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1.
RNA ; 7(11): 1589-602, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11720288

ABSTRACT

An abundant nuclear phosphoprotein, the La autoantigen, is the first protein to bind all newly synthesized RNA polymerase III transcripts. Binding by the La protein to the 3' ends of these RNAs stabilizes the nascent transcripts from exonucleolytic degradation. In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the La protein is required for the normal pathway of tRNA maturation. Experiments in which the human protein was expressed in S. pombe have suggested that phosphorylation of the La protein regulates tRNA maturation. To dissect the role of phosphorylation in La protein function, we used mass spectrometry to identify three sites of serine phosphorylation in the S. cerevisiae La protein Lhp1p. Mutant versions of Lhp1p, in which each of the serines was mutated to alanine, were expressed in yeast cells lacking Lhp1p. Using two-dimensional gel electrophoresis, we determined that we had identified and mutated all major sites of phosphorylation in Lhp1p. Lhp1p lacking all three phosphorylation sites was functional in several yeast strains that require Lhp1p for growth. Northern blotting revealed no effects of Lhp1p phosphorylation status on either pre-tRNA maturation or stabilization of nascent RNAs. Both wild-type and mutant Lhp1 proteins localized to both nucleoplasm and nucleoli, demonstrating that phosphorylation does not affect subcellular location. Thus, although La proteins from yeast to humans are phosphoproteins, phosphorylation does not appear to be required for any of the identified functions of the S. cerevisiae protein.


Subject(s)
Fungal Proteins/metabolism , RNA Stability , RNA, Fungal/biosynthesis , RNA, Transfer/biosynthesis , RNA-Binding Proteins/metabolism , Saccharomyces cerevisiae Proteins , Amino Acid Sequence , Autoantigens/metabolism , Binding Sites , Cell Nucleolus/metabolism , Cell Nucleus/metabolism , Fungal Proteins/genetics , Molecular Sequence Data , Peptide Mapping , Phosphorylation , Protein Isoforms/metabolism , RNA/metabolism , RNA-Binding Proteins/genetics , Ribonucleoproteins/metabolism , Ribonucleoproteins, Small Nuclear/biosynthesis , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , SS-B Antigen
2.
Science ; 293(5533): 1317-20, 2001 Aug 17.
Article in English | MEDLINE | ID: mdl-11509731

ABSTRACT

Organelle transport by myosin-V is down-regulated during mitosis, presumably by myosin-V phosphorylation. We used mass spectrometry phosphopeptide mapping to show that the tail of myosin-V was phosphorylated in mitotic Xenopus egg extract on a single serine residue localized in the carboxyl-terminal organelle-binding domain. Phosphorylation resulted in the release of the motor from the organelle. The phosphorylation site matched the consensus sequence of calcium/calmodulin-dependent protein kinase II (CaMKII), and inhibitors of CaMKII prevented myosin-V release. The modulation of cargo binding by phosphorylation is likely to represent a general mechanism regulating organelle transport by myosin-V.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Calmodulin-Binding Proteins/metabolism , Melanosomes/metabolism , Mitosis , Molecular Motor Proteins/metabolism , Myosin Type V , Nerve Tissue Proteins/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Biological Transport , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Calmodulin-Binding Proteins/chemistry , Calmodulin-Binding Proteins/genetics , Cell Extracts , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Enzyme Inhibitors/pharmacology , Interphase , Mass Spectrometry , Melanophores/metabolism , Melanophores/ultrastructure , Molecular Sequence Data , Mutation , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Ovum , Peptides/pharmacology , Phosphopeptides/analysis , Phosphopeptides/metabolism , Phosphorylation , Phosphoserine/metabolism , Recombinant Fusion Proteins/metabolism , Transfection , Xenopus
3.
Genes Dev ; 15(9): 1078-92, 2001 May 01.
Article in English | MEDLINE | ID: mdl-11331604

ABSTRACT

The budding yeast transcriptional activator Gcn4 is rapidly degraded in an SCF(Cdc4)-dependent manner in vivo. Upon fractionation of yeast extracts to identify factors that mediate Gcn4 ubiquitination, we found that Srb10 phosphorylates Gcn4 and thereby marks it for recognition by SCF(Cdc4) ubiquitin ligase. Srb10 is a physiological regulator of Gcn4 stability because both phosphorylation and turnover of Gcn4 are diminished in srb10 mutants. Gcn4 is almost completely stabilized in srb10Delta pho85Delta cells, or upon mutation of all Srb10 phosphorylation sites within Gcn4, suggesting that the Pho85 and Srb10 cyclin-dependent kinases (CDKs) conspire to limit the accumulation of Gcn4. The multistress response transcriptional regulator Msn2 is also a substrate for Srb10 and is hyperphosphorylated in an Srb10-dependent manner upon heat-stress-induced translocation into the nucleus. Whereas Msn2 is cytoplasmic in resting wild-type cells, its nuclear exclusion is partially compromised in srb10 mutant cells. Srb10 has been shown to repress a subset of genes in vivo, and has been proposed to inhibit transcription via phosphorylation of the C-terminal domain of RNA polymerase II. We propose that Srb10 also inhibits gene expression by promoting the rapid degradation or nuclear export of specific transcription factors. Simultaneous down-regulation of both transcriptional regulatory proteins and RNA polymerase may enhance the potency and specificity of transcriptional inhibition by Srb10.


Subject(s)
Cyclin-Dependent Kinases/metabolism , DNA-Binding Proteins/metabolism , Fungal Proteins/metabolism , Protein Kinases/metabolism , Saccharomyces cerevisiae Proteins , Transcription Factors/metabolism , Amino Acids/metabolism , Cell Nucleus/metabolism , Culture Media , Cyclin-Dependent Kinase 8 , Cyclin-Dependent Kinases/genetics , Mutation , Peptide Synthases/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Protein Subunits , RNA Polymerase II/metabolism , SKP Cullin F-Box Protein Ligases , Trans-Activators/metabolism , Ubiquitins/metabolism , Yeasts/genetics , Yeasts/metabolism
4.
Anal Chem ; 73(3): 393-404, 2001 Feb 01.
Article in English | MEDLINE | ID: mdl-11217738

ABSTRACT

A new, multidimensional electrospray MS-based strategy for phosphopeptide mapping is described which eliminates the need to radiolabel protein with 32P or 33P. The approach utilizes two orthogonal MS scanning techniques, both of which are based on the production of phosphopeptide-specific marker ions at m/z 63 and/or 79 in the negative ion mode. These scan methods are combined with liquid chromatography-electrospray mass spectrometry and nanoelectrospray MS/MS to selectively detect and identify phosphopeptides in complex proteolytic digests. Low-abundance, low-stoichiometry phosphorylation sites can be selectively determined in the presence of an excess of nonphosphorylated peptides, even in cases where the signal from the phosphopeptide is indistinguishable from background in the conventional MS scan. The strategy, which has been developed and refined in our laboratory over the past few years, is particularly well suited to phosphoproteins that are phosphorylated to varying degrees of stoichiometry on multiple sites. Sensitivity and selectivity of the method are demonstrated here using model peptides and a commercially available phosphoprotein standard. In addition, the strategy is illustrated by the complete in vitro and in vivo phosphopeptide mapping of Sic1p, a regulator of the G1/S transition in budding yeast.


Subject(s)
Peptide Mapping/methods , Phosphopeptides/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Amino Acid Sequence , Caseins/chemistry , Chromatography, Liquid , Molecular Sequence Data , Phosphorylation
5.
Biochemistry ; 38(48): 15893-902, 1999 Nov 30.
Article in English | MEDLINE | ID: mdl-10625455

ABSTRACT

The nature of the inhibition of thiol proteases by a new class of mechanism-based inhibitors, 1,5-diacylcarbohydrazides, is described. These potent, time-dependent, active-site spanning inhibitors include compounds that are selective for cathepsin K, a cysteine protease unique to osteoclasts. The 1,5-diacylcarbohydrazides are slow substrates for members of the papain superfamily with inhibition resulting from slow enzyme decarbamylation. Enzyme-catalyzed hydrolysis of 2,2'-N, N'-bis(benzyloxycarbonyl)-L- leucinylcarbohydrazide is accompanied by formation of a hydrazide-containing product and a carbamyl-enzyme intermediate that is sufficiently stable to be observed by mass spectrometry and NMR. Stopped-flow studies yield a saturation limited value of 43 s(-)(1) for the rate of cathepsin K acylation by 2,2'N, N'-bis(benzyloxycarbonyl)-L-leucinylcarbohydrazide. Inhibition potency varies among proteases tested as reflected by 2-3 orders of magnitude differences in K(i) and K(obs)/I, but all eventually form the same stable covalent intermediate. Reactivation rates are equivalent for all enzymes tested (1 x 10(-)(4) s(-)(1)), indicating hydrolysis of a common carbamyl-enzyme form. NMR spectroscopic studies with cathepsin K and 2,2'-N,N'-bis(benzyloxycarbonyl)-L-leucinylcarbohydrazide provide evidence of inhibitor cleavage to generate a covalent carbamyl-enzyme intermediate rather than a tetrahedral complex. The product Cbz-leu-hydrazide does not appear enzyme-bound after cleavage in the NMR spectra, suggesting that the stable inhibited form of the enzyme is the thioester complex. 1, 5-diacylcarbohydrazides represent a new class of unreactive cysteine protease inhibitors that share a common mechanism of action across members of the papain superfamily. Both S and S' subsite interactions are exploited in achieving high selectivity and potency.


Subject(s)
Cathepsins/antagonists & inhibitors , Hydrazines/pharmacology , Protease Inhibitors/pharmacology , Binding Sites , Cathepsin K , Chromatography, High Pressure Liquid , Enzyme Reactivators , Hydrazines/chemistry , Kinetics , Leucine/analogs & derivatives , Leucine/chemistry , Leucine/pharmacology , Magnetic Resonance Spectroscopy , Mass Spectrometry , Papain/antagonists & inhibitors , Spectrophotometry
6.
Science ; 278(5337): 455-60, 1997 Oct 17.
Article in English | MEDLINE | ID: mdl-9334303

ABSTRACT

G1 cyclin-dependent kinase (Cdk)-triggered degradation of the S-phase Cdk inhibitor Sic1p has been implicated in the transition from G1 to S phase in the cell cycle of budding yeast. A multidimensional electrospray mass spectrometry technique was used to map G1 Cdk phosphorylation sites in Sic1p both in vitro and in vivo. A Sic1p mutant lacking three Cdk phosphorylation sites did not serve as a substrate for Cdc34p-dependent ubiquitination in vitro, was stable in vivo, and blocked DNA replication. Moreover, purified phosphoSic1p was ubiquitinated in cyclin-depleted G1 extract, indicating that a primary function of G1 cyclins is to tag Sic1p for destruction. These data suggest a molecular model of how phosphorylation and proteolysis cooperate to bring about the G1/S transition in budding yeast.


Subject(s)
Cyclin-Dependent Kinases/metabolism , Cyclins/metabolism , Fungal Proteins/metabolism , S Phase , Saccharomyces cerevisiae Proteins , Ubiquitin-Protein Ligase Complexes , Yeasts/cytology , Amino Acid Sequence , Anaphase-Promoting Complex-Cyclosome , Cyclin G , Cyclin-Dependent Kinase Inhibitor Proteins , DNA Replication , Enzyme Inhibitors/metabolism , G1 Phase , Ligases/metabolism , Molecular Sequence Data , Mutagenesis , Phenotype , Phosphopeptides/metabolism , Phosphorylation , Recombinant Fusion Proteins/metabolism , Ubiquitin-Protein Ligases , Ubiquitins/metabolism , Yeasts/metabolism
7.
J Biol Chem ; 272(18): 12116-21, 1997 May 02.
Article in English | MEDLINE | ID: mdl-9115281

ABSTRACT

The site of action of a series of pyridinyl imidazole compounds that are selective inhibitors of p38 mitogen-activated protein kinase in vitro and block proinflammatory cytokine production in vivo has been determined. Using Edman sequencing, 125I-SB206718 was shown to cross-link to the nonphosphorylated Escherichia coli-expressed p38 kinase at Thr175, which is proximal to the ATP binding site. Titration calorimetric studies with E. coli-expressed p38 kinase showed that SB203580 bound with a stoichiometry of 1:1 and that binding was blocked by preincubation of p38 kinase with the ATP analogue, FSBA (5'-[p-(fluorosulfonyl)benzoyl]adenosine), which covalently modifies the ATP binding site. The intrinsic ATPase activity of the nonphosphorylated enzyme was inhibited by SB203580 with a Km of 9.6 mM. Kinetic studies of active, phosphorylated yeast-expressed p38 kinase using a peptide substrate showed that SB203580 was competitive with ATP with a Ki of 21 nM and that kinase inhibition correlated with binding and biological activity. Mutagenesis indicated that binding of 125I-SB206718 was dependent on the catalytic residues K53 and D168 in the ATP pocket. These findings indicate that the pyridinyl imidazoles act in vivo by inhibiting p38 kinase activity through competition with ATP and that their selectivity is probably determined by differences in nonconserved regions within or near the ATP binding pocket.


Subject(s)
Adenosine Triphosphate/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Enzyme Inhibitors/metabolism , Imidazoles/metabolism , Mitogen-Activated Protein Kinases , Pyridines/metabolism , Adenosine/analogs & derivatives , Adenosine/pharmacology , Affinity Labels , Amino Acid Sequence , Binding Sites , Binding, Competitive , Calcium-Calmodulin-Dependent Protein Kinases/chemistry , Calcium-Calmodulin-Dependent Protein Kinases/isolation & purification , Calorimetry , Cloning, Molecular , Cross-Linking Reagents , Enzyme Inhibitors/pharmacology , Escherichia coli , HeLa Cells , Humans , Imidazoles/pharmacology , Kinetics , Mutagenesis, Site-Directed , Pyridines/pharmacology , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Saccharomyces cerevisiae , p38 Mitogen-Activated Protein Kinases
8.
Proc Natl Acad Sci U S A ; 94(26): 14249-54, 1997 Dec 23.
Article in English | MEDLINE | ID: mdl-9405598

ABSTRACT

Potent and selective active-site-spanning inhibitors have been designed for cathepsin K, a cysteine protease unique to osteoclasts. They act by mechanisms that involve tight binding intermediates, potentially on a hydrolytic pathway. X-ray crystallographic, MS, NMR spectroscopic, and kinetic studies of the mechanisms of inhibition indicate that different intermediates or transition states are being represented that are dependent on the conditions of measurement and the specific groups flanking the carbonyl in the inhibitor. The species observed crystallographically are most consistent with tetrahedral intermediates that may be close approximations of those that occur during substrate hydrolysis. Initial kinetic studies suggest the possibility of irreversible and reversible active-site modification. Representative inhibitors have demonstrated antiresorptive activity both in vitro and in vivo and therefore are promising leads for therapeutic agents for the treatment of osteoporosis. Expansion of these inhibitor concepts can be envisioned for the many other cysteine proteases implicated for therapeutic intervention.


Subject(s)
Cathepsins/antagonists & inhibitors , Drug Design , Enzyme Inhibitors/chemistry , Binding Sites , Cathepsin K , Cathepsins/chemistry , Crystallography, X-Ray , Enzyme Inhibitors/pharmacology , Humans , Protein Conformation
9.
Anal Biochem ; 239(2): 180-92, 1996 Aug 01.
Article in English | MEDLINE | ID: mdl-8811904

ABSTRACT

We describe a new procedure that enables selective detection and sequencing of Ser-, Thr-, and Tyr-phosphopeptides at the low femtomole level in protein digests. Radiolabeling with 32P is not required, nor is prior chromatographic separation of the peptide mixture. One to two microliters of the unfractionated protein digest is infused at basic pH into an electrospray mass spectrometer at a flow rate of 20-40 nl/min using an ultra-low flow sprayer. A precursor-ion scan of m/z 79 (PO-3) produces a mass spectrum containing only the molecular ions of the phosphopeptides that are present in the sample. In cases where the protein sequence is known, the peptide molecular weights obtained are often sufficient to identify the specific sequences that are phosphorylated. If the protein sequence is not known, tandem MS with collision-induced dissociation of phosphopeptide precursor-ions may be used to obtain the amino acid sequences including the site(s) of phosphorylation. We demonstrate that phosphopeptides may be selectively detected using as little as 3 fmol of a 10 fmol/microl solution and that sequence information for a phosphopeptide in the mixture may be obtained using as little as 3 femtomole of the same solution. In addition, we show that the stoichiometry of phosphorylation at specific sites may be estimated from the ratio of the ion signals for the respective forms of the peptides observed in the normal full-scan mass spectra of the digest. These procedures are illustrated here to identify and sequence phosphopeptides from alpha-casein, a milk-derived protein possessing up to nine phosphorylation-sites. Numerous MS and tandem MS experiments were carried out on a single, 250 fmol/microl loading of the phosphoprotein digest. Phosphopeptides derived from an unexpected variant of the protein were also observed.


Subject(s)
Mass Spectrometry/methods , Phosphopeptides/analysis , Amino Acid Sequence , Caseins/chemistry , Hydrogen-Ion Concentration , Molecular Sequence Data , Reference Standards , Trypsin/metabolism
10.
J Biol Chem ; 271(13): 7752-7, 1996 Mar 29.
Article in English | MEDLINE | ID: mdl-8631817

ABSTRACT

In the preceding report (Ladner, R.D., McNulty, D.E., Carr, S.A., Roberts, G.D., and Caradonna, S.J. (1996) J. Biol. Chem. 271, 7745-7751), we identified two distinct isoforms of dUTPase in human cells. These isoforms are individually targeted to the nucleus (DUT-N) and mitochondria (DUT-M). The proteins are nearly identical, differing only in a short region of their amino termini. Despite the structural differences between these proteins, they retain identical affinities for dUTP (preceding article). In previous work, this laboratory demonstrated that dUTPase is posttranslationally phosphorylated on serine residue(s) (Lirette, R., and Caradonna, S. (1990) J. Cell. Biochem. 43, 339-353). To extend this work and determine if both isoforms of dUTPase are phosphorylated, a more in depth analysis of dUTPase phosphorylation was undertaken. [32P]Orthophosphate-labeled dUTPase was purified from HeLa cells, revealing that only the nuclear form of dUTPase is phosphorylated. Electrospray tandem mass spectrometry was used to identify the phosphorylation site as Ser-11 in the amino-terminal tryptic peptide PCSEETPAIpSPSKR (the NH2-terminal Met is removed in the mature protein). Mutation of Ser-11 by replacement with Ala blocks phosphorylation of dUTPase in vivo. Analysis of the wild type and Ser-11 --> Ala mutant indicates that phosphorylation does not regulate the enzymatic activity of the DUT-N protein in vitro. Additionally, experiments with the Ser-11 --> Ala mutant indicate that phosphorylation does not appear to play a role in subunit association of the nuclear form of dUTPase. The amino acid context of this phosphorylation site corresponds to the consensus target sequence for the cyclin-dependent protein kinase p34(cdc2). Recombinant DUT-N was specifically phosphorylated on Ser-11 in vitro with immunoprecipitated p34(cdc2). Together, these data suggest that the nuclear form of dUTPase may be a target for cyclin-dependent kinase phosphorylation in vivo.


Subject(s)
Cell Nucleus/enzymology , Cyclin-Dependent Kinases/metabolism , Pyrophosphatases/metabolism , Amino Acid Sequence , Base Sequence , CDC2 Protein Kinase/metabolism , Chromatography, High Pressure Liquid , Consensus Sequence , DNA Primers , Escherichia coli/enzymology , HeLa Cells , Humans , Mass Spectrometry , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphates/metabolism , Phosphorylation , Pyrophosphatases/chemistry , Pyrophosphatases/isolation & purification , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Substrate Specificity , Viruses/enzymology
11.
Anal Chem ; 67(22): 4053-6, 1995 Nov 15.
Article in English | MEDLINE | ID: mdl-8633765

ABSTRACT

Growth hormone releasing peptide (GHRP, sequence His-D-Trp-Ala-Trp-D-Phe-Lys-NH2) is a synthetic hexapeptide under consideration for transdermal iontophoretic drug delivery. Cyclic voltammetry, controlled-potential electrolysis, HPLC/UV analysis, LC/MS/MS analysis, and EPR spin-trapping studies indicate that the electrolysis-induced oxidative degradation of GHRP is likely to be mediated by electrogenerated oxygen radicals from the electrolysis of water. Within 2 h and up to 2.5 V versus an Ag/AgCl reference electrode, the peptide backbone remains largely intact. The chemical modifications are selectively on imidazole (histidine) and indole (tryptophan). Strategies for alleviating the electrolysis-induced degradation of GHRP are proposed.


Subject(s)
Growth Hormone-Releasing Hormone/chemistry , Amino Acid Sequence , Electrolysis , Electron Spin Resonance Spectroscopy , Mass Spectrometry , Molecular Sequence Data
12.
Anal Chem ; 65(7): 877-84, 1993 Apr 01.
Article in English | MEDLINE | ID: mdl-8470819

ABSTRACT

Mass spectrometric methods of glycopeptide-specific detection in liquid chromatography/electrospray mass spectrometry (LC/ESMS) of glycoprotein digests are explored using a variety of glycopeptide models and then applied to soluble complement receptor type I, a 240-kDa glycoprotein containing 25 potential sites of N-glycosylation. The most specific method, requiring a triple quadrupole, involves monitoring of sugar oxonium fragment ions during precursor-ion scan ESMS/MS. Signals derived from nonglycosylated peptides are virtually eliminated, resulting in a total-ion current chromatographic trace of only the glycopeptides present in the digest. The corresponding mass spectra yield molecular weight and glycopeptide microheterogeneity information. An alternative and complementary approach that we term collisional-excitation scanning also involves fragmentation of glycopeptides to sugar oxonium ion fragments but does not involve any mass-selection process, permitting the experiment to be performed on a single quadrupole instrument. The resulting total ion chromatogram is similar to the UV chromatogram (215 nm), but a selected-ion chromatogram for carbohydrate-specific ions such as the N-acetylhexosamine oxonium ion (m/z 204) produces a glycopeptide-specific trace. Although there can sometimes be peptide interferences in the spectra of the indicated glycopeptide-containing chromatographic peaks, this latter approach permits peptide mapping to be performed on the same data set that also indicates the location of glycopeptides in the chromatogram. Both methods are suitable for detection of glycopeptides with all common classes of oligosaccharides in either N- or O-linkage to the peptide.


Subject(s)
Chromatography, Liquid , Glycopeptides/analysis , Mass Spectrometry , Carbohydrate Sequence , Flow Injection Analysis , Glycopeptides/chemistry , Glycopeptides/metabolism , Molecular Sequence Data , Molecular Weight , Oligosaccharides/analysis , Oligosaccharides/chemistry
13.
Protein Sci ; 2(2): 183-96, 1993 Feb.
Article in English | MEDLINE | ID: mdl-7680267

ABSTRACT

A mass spectrometry method has been developed for selective detection of glycopeptides at the low (< or = 25) picomole level during chromatography of glycoprotein digests and for differentiation of O-linked from N-linked oligosaccharides. The technique involves observation of diagnostic sugar oxonium-ion fragments, particularly the HexNAc+ fragment at m/z 204, from collisionally excited glycopeptides. Collision-induced fragmentation can be accomplished in either of two regions of a triple quadrupole mass spectrometer equipped with an atmospheric pressure, electrospray (ES) ionization source. If collisions before the first quadrupole are chosen, it is possible to enhance formation of carbohydrate-related fragment ions without distorting the distribution of peptide and glycopeptide signals by increasing the collisional excitation potential only during that portion of each scan in which the low mass carbohydrate-related ions are being detected. This procedure, requiring only a single quadrupole instrument, identifies putative glycopeptide-containing fractions in the chromatogram but suffers from a lack of specificity in the case of co-eluting peptides. Increased specificity is obtained by selectively detecting only those parent ions that fragment in Q2, the second collision region of the triple quadrupole, to produce an ion at m/z 204 (HexNAc+). Only (M + H)+ ions of glycopeptides are observed in these liquid chromatography-electrospray tandem mass spectrometry (LC-ESMS/MS) "parent-scan" spectra. N-linked carbohydrates are differentiated from O-linked by LC-ESMS/MS analysis of the digested glycoprotein prior to and after selective removal of N-linked carbohydrates by peptide N:glycosidase F. These methods, which constitute the first liquid chromatography-mass spectrometry (LC-MS)-based strategies for selective identification of glycopeptides in complex mixtures, facilitate location and preparative fractionation of glycopeptides for further structural characterization. In addition, these techniques may be used to assess the compositional heterogeneity at specific attachment sites, and to define the sequence context of the attachment site in proteins of known sequence. The strategy is demonstrated for bovine fetuin, a 42-kDa glycoprotein containing three N-linked, and at least three O-linked carbohydrates. Over 90% of the fetuin protein sequence was also corroborated by these LC-ESMS studies.


Subject(s)
Glycoproteins/chemistry , Oligosaccharides/chemistry , alpha-Fetoproteins/chemistry , Amino Acid Sequence , Animals , Cattle , Gas Chromatography-Mass Spectrometry/methods , Glycopeptides/analysis , Glycoproteins/drug effects , Glycosylation , Isomerism , Molecular Sequence Data , Trypsin/pharmacology , alpha-Fetoproteins/drug effects
14.
Protein Sci ; 2(1): 86-92, 1993 Jan.
Article in English | MEDLINE | ID: mdl-8443592

ABSTRACT

Four tyrosine residues have been identified as phosphorylation sites in the tyrosine kinase isoform of the heparin-binding fibroblast growth factor receptor flg (FGF-R1). Baculoviral-insect cell-derived recombinant FGF-R1 was phosphorylated and fragmented with trypsin while immobilized on heparin-agarose beads. Phosphotyrosine peptides were purified by chromatography on immobilized anti-phosphotyrosine antibody and analyzed by Edman degradation and electrospray tandem mass spectrometry. Tyrosine residue 653, which is in a homologous spatial position to major autophosphorylation sites in the catalytic domain of the src and insulin receptor kinases, is the major intracellular FGF-R1 phosphorylation site. Residue 766 in the COOH-terminus outside the kinase domain is a secondary site. Tyrosine residues 154 and 307, which are in the extracellular domain of transmembrane receptor isoforms and are in an unusual sequence context for tyrosine phosphorylation, were also phosphorylated.


Subject(s)
Receptors, Fibroblast Growth Factor/chemistry , Amino Acid Sequence , Animals , Binding Sites , Cell Line , Heparin/metabolism , Molecular Sequence Data , Phosphorylation , Protein-Tyrosine Kinases/chemistry , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Receptors, Fibroblast Growth Factor/genetics , Receptors, Fibroblast Growth Factor/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
15.
J Am Soc Mass Spectrom ; 4(9): 710-7, 1993 Sep.
Article in English | MEDLINE | ID: mdl-24225996

ABSTRACT

A mass spectrometry-based method that does not involve the use of radiolabeling was developed for selective detection of phosphopeptides in complex mixtures. Mixtures of phosphorylated and nonphosphorylated peptides at the low picomole level are analyzed by negative ion electrospray liquid chromatography/mass spectrometry using C-18 packed fused-silica columns (≤320-µm i.d.). Peptides and phosphopeptides in the chromatographic eluant undergo collision-induced dissociation in the free-jet expansion region prior to the mass analyzing quadrupole. Using relatively high collisional excitation potentials, phospho|peptides containing phosphoserine, phosphothreonine, and phosphotyrosine fragment to yield diagnostic ions at m/z 63 and 79 corresponding to PO2 (-); and PO3 (-), respectively. Chromatographic peaks containing phosphopeptides are indicated where these diagnostic ions maximize. The highest sensitivity for phosphopeptide detection is obtained using selected-ion monitoring for m/z 63 and 79. Full-scan mass spectra that exhibit the diagnostic phosphopeptide fragment ions, together with pseudomolecular ions, may be obtained by stepping the collisional excitation potential from a high value during the portion of each scan in which the low-mass-to-charge ratio diagnostic marker ions are being detected to a lower value while the upper mass-to-charge ratio range is being scanned. Good sensitivity for phosphopeptide detection was achieved using standard trifluoroacetic acid containing mobile phases for reversed-phase high-performance liquid chromatography. Data illustrating the selectivity and sensitivity of the approach are presented for mixtures of peptides and phosphopeptides containing the three commonly phosphorylated amino acids.

16.
J Biol Chem ; 267(26): 18991-8, 1992 Sep 15.
Article in English | MEDLINE | ID: mdl-1527025

ABSTRACT

Rhodopsin kinase (RK) is a second-messenger-independent protein kinase that is involved in deactivation of photolyzed rhodopsin (Rho*). We have developed a significantly improved method for isolation of RK based on the specific interactions of phosphorylated forms of the enzyme with heparin-Sepharose. Conversion of the dephosphorylated form of RK to the fully phosphorylated enzyme leads to specific elution of the kinase from the resin. Limited proteolysis of RK with endoproteinase Asp-N removes the phosphorylation sites. Peptides containing the autophosphorylation sites were isolated by reverse-phase high performance liquid chromatography and analyzed by Edman degradation and tandem mass spectrometry. The derived amino acid sequence of the peptide containing the major autophosphorylation site yielded the following sequence: DVGAFS488T489VKGVAFEK, where Ser488 and Thr489 are phosphorylated. Additionally, a minor autophosphorylation site was identified at Ser21. A 15-residue peptide (DVGAFSTVKGVAFEK) encompassing the major autophosphorylation site was synthesized and used for phosphorylation and inhibition studies. In contrast to many other protein kinases, the low catalytic activity of RK toward its autophosphorylation site peptide and the poor inhibitory properties of this peptide suggest unique properties of this member of the family of G protein-coupled receptor kinases.


Subject(s)
Eye Proteins , Protein Kinases/metabolism , Amino Acid Sequence , Animals , Cattle , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , G-Protein-Coupled Receptor Kinase 1 , Hydrolysis , Mass Spectrometry , Molecular Sequence Data , Peptide Fragments/genetics , Peptide Mapping , Phosphorylation , Rod Cell Outer Segment/enzymology , Trypsin
17.
J Biol Chem ; 264(35): 21286-95, 1989 Dec 15.
Article in English | MEDLINE | ID: mdl-2592374

ABSTRACT

The primary structure of a soluble form of the CD4 receptor (sCD4) expressed in Chinese hamster ovary cells has been confirmed by mass spectrometric peptide mapping and and tandem mass spectrometry. These studies corroborated 95% of the 369-amino acid-long sequence and established the fidelity of translation of the NH2 and COOH terminal including the absence of "ragged ends." The arrangement of the three disulfide bonds in recombinant sCD4 was also established by mass spectrometry and comparative high performance liquid chromatography mapping and shown to be identical to that expected from previous studies of intrachain disulfide bonding in T4 antigens derived from sheep and mouse. No other arrangements of disulfides were detected. Carbohydrate mapping by mass spectrometry was used to establish that both potential Asn-linked glycosylation sites in sCD4 (Asn271 and Asn300) have oligosaccharides attached. Structural characterization by mass spectrometry and methylation analysis of the heterogeneous family of oligosaccharides at each of the specific attachment sites indicates that the major components of both families of oligosaccharides have the following biantennary structures: (Formula, see text) where m + n = 0-2, and x = 0,1. Minor carbohydrate components having three N-acetylneuraminic acid (NeuAc) groups and an additional hexose-hexosamine unit were detected by high performance anion-exchange chromatography.


Subject(s)
CD4 Antigens , Amino Acid Sequence , Animals , CD4 Antigens/genetics , CD4 Antigens/isolation & purification , Carbohydrate Conformation , Carbohydrate Sequence , Cell Line , Chromatography, High Pressure Liquid , Gene Expression , Genes , Humans , Mass Spectrometry , Methylation , Molecular Sequence Data , Peptide Fragments/isolation & purification , Peptide Mapping , Recombinant Proteins
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