale with recoveries ranging from 92 to 95%. The capillaries have been eluted under conditions compatible with MALDI-MS without any prior desalting step. In another approach, concanavalin A (Con A) or Protein G were immobilized on the epoxy modified latex on the inner wall of the fused silica capillary for the purification of glycoproteins and immunoglobulin, respectively. The design of the capillary and the protocols used for purification permits the direct detection of eluted proteins and peptides with gel electrophoresis or with mass spectrometry. The elution volumes are passed as discrete segments of few microliters over the inner surface of the open-tube capillary, achieving enrichment factors of more than 20-fold from starting samples.

Caseins↗

PubMed2007

Protein phosphorylation and expression profiling by Yin-yang multidimensional liquid chromatography (Yin-yang MDLC) mass spectrometry.

A system which consisted of multidimensional liquid chromatography (Yin-yang MDLC) coupled with mass spectrometry was used for the identification of peptides and phosphopeptides. The multidimensional liquid chromatography combines the strong-cation exchange (SCX), strong-anion exchange (SAX), and reverse-phase methods for the separation. Protein digests were first loaded on an SCX column. The flow-through peptides from SCX were collected and further loaded on an SAX column. Both columns were eluted by offline pH steps, and the collected fractions were identified by reverse-phase liquid chromatography tandem mass spectrometry. Comprehensive peptide identification was achieved by the Yin-yang MDLC-MS/MS for a 1 mg mouse liver. In total, 14 105 unique peptides were identified with high confidence, including 13 256 unmodified peptides and 849 phosphopeptides with 809 phosphorylated sites. The SCX and SAX in the Yin-Yang system displayed complementary features of binding and separation for peptides. When coupled with reverse-phase liquid chromatography mass spectrometry, the SAX-based method can detect more extremely acidic (pI < 4.0) and phosphorylated peptides, while the SCX-based method detects more relatively basic peptides (pI > 4.0). In total, 134 groups of phosphorylated peptide isoforms were obtained, with common peptide sequences but different phosphorylated states. This unbiased profiling of protein expression and phosphorylation provides a powerful approach to probe protein dynamics, without using any prefractionation and chemical derivation.

Amino Acid Sequence↗