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 235 records · Page 13Linked to original sources

PubMed1996

Rapid identification of phosphopeptide ligands for SH2 domains. Screening of peptide libraries by fluorescence-activated bead sorting.

A method for the identification of high-affinity ligands to SH2 domains by fluorescence-activated bead sorting (FABS) was established. Recombinant SH2 domains, expressed as glutathione S-transferase (GST) fusion proteins, were incubated with a phosphotyrosine (Y*)-containing peptide library. 6.4 x 10(5) individual peptides of nine amino acids in length (EPX6Y*X19X7X19X7X6) were each displayed on beads. Phosphopeptide interaction of a given SH2 domain was monitored by binding of fluorescein isothiocyanate-labeled antibodies directed against GST. High-fluorescence beads were isolated by flow cytometric sorting. Subsequent pool sequencing of the selected beads revealed a distinct pattern of phosphotyrosine-containing motifs for each individual SH2 domain: the SH2 domain of the adapter protein Grb2 predominantly selected beads with the sequence Y*ENDP, whereas the C-terminal SH2 domain of the tyrosine kinase Syk selected Y*EELD, each motif representing the most frequently found residues C-terminal to the phosphotyrosine. For deconvolution studies, soluble phosphopeptides comprising variations of the Grb2 motifs were resynthesized and analyzed by surface plasmon resonance.

Amino Acid Sequence↗

PubMed1997

pH titration studies of an SH2 domain-phosphopeptide complex: unusual histidine and phosphate pKa values.

Electrostatic interactions in a complex of the phospholipase C-gamma 1 C-terminal SH2 domain with a high-affinity binding phosphopeptide representing the sequence around Tyr 1021 of the beta platelet-derived growth factor receptor were studied by pKa determination of various titratable groups over the pH range of 5 to 8. A histidine residue that is highly conserved among SH2 domains (His beta D4) and the phosphotyrosine (pTyr) phosphate group show pKa values significantly lower than average for these residue types in proteins. The reduced pKa of these two groups is due to the proximity of the highly positively charged pTyr binding pocket. The unusual pKa of His beta D4 is also due to burial from solvent in a hydrogen-bonding network that appears necessary for the positioning of arginine residues involved in pTyr binding. Mutation of the analogous histidine in other SH2 domains has been shown to abrogate pTyr binding. In addition to these large shifts in pKa values, smaller effects were observed for the titratable groups of a glutamic acid and histidine near the C-terminus of the the second helix due to its helical dipole. Finally, exchange behavior of arginine guanidinium protons with solvent as a function of pH in this SH2 domain-phosphopeptide complex confirms previous descriptions of the roles of different arginines in the structure and function of this protein.

Amides↗

PubMed1987

Differences between glycogen synthases from rat and rabbit skeletal muscle as indicated by phosphopeptide maps.

Glycogen synthase I was purified from rat skeletal muscle. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, the enzyme migrated as a major band with a subunit Mr of 85,000. The specific activity (24 units/mg protein), activity ratio (the activity in the absence of glucose-6-P divided by the activity in the presence of glucose-6-P X 100) (92 +/- 2) and phosphate content (0.6 mol/mol subunit) were similar to the enzyme from rabbit skeletal muscle. Phosphorylation and inactivation of rat muscle glycogen synthase by casein kinase I, casein kinase II (glycogen synthase kinase 5), glycogen synthase kinase 3 (kinase FA), glycogen synthase kinase 4, phosphorylase b kinase, and the catalytic subunit of cAMP-dependent protein kinase were similar to those reported for rabbit muscle synthase. The greatest decrease in rat muscle glycogen synthase activity was seen after phosphorylation of the synthase by casein kinase I. Phosphopeptide maps of glycogen synthase were obtained by digesting the different 32P-labeled forms of glycogen synthase by CNBr, trypsin, or chymotrypsin. The CNBr peptides were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the tryptic and chymotryptic peptides were separated by reversed-phase HPLC. Although the rat and rabbit forms of synthase gave similar peptide maps, there were significant differences between the phosphopeptides derived from the N-terminal region of rabbit glycogen synthase and the corresponding peptides presumably derived from the N-terminal region of rat glycogen synthase. For CNBr peptides, the apparent Mr was 12,500 for rat and 12,000 for the rabbit. The tryptic peptides obtained from the two species had different retention times. A single chymotryptic peptide was produced from rat skeletal muscle glycogen synthase after phosphorylation by phosphorylase kinase whereas two peptides were obtained with the rabbit enzyme. These results indicate that the N-terminus of rabbit glycogen synthase, which contains four phosphorylatable residues (Kuret et al. (1985) Eur. J. Biochem. 151, 39-48), is different from the N-terminus of rat glycogen synthase.

Animals↗

PubMed1986

Distinct specificities of repressible acid phosphatase from yeast toward phosphoseryl and phosphotyrosyl phosphopeptides.

By using [32P]-labeled phosphoaminoacids it has been shown that, at mu molar range concentrations, Tyr-32P but neither Ser-32P nor Thr-32P can be significantly dephosphorylated by highly purified repressible acid phosphatase from Saccharomyces cerevisiae. The phosphopeptide Arg-Arg-Ala-Ser(32P)-Val-Ala however, reproducing the phosphorylation site of pyruvate kinase and previously phosphorylated by cAMP-dependent protein kinase, can be very readily dephosphorylated with favourable kinetic constants (Km 0.28 microM, Vmax = 62 units/micrograms) while its derivatives Ala-Ser(32P)-Val-Ala, Arg-Arg-Ala-Thr(32P)-Val-Ala, Arg-Arg-Pro-Ser(32P)-Pro-Ala as well as other peptides and protein substrates phosphorylated by either protein kinase-C or casein kinase-2 are either unaffected or very slowly dephosphorylated by the phosphatase. Conversely Tyr-32P containing angiotensin, poly (Glu, Tyr) 4:1 and the phosphopeptide Asp-Ala-Glu-Tyr(32P)-Ala-Ala-Arg-Arg-Arg-Gly are all dephosphorylated with kinetic constants comparable to those of free phosphotyrosine (Km 0.2-1 microM; Vmax = 4-10 units/micrograms). It is proposed that, while acid phosphatase exhibits a broad specificity toward phosphotyrosine and phosphotyrosyl polypeptides, it is highly selective toward phosphoseryl sites fulfilling definite structural requirements which are reminiscent of those determining phosphorylation by cAMP-dependent protein kinase.

Acid Phosphatase↗

PubMed1998

Calorimetric investigation of proton linkage by monitoring both the enthalpy and association constant of binding: application to the interaction of the Src SH2 domain with a high-affinity tyrosyl phosphopeptide.

The binding of Src homology 2 (SH2) domains to tyrosyl phosphopeptides depends on electrostatic interactions between the phosphotyrosine and its binding site. To probe the role of these interactions, we have used isothermal titration calorimetry to study the pH dependence of the binding of the SH2 domain of the Src kinase to a high-affinity tyrosyl phosphopeptide. Two independent approaches were employed. In a first series of experiments that focused on determining the peptide's association constant between pH 5.0 and 9.0, two ionizable groups were characterized. One group, with free and bound pKas of 6.2 and 4.4, respectively, could be identified as the phosphate in the phosphotyrosine while the other group, with free and bound pKas of 8.2 and 8.5, respectively, could be only tentatively assigned to a cysteine in the phosphotyrosine binding pocket. Further information on the linkage between peptide binding and protonation of the phosphotyrosine was obtained from a second series of experiments, which focused on determining the peptide binding enthalpy at low values of pH in several buffers with different ionization enthalpies. These data provided free and bound pKa values for the phosphotyrosine identical to those derived from the first series of experiments, and hence demonstrated for the first time that the two approaches provide identical information regarding proton linkage. In addition, the second series of experiments also determined the intrinsic enthalpy of binding of both the protonated and deprotonated phosphate forms of the peptide. These two sets of experiments provided a complete energetic profile of the linkage between phosphate ionization and peptide binding. From this profile, it was determined that the PO32- form of the peptide binds 2.3 kcal mol-1 more favorably than the PO3H1- form due entirely to a more favorable entropy of binding.

Animals↗

PubMed2003

Incorporation of casein phosphopeptide-amorphous calcium phosphate into a glass-ionomer cement.

Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) nanocomplexes have been shown to prevent demineralization and promote remineralization of enamel subsurface lesions in animal and in situ caries models. The aim of this study was to determine the effect of incorporating CPP-ACP into a self-cured glass-ionomer cement (GIC). Incorporation of 1.56% w/w CPP-ACP into the GIC significantly increased microtensile bond strength (33%) and compressive strength (23%) and significantly enhanced the release of calcium, phosphate, and fluoride ions at neutral and acidic pH. MALDI mass spectrometry also showed casein phosphopeptides from the CPP-ACP nanocomplexes to be released. The release of CPP-ACP and fluoride from the CPP-ACP-containing GIC was associated with enhanced protection of the adjacent dentin during acid challenge in vitro.

Analysis of Variance↗

PubMed2006

RNA polymerase II CTD phosphopeptides compete with RNA for the interaction with Pcf11.

In Saccharomyces cerevisiae, the cleavage/polyadenylation factor Pcf11 is an important regulatory factor required for recruiting the polyadenylation machinery to the elongating RNA polymerase II (RNAPII) and is necessary for correct transcriptional termination. The interaction with RNAPII is mediated by a CTD-interacting domain (CID) located in the N-terminal region of Pcf11 that binds in a phospho-dependent manner the heptad repeats in the RNAPII CTD. We have previously investigated this protein-protein interaction. We examine here the interaction of the CID with different RNA sequences and look at the effect of phosphopeptides derived from the CTD heptad repeats on the RNA-protein interaction. Our findings demonstrate that the CID displays weak RNA-binding activity, but with some degree of sequence preference, the RNA-protein and peptide-protein interfaces overlap and the CTD-derived phosphopeptides and RNA compete for the binding site. We propose that competition between the protein-peptide and the protein-RNA interaction is important mechanistically and required for the disengagement of polyadenylation factors from RNAPII.

Base Sequence↗

PubMed2003

Stimulatory effect of a dietary casein phosphopeptide preparation on the mucosal IgA response of mice to orally ingested lipopolysaccharide from Salmonella typhimurium.

The effect on immunoglobulin production of a commercially available casein phosphopeptide preparation (CPP-III) consisting mainly of bovine alpha s2-casein (1-32) and beta-casein (1-28) in mice that had orally ingested lipopolysaccharide (LPS) from Salmonella typhimurium was investigated. No significant difference in body weight gain was observed between the mice fed on the CPP-III-added diet and those fed on the control diet. The mice fed on the CPP-III-added diet exhibited similar serum and intestinal IgG, IgM, and IgE responses towards LPS to those fed on the control diet. In contrast, fecal and intestinal anti-LPS IgA and total IgA in mice fed on the CPP-III-added diet were significantly higher than in those fed on the control diet. Spleen cells from mice fed on the CPP-III-added diet produced larger amounts of IgA, IL-5, and IL-6 than cells from mice fed on the control diet. These results suggest that dietary casein phosphopeptide may protect a host from invasion of the intestinal mucosa by food-born pathogenic microorganisms.

Adjuvants, Immunologic↗

PubMedDate not supplied

Adding casein phosphopeptide-amorphous calcium phosphate to sports drinks to eliminate in vitro erosion.

PURPOSE: Enamel erosion can occur with frequent consumption of sports drinks. The purpose of this study was to determine a minimal concentration of casein phosphopeptide-stabilized amorphous calcium phosphate (CPP-ACP) which when added to a sports drink would eliminate such erosion in vitro. METHODS: Human enamel specimens were immersed in: (1) the sports drink Powerade; (2) Poweradeplus 4 concentrations of CPP-ACP (0.063%, 0.09%, 0.125%, 0.25%); or (3) double deionized water. Windows of test and control enamel were profiled, and the enamel surface characteristics were examined under scanning electron microscopy (SEM). RESULTS: The pH of test solutions increased and the titratable acidity decreased with increasing CPP-ACP concentrations. Erosive step lesions occurred in specimens immersed in Powerade (mean depth=38.70kA +/- 5.60), which were eliminated by the addition of CPP-ACP to Powerade at all test concentrations except 0.063% CPP-ACP. Microscopic surface irregularities on test enamel were observed, apparent as adherent granules or globules. These may represent redeposited mineral phases following mobilization of calcium and phosphate from CPP-ACP. Tasters in a taste panel could not distinguish Powerade from Powerade plus 0.125% CPP-ACP. CONCLUSIONS: Adding casein phosphopeptide-stabilized amorphous calcium phosphate to the sports drink Powerade significantly reduced the beverage's erosivity without affecting the product's taste.

Analysis of Variance↗

PubMed1987

Dephosphorylation of phosphoproteins and synthetic phosphopeptides. Study of the specificity of the polycation-stimulated and MgATP-dependent phosphorylase phosphatases.

The substrate specificity of different forms of polycation-stimulated (PCSH, PCSL, and PCSC) phosphorylase phosphatases and of the catalytic subunit of the MgATP-dependent protein phosphatase from rabbit skeletal muscle was investigated. This was done, with phosphorylase a as the reference substrate, using the synthetic phosphopeptides patterned after the phosphorylated sites of pyruvate kinase (type L) (Arg2-Ala-Ser(32P)-Val-Ala (S2), and its Thr(32P) substitute (T4)), inhibitor-1 (Arg4-Pro-Thr(32P)-Pro-Ala (T5), Arg2-Pro-Thr(32P)-Pro-Ala (T1), and its Ser(32P) substitute (S1)), and some modified phosphopeptides (Arg2-Ala-Thr(32P)-Pro-Ala (T2) and Arg2-Pro-Thr(32P)-Val-Ala (T3)), all phosphorylated by cyclic AMP-dependent protein kinase. In addition, casein(Thr-32P), phosphorylated by casein kinase-2, was also tested. The PCS phosphatases show a striking preference for the T4 configuration, PCSC being the least efficient. The catalytic subunit of the MgATP-dependent phosphatase was almost completely inactive toward all these substrates. As shown for the PCSH phosphatase, and comparing with T4, the two proline residues flanking the Thr(P) in T1 and T5, just as in inhibitor-1, drastically imparied the dephosphorylation by lowering the Vmax and not by affecting the apparent Km. The C-terminal proline (as in T2) by itself represents a highly unfavorable factor in the dephosphorylation. The critical effect of the sequence X-Thr(P)-Pro or Pro-Thr(P)-Pro (T1, T2, T5, and inhibitor-1) can be overcome by manganese ions. The additional finding that this is not the case with the Pro-Ser(P)-Pro sequence (S1) suggests that the effect of Mn2+ is highly substrate specific. These observations show the considerable importance of the primary structure of the substrate in determining the specificity of the protein phosphatases.

Adenosine Triphosphate↗

PubMed1982

Phosphoproteins in the parotid saliva from the subhuman primate Macaca fascicularis. Isolation and characterization of a proline-rich phosphoglycoprotein and the complete covalent structure of a proline-rich phosphopeptide.

Parotid saliva from the cynomolgus monkey (Macaca fascicularis) and from pooled human collections displayed the same groups of proteins when fractionated by anion exchange and gel filtration chromatography. We have isolated and characterized a proline-rich phosphoglycoprotein (MPRP) and a proline-rich phosphopeptide (M-statherin) from macaque parotid saliva. MPRP has an apparent molecular weight of 16,900 and displays an unusual chemical composition. It is enriched in proline, glycine, and acidic amino acids, but lacks cysteine, methionine, and tyrosine. MPRP contains 25% (w/w) carbohydrate with 7.0 mol of neutral hexoses, 5.3 mol of galactosamine, 5.9 mol of sialic acid, and 3 mol of phosphorus/mol of protein. M-statherin is a 42-residue phosphopeptide with a high proline, glutamic acid, and tyrosine content, but which lacks threonine, valine, cysteine, methionine, isoleucine, and histidine. The complete covalent structure of M-statherin (Mr = 5,368) is: NH2-Asp-Pse-Pse-Glu-Glu-Lys-Phe-Leu-Arg-Arg-Leu-Arg-Arg-Phe-Asp-Glu-Gly-Arg-Tyr- -Gly-Pro-Tyr-Gln-Pro-Phe-Ala-Pro-Gln-Pro-Leu-Tyr-Pro-Gln-Pro-Tyr-Gln-Pro-Tyr-Gln-Pro-Gln-Tyr-COOH This is the first complete amino acid sequence of a component in the salivary secretion of a subhuman primate. Phosphoserine occurs at residues 2 and 3. All 13 acidic and basic amino acids are located in the NH2-terminal half of the molecule. The carboxyl-terminal half of the molecule is hydrophobic where the tripeptide Tyr-Gln-Pro is repeated three times, the dipeptide Gln-Pro occurs twice, and the tripeptides Tyr-Gly-Pro, Phe-Ala-Pro, and Leu-Tyr-Pro occur once. Evaluation of secondary structure by the Chou-Fasman method predicts an alpha helix in the NH2-terminal half (residue 4-16) and a beta pleated sheet in the carboxyl-terminal half (residues 22-26; 38-42) of the molecule. Both MPRP and M-statherin inhibit spontaneous and seeded precipitation from solutions supersaturated with respect to calcium phosphate salts. This suggests that these macaque compounds may function by maintaining saliva supersaturated with respect to calcium phosphate salts, a necessary requirement for stabilization of hydroxyapatite in the surface layers of teeth.U

Amino Acid Sequence↗

PubMed2000

Enzymatic protecting group techniques for glyco- and phosphopeptide chemistry: synthesis of a glycophosphopeptide from human serum response factor.

The covalent modification of proteins by phosphorylation and by glycosylation with GlcNAc residues are important regulatory processes which mediate biological signal transduction. For the study of such biological phenomena in molecular detail characteristic peptides which embody both types of modification may serve as efficient tools. However, their synthesis is complicated by their pronounced acid and base lability as well as their multifunctionality. For this purpose the enzyme-labile choline ester was developed. The choline ester can be removed selectively and in high yields from various GlcNAc-glycopeptides and phosphopeptides at pH 6.5 and 37 degrees C. The conditions under which the enzymatic deprotections proceed are so mild that no undesirable side reactions are observed (i.e., no cleavage or anomerization of the glycosidic bonds and no beta-elimination of the phosphate or the carbohydrate occur). The specificity of the biocatalyst guarantees that neither the peptide bonds nor the other protecting groups present are being attacked. When this enzymatic protecting group technique was combined with the enzyme-labile 4-(phenylacetoxy)benzyloxycarbonyl (PhAcOZ) urethane protecting group a complex glycophosphopeptide could be built up. The glycopeptide is equipped with a biotin label by which it can be traced in biological systems. This peptide represents a characteristic partial structure of a glycosylated and phosphorylated sequence from the transactivation domain of serum response factor (SRF), a widely occurring human transcription factor.

Amino Acid Sequence↗

PubMed2002

Cytochemical assessment of phosphopeptides derived from casein as potential ingredients for functional food.

Phosphopeptides derived from casein hydrolysis are suggested to have beneficial effects beyond their mere nutritive value by enhancing availability of dietary minerals, especially calcium, iron and zinc. Apart from a possible positive action the potential may exist for adverse effects that could impose restrictions to their widespread application in functional foods for human nutrition. In the present work, various case-inophosphopeptide (CPP) preparations were assessed using different human cell culture model systems in order to estimate their cytotoxic potential and their influence on epithelial properties and differentiation of human intestinal cells (Caco-2). The general cytotoxic potential of CPPs was tested using the AlphaTox NR assay. Structural and functional differentiation of intestinal Caco-2 cells was determined by measuring transepithelial electrical resistance and activity of brush border associated alkaline phosphatase over an extended cultivation period. No loss in cell viability with respect to membrane integrity could be detected, as uptake and retention of neutral red dye (AlphaTox assay) into HeLa cells was not affected by the presence of CPPs. Cytochemical assays conducted on epithelial cells (Caco-2) showed no disturbance of normal cell development and differentiation with respect to structural as well as functional differentiation markers. As all CPPs tested did not provoke any adverse effects in terms of cytotoxicity and showed no impairment of cell differentiation and monolayer integrity of human intestinal cells, it may be supposed that CPPs do not provoke a cytotoxic response in vivo in the cells assayed in the present study.

Alkaline Phosphatase↗

PubMed1995

High performance capillary electrophoresis of casein phosphopeptides containing 2-5 phosphoseryl residues: relationship between absolute electrophoretic mobility and peptide charge and size.

Multiple phosphoseryl-containing peptides from casein containing the cluster sequence -Ser(P)-Ser(P)-Ser(P)-Glu-Glu- stabilize amorphous calcium phosphate at neutral and alkaline pH and have been shown to be anticariogenic in various in vitro, animal and human experiments. In an approach to obtain insight into the structure and function of these peptides, we previously developed a method for their analysis using high-performance capillary electrophoresis (HPCE). A linear relationship was obtained between absolute electrophoretic mobility and q/M2/3 where q is the net negative charge of the peptide calculated using relevant pKa values and M is the molecular mass. M2/3 is a measure of the surface area of a sphere that has a volume proportional to the M of the peptide and relates to the frictional drag exerted on the peptide during electrophoretic migration. To examine this relationship further, we have analyzed a range of high-performance liquid chromatography (HPLC)-purified cluster, di- and triphosphorylated peptides from casein enzymic (trypsin, alcalase and pancreatin) digests using HPCE and attempted to relate their absolute electrophoretic mobility to various models incorporating peptide size and charge. Absolute electrophoretic mobility of casein phosphopeptides containing the cluster sequence-Ser(P)-Ser(P)-Ser(P)-Glu-Glu- was most closely correlated with q/M2/3. Di- and triphosphorylated peptide absolute electrophoretic mobility correlated with both q/M2/3 and ln(q+l)/n0.43. However, for both the q/M2/3 and In(q+l)/n0.43 relationships with absolute electrophoretic mobility, the di- and triphosphorylated peptides formed a separate linear relationship to that of the cluster peptides. From these relationships, a di- or triphosphorylated peptide exhibited a greater absolute electrophoretic mobility than a corresponding cluster peptide with sam q/M2/3 or In(q+l)/n0.43 value.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1986

Activation of ribosomal protein S6 phosphorylation during meiotic maturation of Xenopus laevis oocytes: in vitro ordered appearance of S6 phosphopeptides.

During meiotic maturation of Xenopus laevis stage 6 oocytes into unfertilized eggs, 40S ribosomal protein S6 undergoes multiple phosphorylation. Extracts prepared from unfertilized eggs are up to 10-fold more efficient in phosphorylating S6 than those prepared from immature oocytes. When analyzed by DEAE chromatography the S6 kinase activity elutes as a single peak. If extracts from unfertilized eggs are prepared in the absence of beta-glycerol phosphate, a putative phosphatase inhibitor, there is a severe reduction in recovered S6 kinase activity. Under optimal conditions, incubation of unfertilized egg extracts with 40S ribosomes in the presence of ATP leads to the average incorporation of 3.5 mol of phosphate/mol of S6. Prior incubation of these extracts with the cAMP-dependent protein kinase inhibitor does not inhibit S6 phosphorylation indicating that another kinase is responsible. Analysis of the in vitro phosphorylated peptides demonstrates that they migrate to the equivalent position of those observed previously in vivo and in vitro. More strikingly, if each of the increasingly phosphorylated derivatives of S6 is analyzed independently, it is found that the phosphopeptides appear in a specific order.

Animals↗

PubMed1993

CD45 specifically modulates binding of Lck to a phosphopeptide encompassing the negative regulatory tyrosine of Lck.

CD45 is a tyrosine phosphatase expressed in all hematopoietic cells which is important for signal transduction through the T cell antigen receptor (TCR). Studies using CD45-deficient cells have revealed that Lck, a tyrosine kinase thought to be essential for TCR signaling, is hyperphosphorylated on Y505 in the absence of CD45. This site of tyrosine phosphorylation negatively regulates the function of the Src family of kinases. Here we provide evidence that CD45 can modulate the binding of the Lck to an 11 amino acid tyrosine phosphorylated peptide containing the carboxy-terminus of Lck (lckP). Significantly, CD45 did not influence the binding of Fyn, PLC gamma 1, GAP and Vav to the same phosphopeptide. Lck protein which bound the peptide was dephosphorylated on Y505 and consisted of only 5-10% of the total cellular Lck. Interestingly, there was a marked increase in binding 15-30 min after CD4 or TCR cross-linking. Taken together, our data suggest that CD45 specifically modulates the conformation of Lck in a manner consistent with the intramolecular model of regulation of Src-like kinases.

Amino Acid Sequence↗

PubMed2002

Phosphopeptide detection and sequencing by matrix-assisted laser desorption/ionization quadrupole time-of-flight tandem mass spectrometry.

A prototype matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI-TOF) tandem mass spectrometer was used to sequence a series of phosphotyrosine-, phosphothreonine- and phosphoserine-containing peptides. The high mass resolution and mass accuracy of the instrument allowed the localization of one, three or four phosphorylated amino acid residues in phosphopeptides up to 3.1 kDa. Tandem mass spectra of two different phosphotyrosine peptides permitted amino acid sequence determination and localization of one and three phosphorylation sites, respectively. The phosphotyrosine immonium ion at m/z 216.04 was observed in these MALDI low-energy CID tandem mass spectra. Elimination of phosphate groups was evident from the triphosphorylated peptide but not from the monophosphorylated species. The main fragmentation pathway for the synthetic phosphothreonine-containing peptide and for phosphoserine-containing peptides derived from beta-casein and ovalbumin was the beta-elimination of phosphoric acid with concomitant conversion of phosphoserine to dehydroalanine and phosphothreonine to 2-aminodehydrobutyric acid. Peptide fragment ions of the b- and y-type allowed, in all cases, the localization of phosphorylation sites. Ion signals corresponding to (b-17), (b-18) and (y-17) fragment ions were also observed. The abundant neutral loss of phosphoric acid (-98 Da) is useful for femtomole level detection of phosphoserine-peptides in crude peptide mixtures generated by gel in situ digestion of phosphoproteins.

Amino Acid Sequence↗

PubMed2007

Optimization of phosphopeptide elution conditions in immobilized Fe(III) affinity chromatography.

While immobilized metal affinity chromatography (IMAC) has been widely used for affinity purification of phosphopeptides, the technique suffers from insufficient specificity. Therefore, there is an urgent need for IMAC optimization to yield the selectivity and sensitivity that is required for more challenging analyses. Recently, 2,5-dihydroxybenzoic acid (DHB) and phosphoric acid mixture has been reported as an efficient IMAC eluant. The disadvantage of DHB is that is not suitable for electrospray ionization-mass spectrometry. While further developing the IMAC elution protocol to overcome this problem, we noticed that DHB is not necessary and found a novel combination of phosphoric acid and acetonitrile to be more efficient. The purification efficacy of the novel protocol is superior to all previously described methods, while still being compatible with the most commonly used mass-spectrometric techniques in phosphoproteomics.

Chromatography, Affinity↗