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PubMed2005

Iodoacetamide-alkylated methionine can mimic neutral loss of phosphoric acid from phosphopeptides as exemplified by nano-electrospray ionization quadrupole time-of-flight parent ion scanning.

Formation of S-carbamidomethylmethionine (camMet) occurs as a side reaction during cysteine alkylation with iodoacetamide (IAA). In collision-induced dissociation, peptides with camMet show an abundant neutral loss of 2-(methylthio)acetamide (C3H7NOS = 105.025 Da) at moderate collision offset values which are similar to those optimal for loss of phosphoric acid (H3PO4 = 97.977 Da). Neutral loss analysis is used for spotting of phosphopeptides which contain phosphoserine (pSer) or phosphothreonine (pThr) residues. In the case where precursor ions cannot be accurately assigned in the survey spectrum (e.g. due to low ion abundance or signal overlap), the mass accuracy of a neutral loss tandem mass spectrometry (MS/MS) analysis depends on the precursor ion isolation window. For the charge states 2+, 3+ or 4+, a typical 3.5 Da precursor isolation window results in neutral loss windows of 7, 10.5 or 14 Da, respectively. Consequently, neutral loss of 105 Da from alkylated methionine residues can mimic the phosphoserine/phosphothreonine-specific neutral loss of 98 Da. In the evaluation of quadrupole time-of-flight (QTOF) parent ion scan data for neutral loss of H3PO4, this interference was frequently observed. It is illustrated in this study using the analysis of ovalbumin phosphorylation as an example. The +80 Da molecular weight shift connected with phosphorylation at serine or threonine may also be mimicked by carbamidomethylation of methionine through a combination with sodium adduction (+57 Da +22 Da = +79 Da). For highly sensitive neutral loss analysis of serine and threonine phosphorylation, careful data inspection is recommended if reduction and alkylation by IAA is employed.

Alkylation↗

PubMed2001

Selective analysis of phosphopeptides within a protein mixture by chemical modification, reversible biotinylation and mass spectrometry.

A new method combining chemical modification and affinity purification is described for the characterization of serine and threonine phosphopeptides in proteins. The method is based on the conversion of phosphoserine and phosphothreonine residues to S-(2-mercaptoethyl)cysteinyl or beta-methyl-S-(2-mercaptoethyl)cysteinyl residues by beta-elimination/1,2-ethanedithiol addition, followed by reversible biotinylation of the modified proteins. After trypsin digestion, the biotinylated peptides were affinity-isolated and enriched, and subsequently subjected to structural characterization by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Database searching allowed for automated identification of modified residues that were originally phosphorylated. The applicability of the method is demonstrated by the identification of all known phosphorylation sites in a mixture of alpha-casein, beta-casein, and ovalbumin. The technique has potential for adaptations to proteome-wide analysis of protein phosphorylation.

Amino Acid Sequence↗

PubMed2000

Phosphopeptide sequencing by in-source decay spectrum in delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Protein phosphorylation underlies numerous cellular signaling processes. Since a reliable prediction of phosphorylation site(s) based on a consensus amino acid sequence is rather difficult to date, determination of phosphorylation site(s) in phosphoproteins is a crucial step toward the understanding of their function at the molecular level. A conventional protocol for the determination of phosphorylation sites utilizes radioactive labeling of a phosphoprotein by (32)P and purification of digested peptides carrying radioactivity, followed by Edman degradation. This method is not only tedious, but also indirect because the evidence will be based on disappearance of a phenylthiohydantoin signal from the degradation cycle where the (32)P radioactivity is eluted. Several methodologies have been developed to determine the phosphorylation sites directly by using mass spectrometry. These include collision-induced dissociation (CID) and post-source decay (PSD), both of which tend to produce fragment ions less efficiently as the number of residues exceeds 20. Moreover, in both decay processes, there is a tendency for the phosphate group to be removed during the breakdown of the main peptide chain. We report a method that allows direct observation of phosphorylated peptide fragments of phosphopeptides exceeding 20 residues by using an in-source decay fragmentation by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, yielding results which are difficult or impossible to obtain by existing methods using CID or PSD.

Animals↗

PubMed1999

Structure and function of a new phosphopeptide-binding domain containing the FHA2 of Rad53.

The forkhead-associated (FHA) domain is a 55-75 amino acid residue module found in >20 proteins from yeast to human. It has been suggested to participate in signal transduction pathways, perhaps via protein-protein interactions involving recognition of phosphopeptides. Neither the structure nor the ligand of FHA is known. Yeast Rad53, a checkpoint protein involved in DNA damage response, contains two FHA domains, FHA1 (residues 66-116) and FHA2 (residues 601-664), the second of which recognizes phosphorylated Rad9. We herein report the solution structure of an "FHA2-containing domain" of Rad53 (residues 573-730). The structure consists of a beta-sandwich containing two antiparallel beta-sheets and a short, C-terminal alpha-helix. Binding experiments suggested that the FHA2-containing domain specifically recognizes pTyr and a pTyr-containing peptide from Rad9, and that the binding site involves residues highly conserved across FHA domains. The results, along with other recent reports, suggest that FHA domains could have pTyr and pSer/Thr dual specificity.

Amino Acid Sequence↗

PubMed2002

Dissection of the energetic coupling across the Src SH2 domain-tyrosyl phosphopeptide interface.

Src Homology (SH2) domains play critical roles in signaling pathways by binding to phosphotyrosine (pTyr)-containing sequences, thereby recruiting SH2 domain-containing proteins to tyrosine-phosphorylated sites on receptor molecules. Investigations of the peptide binding specificity of the SH2 domain of the Src kinase (Src SH2 domain) have defined the EEI motif C-terminal to the phosphotyrosine as the preferential binding sequence. A subsequent study that probed the importance of eight specificity-determining residues of the Src SH2 domain found two residues which when mutated to Ala had significant effects on binding: Tyr beta D5 and Lys beta D3. The mutation of Lys beta D3 to Ala was particularly intriguing, since a Glu to Ala mutation at the first (+1) position of the EEI motif (the residue interacting with Lys beta D3) did not significantly affect binding. Hence, the interaction between Lys beta D3 and +1 Glu is energetically coupled. This study is focused on the dissection of the energetic coupling observed across the SH2 domain-phosphopeptide interface at and around the +1 position of the peptide. It was found that three residues of the SH2 domain, Lys beta D3, Asp beta C8 and AspCD2 (altogether forming the so-called +1 binding region) contribute to the selection of Glu at the +1 position of the ligand. A double (Asp beta C8Ala, AspCD2Ala) mutant does not exhibit energetic coupling between Lys beta D3 and +1 Glu, and binds to the pYEEI sequence 0.3 kcal/mol tighter than the wild-type Src SH2 domain. These results suggest that Lys beta D3 in the double mutant is now free to interact with the +1 Glu and that the role of Lys beta D3 in the wild-type is to neutralize the acidic patch formed by Asp beta C8 and AspCD2 rather than specifically select for a Glu at the +1 position as it had been hypothesized previously. A triple mutant (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) has reduced binding affinity compared to the double (Asp beta C8Ala, AspCD2Ala) mutant, yet binds the pYEEI peptide as well as the wild-type Src SH2 domain. The structural basis for such high affinity interaction was investigated crystallographically by determining the structure of the triple (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) mutant bound to the octapeptide PQpYEEIPI (where pY indicates a phosphotyrosine). This structure reveals for the first time contacts between the SH2 domain and the -1 and -2 positions of the peptide (i.e. the two residues N-terminal to pY). Thus, unexpectedly, mutations in the +1 binding region affect binding of other regions of the peptide. Such additional contacts may account for the high affinity interaction of the triple mutant for the pYEEI-containing peptide.

Binding Sites↗

PubMed1993

The effect of casein phosphopeptides on calcium utilization in young ovariectomized rats.

The effect of casein phosphopeptides (CPPs) on Ca utilization in ovariectomized (OVX) rats was studied. A mixture of CPPs corresponding to the amino acid sequences 1-25 and 1-28 in the beta-casein was isolated from the tryptic digest of beta-casein (beta CPP). After being fed a low Ca diet for 30 days, OVX rats were fed experimental diets of which the Ca level was 0.1%, 0.3% or 0.5% with or without 0.15% beta CPP for 28 days. During days 1-3 of the Ca refeeding period, rats fed beta CPP with 0.5% Ca showed a higher Ca absorption than control rats not supplemented with beta CPP. During days 7-9 and 26-28, there were no significant differences in Ca and P balances between the beta CPP group and the control group for each dietary Ca level. Femoral Ca and P contents from rats fed beta CPP tended to be higher than those from control rats. These results suggest that beta CPP supplementation could have an effect on Ca absorption at a certain degree of Ca deficiency.

Absorption↗

PubMed1995

Characterization of trypsin immobilized on oxirane-acrylic beads for obtaining phosphopeptides from casein.

The aim of the study was to characterize the proteolytic properties of immobilized trypsin for obtaining phosphopeptide-rich fractions from casein. Trypsin was covalently bound to oxirane-acrylic beads. After incubation for 48 h immobilization degrees of about 85% were achieved. 20% of the immobilized enzyme exhibited no activity towards the substrate N-benzoyl-L-arginine ethyl ester. Compared with homogeneous catalysis with the soluble enzyme a 25% lower degree of proteolysis was calculated and a modified peptide pattern of the resulting proteolysates established. A caseinophosphopeptide (CPP) from alpha s1-CN (59-79) was not detectable after incubation with the carrier-bound enzyme. At a substrate concentration (S) of 15% (w/w) substrate saturation of the enzyme (E) was achieved. Increasing the substrate concentration to 20% (w/w) decreased the conversion rates (content of soluble amino-N) and the liberation of CPPs. Proteolysis of small (1% w/w) and partly also large (20% w/w) substrate concentrations (E/S = 1/100) is subject to changed kinetic conditions. The same was true for small and large enzyme concentrations (S = 5% w/w). Compared with enzyme saturation (E/S = 1/50), lack of enzyme (E/S = 1/800) led to a disproportional decrease in the proteolysis rate and to a markedly decreased content of hydrophobic peptides in the resulting proteolysates. Increasing the pH from 7.8 to 8.8 and the temperature from 37 degrees to 47 degrees C caused only a slight increase in conversion rates, but an overproportional liberation of CPPs (pH 8.8 = + 47%, 47 degrees C = +89%), in particular from beta-casein. Repeated use of immobilized trypsin resulted after nine runs in a loss in proteolytic activity and in CPP yields of approximately 25%, while the peptide pattern of the proteolysates remained qualitatively unchanged. Light microscopy shows that the oxirane-acrylic beads disintegrate to a large extent after nine repetitions.

Acrylates↗

PubMed1986

Casein phosphopeptides: their effect on calcification of in vitro cultured embryonic rat bone.

The effect of casein phosphopeptides (CPP) and enzymatically dephosphorylated, otherwise identical peptides (DPP) on Ca solubility and mineralization was studied in an in vitro model. The model consisted of explanted embryonic rat bone rudiments (femora, tibiae, and metatarsals), cultured in Bigger's medium at two different concentrations of total calcium (1.78 mM and 3.24 mM). The medium enabled growth of the explants, but minor or no mineralization took place during a culture period of 11 days. The medium was unstable with respect to Ca solubility, and measurements with a selective electrode showed that during most of the time the actual concentration of soluble, ionic Ca2+ was not higher than about 0.7 mM. CPP added at a concentration of 10 mg/100 ml of the medium vastly improved Ca-solubility under culture conditions. The mean concentration of soluble, ionic Ca2+ in the presence of the peptides was 1.4 mM at total calcium = 1.78 mM and 2.3 mM at the higher total calcium level (3.24 mM). Rudiments cultured in the presence of CPP showed impressive increases in calcification of their diaphyseal area. This progress in mineralization was quantitatively assayed by measuring the projected dark diaphyseal area at the beginning and at the end of the culture period. It was also verified by histological sections of the rudiments. DDP did not enhance Ca solubility of the culture medium, nor did explants cultured in the presence of 10 mg/100 ml of DPP show any significant progress in mineralization. This indicated that the activity of CPP was dependent on the presence of serine-linked phosphate groups.

Animals↗

PubMed1995

The synthesis and compositional analysis of phosphopeptides.

Protein phosphorylation plays a critical role in the regulation of cell growth and differentiation. There is considerable interest, therefore, in the facile synthesis of peptides that possess selectively phosphorylated residues for use as molecular probes in mechanistic studies of the biological consequences of phosphorylation. This work will review the various synthetic protocols used in the generation of phosphopeptides and will discuss their characterization by amino acid compositional analysis.

Amino Acids↗

PubMed2005

Speciation analysis of calcium, iron, and zinc in casein phosphopeptide fractions from toddler milk-based formula by anion exchange and reversed-phase high-performance liquid chromatography-mass spectrometry/flame atomic-absorption spectroscopy.

Casein phosphopeptides (CPP) are phosphorylated casein-derived peptides that can be released by in-vitro or in-vivo enzymatic hydrolysis of alpha(s1)-casein, alpha(s2)-casein, and beta-casein (CN). Many of these peptides contain a highly polar acidic sequence of three phosphoseryl groups followed by two glutamic acid residues. These domains are binding sites for minerals such as calcium, iron, and zinc and play an important role in mineral bioavailability. The aim of this study was speciation analysis of calcium, iron, and zinc in CPP fractions from the soluble fraction of a toddler milk-based formula. Methods for CPP separation by anion-exchange high-performance liquid chromatography (AE-HPLC) were combined with CPP identification by reversed-phase high performance liquid chromatography-electrospray ionization mass spectrometry and determination of the calcium, iron, zinc, and phosphorus content of the fractions obtained by AE-HPLC. Calcium and phosphorus were detected in all the analyzed AE-HPLC fractions. Calcium and zinc could be bound to CPP derived from alpha(s1)-CN and alpha(s2)-CN in fraction 3. Iron could be bound to CPP in fraction 4 in which beta-CN(15-34)4P was present with the cluster sequence S(P)S(P)S(P)EE. The results obtained prove the different distribution of calcium, iron, and zinc in heterogeneous CPP fractions.

Amino Acid Sequence↗

PubMed1976

A study of the enzymic dephosphorylation of beta-casein and a derived phosphopeptide.

beta-Casein, and the phosphate containing peptide derived from it by tryptic digestion, have been dephosphorylated by the action of two phosphatases. Escherichia coli alkaline phosphatase (EC 3.1.3.1) has been shown to remove the phosphates from these substrates in two distinct stages. Substrate molecules retaining three of the original phosphoseryl residues accumulate during the reaction and are resistant to further dephosphorylation at low enzyme concentrations. In contrast bovine spleen phosphoprotein phosphatase (EC 3.1.3.16) achieves complete dephosphorylation of these substrates sequentially without any of the intervening species showing resistance to the action of the enzyme. The phosphopeptide has been partially dephosphorylated by the action of the two phosphatases and the resultant peptides containing three phosphoseryl residues compared in their reactivity toward the E. coli alkaline phosphatase. The results obtained are discussed in relation to the mode of action of the two enzymes.

Alkaline Phosphatase↗

PubMed1989

Characterization of phosphopeptides released during dialysis of EDTA-reacted highly purified DNA prepared from calf thymus nuclei.

The treatment of highly purified DNA obtained from calf thymus nuclei (N-DNA) with a chelating agent and subsequent repeated dialyses led to release of phosphopeptides (PPs) into the dialysates. By means of anion exchange column chromatography, the PPs were separated into 9 main fractions. Two of them (P1 and P5) contained the amino acids phosphoserine, asp, thr, ser, glu, gly, ala, val, ile, leu, and arg, as well as metal ion complexes of phosphoserine. The complexes were dissociated by deionization with nitrilotriacetate + Chelex. The proportion of phosphoserine was about twice as great in P5 as in P1. Whereas P1 and P5 contained essentially no nucleotide material, the other fractions contained ribonucleotides and deoxynucleotides. The deoxynucleotide content was less than 10% of that of total nucleotides. After a deionizing treatment, the amounts of nucleotides in these fractions were reduced to a level corresponding to 1 nucleotide per peptide of 5-15 amino acids.

Amino Acids↗

PubMed1990

Identification of phosphopeptides by mass spectrometry.

Phosphopeptides can be identified by ion spray mass spectrometry. The method was tested with phosphokemptide and with a proteolytic digest of one subunit (delta-subunit) of the nicotinic acetylcholine receptor. In the latter one peptide containing tyrosine phosphate, one with two serine phosphates, and two different peptides each containing one serine phosphate were unambiguously identified. Thus it is proven that ion spray mass spectrometry can be applied for the localization of phosphorylation sites in a known primary structure.

Chromatography, High Pressure Liquid↗

PubMed1995

Synthetic phosphopeptide from rhodopsin sequence induces retinal arrestin binding to photoactivated unphosphorylated rhodopsin.

A synthetic heptaphosphopeptide comprising the fully phosphorylated carboxyl terminal phosphorylation region of bovine rhodopsin, residues 330-348, was found to induce a conformational change in bovine arrestin. This caused an alteration of the pattern of limited proteolysis of arrestin similar to that induced by binding phosphorylated rhodopsin or heparin. Unlike heparin, the phosphopeptide also induced light-activated binding of arrestin to both unphosphorylated rhodopsin in disk membranes as well as to endoproteinase Asp-N-treated rhodopsin (des 330-348). These findings suggest that one function of phosphorylation of rhodopsin is to activate arrestin which can then bind to other regions of the surface of the photoactivated rhodopsin.

Amino Acid Sequence↗

PubMed1995

Insulin resistance is associated with abnormal dephosphorylation of a synthetic phosphopeptide corresponding to the major autophosphorylation sites of the insulin receptor.

Insulin resistance in the ob/ob mouse model is associated with a reduction in insulin-induced protein-tyrosine phosphorylation in tissues such as liver. To ascertain whether this decrease in phosphorylation may be due to increased phosphatase activity, protein-tyrosine phosphatase (PTPase) activity was determined in particulate and soluble fractions from livers of 5- to 23-week-old ob/ob mice and age-matched lean littermates. PTPase activity was measured using a synthetic phosphopeptide, TRDIY(P)ETDY(P)Y(P)RK, as the substrate, corresponding to residues 1142 to 1153 of the insulin receptor and containing the major autophosphorylation sites of the regulatory domain. The ob/ob mice were hyperinsulinemic across all age groups, but only the youngest mice (aged 5 to 7 weeks) were hyperglycemic. Most PTPase activity was present in the liver particulate fraction and was 19% to 114% greater in ob/ob mice as compared with controls. PTPase activity in the liver soluble fraction was 26% less than control values in the youngest ob/ob mice (5 to 7 weeks), but increased with age and was 41% and 131% above control values at 21 to 23 and 25 to 27 weeks of age, respectively. Oral administration of the PTPase inhibitor sodium orthovanadate (0.6 mg/mL in drinking water for 2 weeks) to young ob/ob mice caused a significant reduction in the elevated particulate PTPase activity, with concomitant decreases in plasma insulin and plasma glucose. Assessment of PTPase activity with a monophosphate form of the same synthetic peptide, TRDIY(P)ETDYYRK, showed lower PTPase activities as compared with the triphosphate form and no significant differences between ob/ob and control preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1991

Characterization of phosphopeptide derived from bovine beta-casein: an inhibitor to intra-intestinal precipitation of calcium phosphate.

Casein phosphopeptide (CPP), a highly phosphorylated peptide fragment which inhibits the formation of hydroxyapatite crystal was isolated from pooled ileal contents of rats fed a semi-synthetic diet containing bovine beta-casein. The estimated amino acid sequence of the CPP was shorter than that of the trypsin-digested beta-casein but the core region consisting of consecutive bindings of phosphoserine was fully conserved. A moderate and exchangeable binding to Ca2+ of the CPP molecule well substantiates the high absorbability of calcium from milk and dairy products.

Amino Acid Sequence↗

PubMed1991

The use of phosphopeptides to distinguish between protein phosphatase and acid/alkaline phosphatase activities: opposite specificity toward phosphoseryl/phosphothreonyl substrates.

The four main classes of protein phosphatases (PP-1, 2A, 2B and 2C), although differing in their ability to dephosphorylate phosphopeptide substrates, invariably display a marked preference toward phosphothreonyl peptides over their phosphoseryl counterparts. Conversely, all the acidic and alkaline phosphatases tested so far dephosphorylate phosphoseryl derivatives far more readily than phosphothreonyl ones. This opposite behaviour provides a criterion for discriminating between protein dephosphorylating activity due to authentic protein phosphatases as compared to nonspecific acid and/or alkaline phosphatases. In particular the phosphothreonyl peptides RRATPVA and RRREEETPEEEAA appear to be especially suited for detecting the activity of PP-2C and PP-2A, since they are hardly dephosphorylated by acid and alkaline phosphatases. Conversely, the phosphoseryl peptides SPEEEEE and RRASPVA can provide a sensitive evaluation of the majority of acid and alkaline phosphatases, while being refractory to protein phosphatases.

Acid Phosphatase↗

PubMed1995

Selective determination of phosphopeptide beta-CN(1-25) in a beta-casein digest by adding iron: characterization by liquid chromatography with on-line electrospray-ionization mass spectrometric detection.

A beta-casein tryptic digest has been analysed by reversed-phase high-performance liquid chromatography (RP-HPLC) with on-line electrospray-ionization mass spectrometry (ESI-MS). Analyses of peptides were carried out before and after addition of iron(II) to the peptides in solution. In both cases, the majority of peptides were identified by the determination of molecular masses by ESI-MS and by prior knowledge of the amino acid sequence of beta-casein, and thus of its corresponding tryptic peptides. In the presence of iron(II), only phosphopeptide beta-CN(1-25) was able to bind iron to form different complexes that have increased retention times on the RP-HPLC column and that also absorbed at 280 nm. The method presented here appears to be selective for peptides containing phosphoseryl cluster(s).

Caseins↗