SEARCH · PubMed Health

Results for “PHOSPHOPEPTIDES”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 91 records · Page 5Linked to original sources

PubMed1996

Phosphopeptides interacting with colloidal calcium phosphate isolated by tryptic hydrolysis of bovine casein micelles.

After extended tryptic hydrolysis of large bovine casein micelles, a mineral-rich peptide fraction was recovered by ultracentrifugation. Its mineral part contained 72% of the colloidal Ca and 49% of the colloidal P1 originally present in the native micelle. Colloidal nitrogenous components were also present, amounting to 27% of the original N content. They contained most of the phosphopeptides and 82% of the micellar phosphoseryl residues. These tryptic peptides were characterized by reversed-phase HPLC on-line electrospray ion source-mass spectrometry analysis. Among the peptides produced 14 phosphopeptides were identified: alpha s2-CN(1-24), alpha s2-CN(1-21), alpha s1-CN(43-79), alpha s1-CN(35-79)7P, alpha s1-CN(35-79)8P, alpha s1-CN(37-79), alpha s1-CN(104-119), alpha s1-CN(104-124), beta-CN(1-25), beta-CN(1-28), beta-CN(1-29), beta-CN(30-97), beta-CN(33-97) and beta-CN(29-97). The proportion of the phosphopeptides interacting with colloidal calcium phosphate was correlated with their relative content of phosphoserine residues, since phosphopeptides containing more than four phosphoserine residues were consistently present within this fraction. It also appeared that other types of peptides, some of them hydrophobic in nature, were also partly or completely present within the colloidal fraction, including alpha s1-CN(91-100), alpha s1-CN(152-193), alpha s1-CN(23-34), alpha s1-CN(125-193), alpha s1-CN(125-199), beta-CN(177-209). beta-CN(184-209), beta-CN(114-169) and beta-CN(108-169). Their possible involvement in the micellar backbone is discussed.

Animals↗

PubMed1997

Relationship between degree of casein hydrolysis and phosphopeptide release.

In an approach to develop a commercial-scale process for the production of casein phosphopeptides containing the cluster sequence-SerP-SerP-SerP-Glu-Glu-, we have studied the relationship between casein hydrolysis and phosphopeptide release. The degrees of hydrolysis (DH) of casein using Novo trypsin PTN 3.0 S and pancreatin 4NF independently, at enzyme to substrate (E:S) ratios of 1:50-1:1600 (by weight), were determined using the pH-stat method. Casein phosphopeptides (CPP) were selectively precipitated using Ca2+ and ethanol from the acid-clarified hydrolysates. The precipitates were analysed by high performance capillary electrophoresis to calculate individual phosphopeptide yields based on extinction coefficients of the purified peptides. Individual peptides were purified by reversed-phase HPLC and anion-exchange FPLC and characterized by MALDI-TOF mass spectrometry and amino acid sequence analysis. For both enzymes, lowering the E:S ratio resulted in reductions in the DH and the release of the CPP, and an increase in peptide chain length. The longer chain length offset the reduction in release such that the gravimetric yields of CPP preparations remained relatively constant. For Novo trypsin the highest yields of the major cluster peptides (beta-casein(CN)f(1-25), alpha s1-CNf(59-79), alpha s2-CNf(1-21), alpha s2-CNf(46-70) and related peptides) in the selective precipitates were obtained at a casein DH of 17%. At lower DH values (9-15%), there was a decrease in yield of the peptides derived from alpha s1-CN and alpha s2-CN while the yield of the beta-CN-derived cluster peptides remained relatively constant. The CPP produced using pancreatin were found to be truncated at all E:S ratios, relative to the tryptic CPP, owing to higher levels of chymotryptic and carboxypeptidase activities in pancreatin. The highest yields of the truncated forms of the major cluster peptides using pancreatin were obtained at a casein DH of 19-23%.

Amino Acid Sequence↗

PubMed2001

Selective, sensitive, and rapid phosphopeptide identification in enzymatic digests using ESI-FTICR-MS with infrared multiphoton dissociation.

Rapid screening for phosphopeptides within complex proteolytic digests involving electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) in the negative ion mode with infrared multiphoton dissociation (IRMPD) accompanied by improved phosphopeptide sensitivity and selectivity is demonstrated with the tryptic digests of the naturally phosphorylated proteins bovine alpha- and beta-casein. All peptides in a complex proteolytic digest can be examined simultaneously for phosphorylation with a 4-s IR laser pulse at 7-11 W where phosphopeptide signature ions form upon irradiation, as the low energy of activation phosphate moiety cleavage transpires without the dissociation of the unphsophorylated peptide population. The tyrosine phosphorylated peptide HGLDN-pY-R, its nonphosphorylated analogue HGLDNYR, the kinase domain of insulin receptor unphosphorylated TRDIYETDYYRK, monophosphorylated TRDIYED-pY-YRK, and triphosphorylated TRDI-pY-ETD-pY-pY-RK were also used as model peptides in this research. The sensitivity and selectivity of phosphopeptides is shown to greatly improve when the volatile base piperidine is used to adjust the pH of th

Amino Acid Sequence↗

PubMed2004

Phosphoric acid as a matrix additive for MALDI MS analysis of phosphopeptides and phosphoproteins.

Phosphopeptides are often detected with low efficiency by MALDI MS analysis of peptide mixtures. In an effort to improve the phosphopeptide ion response in MALDI MS, we investigated the effects of adding low concentrations of organic and inorganic acids during peptide sample preparation in 2,5-dihydroxybenzoic acid (2,5-DHB) matrix. Phosphoric acid in combination with 2,5-DHB matrix significantly enhanced phosphopeptide ion signals in MALDI mass spectra of crude peptide mixtures derived from the phosphorylated proteins alpha-casein and beta-casein. The beneficial effects of adding up to 1% phosphoric acid to 2,5-DHB were also observed in LC-MALDI-MS analysis of tryptic phosphopeptides of B. subtilis PrkC phosphoprotein. Finally, the mass resolution of MALDI mass spectra of intact proteins was significantly improved by using phosphoric acid in 2,5-DHB matrix.

Amino Acid Sequence↗

PubMed2006

Metal affinity capture tandem mass spectrometry for the selective detection of phosphopeptides.

We report a new method called metal affinity capture that when coupled with tandem mass spectrometry (MAC-MSMS) allows for the selective detection and identification of phosphopeptides in complex mixtures. Phosphopeptides are captured as ternary complexes with Ga(III) or Fe(III) and N(alpha),N(alpha)-bis(carboxymethyl)lysine (LysNTA) in solution and electrosprayed as doubly or triply charged positive ions. The gas-phase complexes uniformly dissociate to produce a common (LysNTA + H)+ ion that is used as a specific marker in precursor ion scans. The advantages of MAC-MSMS over the current methods of phosphopeptide detection are as follows. (1) MAC-MSMS uses metal complexes that self-assemble in solution at pH <5, which is favorable for the production of positive ions by electrospray. (2) Phosphorylation at tyrosine, serine, and threonine is detected by MAC-MSMS. (3) The phosphopeptide peaks in the mass spectra are encoded with the 69Ga-71Ga isotope pattern for selective recognition in mixtures. Detection by MAC-MSMS of singly and multiply phosphorylated peptides in tryptic digests is demonstrated at low-nanomolar protein concentrations.

Amino Acid Sequence↗

PubMed1996

Phosphopeptide analysis by matrix-assisted laser desorption time-of-flight mass spectrometry.

In this paper we present methods for identifying and sequencing phosphopeptides in simple mixtures, such as HPLC fractions, at the subpicomole level by (+) ion matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-MS). Data are presented which indicate that when a reflectron time-of-flight mass spectrometer is used, MALDI can distinguish tyrosine phosphorylation from serine and threonine phosphorylation for peptides containing a single phosphate group. Phosphopeptides are identified in the (+) ion MALDI reflector spectrum by the presence of [MH-H3PO4]+ and [MH-HPO3]+ fragment ions formed by metastable decomposition. An abundant [MH-H3PO4]+ ion, accompanied by a weaker [MH-HPO3]+ ion indicates that the peptide is most likely phosphorylated on serine or threonine. In contrast, phosphotyrosine-containing peptides generally exhibit [MH-HPO3]+ fragment ions and little, if any [MH-H3PO4]+. Ambiguities do arise, most often with phosphopeptides that contain residues which readily lose water (such as unmodified serine), but these can often be resolved by recording a complete metastable fragment ion (postsource decay) spectrum. Postsource decay is shown here to be a viable technique for sequencing phosphopeptides. It can be used to distinguish between serine/ threonine and tyrosine phosphorylation and in many cases can be used to determine the exact site of phosphorylation in a peptide sequence. Nearly complete sequence coverage and phosphorylation site mapping is generally possible using approximately 300 fmol of peptide.

Chromatography, High Pressure Liquid↗

PubMed1994

Thermodynamic studies of tyrosyl-phosphopeptide binding to the SH2 domain of p56lck.

The interaction between SH2 domains and tyrosine-phosphorylated proteins is essential in several cytosolic signal transduction pathways. Here we report thermodynamic studies of the interaction of the p56lck (lck) SH2 domain with several phosphopeptides, using the technique of isothermal titration calorimetry (ITC). This is the first report of the use of ITC to study SH2 domain binding reactions. The free energy of binding of the SH2 domain of lck to a phosphopeptide corresponding to the autoregulatory C-terminus of the protein (pY505) was found to be similar to that measured for a phosphopeptide modeled on the C-terminus of the epidermal growth-factor receptor (delta G degrees approximately -7.0 kcal mol-1 at pH 6.8), although significant differences in the enthalpy and entropy were observed. Binding of a phosphopeptide modeled on the C-terminus of p185neu was weaker (delta G degrees approximately -5.4 kcal mol-1 at pH 6.8). Lowering the pH to 5.5 reduced the binding affinity of pY505 by approximately 1 order of magnitude. We ascribe this to the protonation of a histidine side chain in the SH2 domain (H180), which is involved in a hydrogen-bonding network that optimizes the binding site geometry. No difference in affinity was observed between portions of lck corresponding to the SH3-SH2 (residues 63-228) and SH2 (residues 123-228) domains for the pY505 peptide. We also studied the effect upon pY505 peptide binding of mutations at two highly conserved arginine residues in the lck SH2 domain (R134 and R154).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2002

Gas-phase ion unimolecular dissociation for rapid phosphopeptide mapping by IRMPD in a Penning ion trap: an energetically favored process.

This communication discusses the efficient detection of the most important and common protein modification, phosphorylation, using ESI-FTICR-MS and IRMPD. Preliminary studies have demonstrated that within a complex protein digest, all phosphopeptides can be identified by a single IR laser irradiation event due to preferential dissociation of the modified peptides. This research demonstrates that the energy of activation for dissociation of the phosphopeptides is lower than that of the unmodified analogues providing the basis for the success of this technique. However, the P-O stretch (9.6-11 mum or 1042-909 cm-1) of this posttranslational modification is in direct resonance with the CO2 IR laser (10.6 mum or 943 cm-1) used for IRMPD. Therefore, the vibrational frequency of the phosphate moiety may be an additional factor in the rapid first-order decay of phosphopeptides. Based upon the energetics of dissociation discussed in this manuscript, IRMPD of ions in a Penning ion trap is an ideal platform for rapid phosphopeptide mapping.

Amino Acid Sequence↗

PubMed2004

Purification and identification of water-soluble phosphopeptides from cheese using Fe(III) affinity chromatography and mass spectrometry.

Water-soluble phosphopeptides from cheese were isolated using immobilized metal affinity chromatography (IMAC). Phosphopeptides from aqueous cheese extracts were completely retained on iminodiacetic acid (IDA) Sepharose equilibrated with FeCl3 and subsequently eluted with ammonium dihydrogen phosphate. Peptides in the eluate from the IMAC-Fe(III) column were identified using reversed phase liquid chromatography-electronic spray identification-tandem mass spectrometry. Phosphopeptides from two different cheeses were analyzed using the described method: a 10-month-old semihard Herrgard cheese made with mesophilic starter and a 24-month-old Parmigiano Reggiano cheese made with thermophilic starter. Elution of the IMAC-Fe(III) column with a gradient of ammonium dihydrogen phosphate resulted in three distinct peaks for Herrgard cheese corresponding to peptides carrying one, two, and four phosphorylated serine residues, respectively. Sixty-five different phosphopeptides were identified from the Herrgard, whereas only 9 from the Parmigiano Reggiano.

Cheese↗

PubMed2004

Caged phosphopeptides reveal a temporal role for 14-3-3 in G1 arrest and S-phase checkpoint function.

Using classical genetics to study modular phosphopeptide-binding domains within a family of proteins that are functionally redundant is difficult when other members of the domain family compensate for the product of the knocked-out gene. Here we describe a chemical genetics approach that overcomes this limitation by using UV-activatable caged phosphopeptides. By incorporating a caged phosphoserine residue within a consensus motif, these reagents simultaneously and synchronously inactivate all phosphoserine/phosphothreonine-binding domain family members in a rapid and temporally regulated manner. We applied this approach to study the global function of 14-3-3 proteins in cell cycle control. Activation of the caged phosphopeptides by UV irradiation displaced endogenous proteins from 14-3-3-binding, causing premature cell cycle entry, release of G1 cells from interphase arrest and loss of the S-phase checkpoint after DNA damage, accompanied by high levels of cell death. This class of reagents will greatly facilitate molecular dissection of kinase-dependent signaling pathways when applied to other phosphopeptide-binding domains including SH2, Polo-box and tandem BRCT domains.

14-3-3 Proteins↗

PubMed1994

Site-specific anti-phosphopeptide antibodies: use in assessing insulin receptor serine/threonine phosphorylation state and identification of serine-1327 as a novel site of phorbol ester-induced phosphorylation.

Rabbit antisera were raised against synthetic phosphopeptides corresponding to defined or putative sites of insulin receptor serine/threonine phosphorylation (Ser-1305, Ser-1327, Thr-1348). All of these antibodies bound specifically to the immunogenic phosphopeptide but not to the non-phosphorylated form of the peptide or to other phosphopeptides, in a microtitre plate competition enzyme-linked immunosorbent assay. Anti-PS1327 antibody reacted well with native insulin receptor prepared from phorbol ester-treated transfected CHO.T cells, but showed little reaction with receptor from untreated cells. Anti-PT1348 antibody in crude form reacted substantially with receptor from both phorbol 12-myristate 13-acetate-treated and untreated cells, but displayed specificity for phosphoreceptor after adsorption to remove antibodies reactive with dephosphopeptide. The ability to discriminate between receptor from cells treated with or without phorbol ester was retained when these antibodies were used to probe denatured receptor on Western blots. Thus anti-PS1327 and anti-PT1348 react with insulin receptor in a site-specific and phosphorylation-state-dependent manner. Anti-PT1348, but not anti-PS1327, also showed increased reactivity with receptor prepared from insulin-treated cells. The third antibody, anti-PS1305, did not react with intact insulin receptor under any conditions. It is concluded that serine 1327 is a major, previously unrecognized, site of phorbol ester-induced receptor phosphorylation, and that anti-phosphopeptide antibodies will be valuable reagents with which to examine the serine/threonine phosphorylation state of receptor extracted from tissues.

Amino Acid Sequence↗

PubMed2006

Identification of class I MHC-associated phosphopeptides as targets for cancer immunotherapy.

Alterations in phosphorylation of cellular proteins are a hallmark of malignant transformation. Degradation of these phosphoproteins could generate cancer-specific class I MHC-associated phosphopeptides recognizable by CD8+ T lymphocytes. In a comparative analysis of phosphopeptides presented on the surface of melanoma, ovarian carcinoma, and B lymphoblastoid cells, we find 5 of 36 that are restricted to the solid tumors and common to both cancers. Differential presentation of these peptides can result from differential phosphorylation of the source proteins. Recognition of the peptides on cancer cells by phosphopeptide-specific CD8+ T lymphocytes validates the potential of these phosphopeptides as immunotherapeutic targets.

Amino Acid Sequence↗

PubMed1982

Analysis of desmin and vimentin phosphopeptides in cultured avian myogenic cells and their modulation by 8-bromo-adenosine 3',5'-cyclic monophosphate.

The intermediate filament proteins desmin and vimentin are two of the major 32P phosphate acceptors in chicken myotubes differentiating in tissue culture. Analysis of the desmin and vimentin phosphopeptides by two-dimensional tryptic peptide mapping shows that both proteins are phosphorylated at multiple sites, giving rise to 5 phosphopeptides in desmin and as many as 11 in vimentin. Addition of the cAMP analogue 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) to the culture medium of mature (8-day-old) myotubes results in a 2- to 3-fold increase in PO4 incorporation into desmin and vimentin. Two-dimensional tryptic analysis of desmin and vimentin from 8-BrcAMP-treated myotubes shows increased 32PO4 incorporation into a subset of the phosphopeptides observed in control cells. Comparison of phosphopeptides from the two proteins shows the presence of at least three comigrating peptides. All three comigrating peptides exhibit cAMP-dependent increases in 32PO4 incorporation in vimentin, while only two of the three exhibit 8-BrcAMP-dependent responses in desmin. While these peptides are the only two that are sensitive to 8-BrcAMP in desmin, vimentin contains additional peptides that exhibit increased 32PO4 incorporation in response to 8-BrcAMP. This result suggests the existence of both common and distinct phosphorylation sites between desmin and vimentin that may be differentially regulated by cAMP. Thus, desmin and vimentin, even though structurally related, may be capable of responding differently to physiological stimuli.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2003

Identification of novel phosphorylation sites on Xenopus laevis Aurora A and analysis of phosphopeptide enrichment by immobilized metal-affinity chromatography.

Mass spectrometric analysis of proteolytically derived phosphopeptides has developed into a widespread technique for the identification of phosphorylated amino acids. Using liquid chromatography-electrospray ionization tandem mass spectrometry, 14 phosphorylation sites were identified on Xenopus laevis His6-Aurora A, a highly conserved regulator of centrosome maturation and cell division. These included seven novel phosphorylation sites, Ser-12, Thr-21, Thr-103, Ser-116, Thr-122, Tyr-155, and Thr-294, as well as the previously identified regulatory sites, Ser-53, Thr-295, and Ser-349. The identification of these novel phosphorylation sites will be important for future studies aimed at elucidating the mechanisms of Aurora A regulation by phosphorylation. Furthermore, we demonstrate that a "kinase-inactive" mutant of Aurora A, K169R, still retains 10% of activity of the wild-type enzyme in vitro along with occupancy of Thr-295 and Ser-12. However, mutation of Asp-281 to Ala completely abolishes activity of the enzyme and should therefore be used preferentially as a genuine kinase-dead construct. Because of the abundance of phosphorylated residues on His6-Aurora A, we found this protein to be an ideal tool for the characterization of immobilized metal-affinity chromatography (IMAC) as a method for phosphopeptide enrichment from complex mixtures. We present a detailed analysis of the binding and elution properties of both the phosphopeptides and unphosphorylated peptides of His6-Aurora A to Fe3+-IMAC before and after methyl esterification. Moreover, we demonstrate a significant difference in enrichment of phosphopeptides when different resins are used for Fe3+-IMAC and characterize the strengths and limitations of this methodology for the study of phosphoproteomics.

Amino Acid Sequence↗

PubMed2001

Substrate specificity of Ca(2+)/calmodulin-dependent protein kinase phosphatase: kinetic studies using synthetic phosphopeptides as model substrates.

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKPase) dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases. In order to elucidate the mechanism of substrate recognition by CaMKPase, we chemically synthesized a variety of phosphopeptide analogs and carried out kinetic analysis using them as CaMKPase substrates. This is the first report using systematically synthesized phosphopeptides as substrates for kinetic studies on substrate specificities of protein Ser/Thr phosphatases. CaMKPase was shown to be a protein Ser/Thr phosphatase having a strong preference for a phospho-Thr residue. A Pro residue adjacent to the dephosphorylation site on the C-terminal side and acidic clusters around the dephosphorylation site had detrimental effects on dephosphorylation by CaMKPase. Deletion analysis of a model substrate peptide revealed that the minimal length of the substrate peptide was only 2 to 3 amino acid residues including the dephosphorylation site. The residues on the C-terminal side of the dephosphorylation site were not essential for dephosphorylation, whereas the residue adjacent to the dephosphorylation site on the N-terminal side was essential. Ala-scanning analysis suggested that CaMKPase did not recognize a specific motif around the dephosphorylation site. Myosin light chain phosphorylated by protein kinase C and Erk2 phosphorylated by MEK1 were poor substrates for CaMKPase, while a synthetic phosphopeptide corresponding to the sequence around the phosphorylation site of the former was not dephosphorylated by CaMKPase but that of the latter was fairly good substrate. These data suggest that substrate specificity of CaMKPase is determined by higher-order structure of the substrate protein rather than by the primary structure around its dephosphorylation site. Use of phosphopeptide substrates also revealed that poly-L-lysine, an activator for CaMKPase, activated the enzyme mainly through increase in the V(max) values.

Amino Acid Sequence↗

PubMed2000

A new practical strategy for the synthesis of long-chain phosphopeptide.

A new practical strategy has been developed for the synthesis of long-chain phosphopeptide. Both the 2-chlorobenzyloxycarbonyl (CIZ) group for Lys and methyl (Me) for phosphoamino acids remained intact, while other commonly used side-chain protecting groups were cleaved quantitatively, during the reaction using a highly acidic trifluoromethanesulfonic acid (TFMSA)-based reagent system (High TFMSA: TFMSA-TFA-m-cresol=1:9:1, v/v). Selective deprotection of the CIZ and Me group-containing protected phosphopeptide resin with the High TFMSA gave a partially protected phosphopeptide fragment suitable for thioester-mediated fragment condensation. A deprotection protocol of the 9-fluorenylmethyloxycarbonyl (Fmoc) group, which evades significant side reaction toward the protected phosphoamino acid, was also developed. These two new findings enabled us to synthesize long-chain phosphopeptide via thioester-mediated fragment condensation.

Amino Acid Sequence↗

PubMed2004

Identification of G-protein-coupled receptor phosphorylation sites by 2D phosphopeptide mapping.

Reversible phosphorylation is important for G-protein-coupled receptor (GPCR) signaling, desensitization, and endocytosis, yet the precise location and role of in vivo phosphorylation sites is unknown for most receptors. This chapter describes a powerful analytical method for the direct identification of GPCR phosphorylation sites by two-dimensional (2D) phosphopeptide mapping. The GPCR of interest is isolated from 32P-labeled cells by immunoprecipitation and transferred to nitrocellulose membranes. In situ cleavage by trypsin releases phosphopeptides that are separated by a combination of high-voltage electrophoresis and chromatography. Phosphoamino acid analysis and Edman sequencing of isolated phosphopeptides reveals information that can lead to the direct identification of GPCR phosphorylation sites. Furthermore, the 2D phosphopeptide mapping technique allows the analysis of temporal and positional changes in the GPCR phosphorylation pattern under different physiological conditions.

Animals↗

PubMed2004

Titania as a chemo-affinity support for the column-switching HPLC analysis of phosphopeptides: application to the characterization of phosphorylation sites in proteins by combination with protease digestion and electrospray ionization mass spectrometry.

A method for the determination of phosphorylation sites in phosphoproteins based on column-switching high-performance liquid chromatography (HPLC) has been developed. The HPLC system consisted of a titania precolumn for the selective adsorption of phosphopeptides, an anion-exchange analytical column and a UV detector (215 nm). Rabbit muscle phosphorylase a (RPa) and porcine stomach pepsin (PSP) were tested as model phosphoproteins. After protease digestion, the resulting phosphopeptides were successfully isolated by column-switching HPLC. The phosphopeptide fractions were analyzed by electrospray ionization mass spectrometry with a positive or negative ion mode after purification by reversed-phase HPLC. Pseudo-molecular ion peaks corresponding to Gln-Ile-Ser(p)-Val-Arg (MW 681.7) and Glu-Ala-Thr-Ser(p)-Gln-Glu-Leu (MW 856.8) were detected from the tryptic digest of RPa and chymotryptic digest of PSP, respectively, which agreed with the theoretically expected phosphopeptide fragments.

Alkaline Phosphatase↗