Epitope mapping by a combination of epitope excision and MALDI-MS.
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Amino Acid Sequence↗
Biomedical subjects
K B Tomer
Publications and source records attributed to K B Tomer.
Direct analysis of the products of sequential cleavages of peptides and proteins affinity-bound to immobilized metal ion beads by matrix-assisted laser desorption/ionization mass spectrometry.
Consecutive enzymatic reactions on analytes affinity-bound to immobilized metal ion beads with subsequent direct analysis of the products by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry have been used for detecting protein synthesis errors occuring at the N-terminus. The usefulness of this method was demonstrated by analyzing two commercially available recombinant HIV proteins with affinity tags at the N-terminus, and histatin-5, a peptide with multiple histidine residues. The high specificity, sensitivity, and speed of analysis make this method especially useful in obtaining N-terminal sequencing information of histidine-tagged recombinant proteins.
Amino Acid Sequence↗
Characterization of the noncovalent complex of human immunodeficiency virus glycoprotein 120 with its cellular receptor CD4 by matrix-assisted laser desorption/ionization mass spectrometry.
The initial event in infection by the human immunodeficiency virus type 1 (HIV-1) is the interaction of the viral envelope glycoprotein (HIV-gp120) with its primary cellular receptor, the glycoprotein CD4. Molecular structure information about the HIV-gp120/CD4 complex can provide information relevant to an understanding of the basic processes occurring in HIV infection and to development of therapies that can inhibit AIDS. Previous studies by sugar gradient sedimentation of the interaction of HIV-gp120 with a cytoplasmic domain truncated soluble CD4 (sCD4) suggested that a one-to-one complex was formed. The stoichiometry, however, of the sCD4/HIV-gp120 complex remained to be confirmed by an independent method because (i) recent X-ray examination revealed dimerization of sCD4 and (ii) the low resolution and low accuracy of molecular weight determination by sugar gradient sedimentation can lead to artifactual data. Therefore, in this study matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to determine the molecular mass of the complex of fully glycosylated HIV-gp120 and sCD4. A mass of 145 kDa was measured, which is exactly the sum of the molecular masses of one HIV-gp120 and one sCD4 molecule. Complexes of higher order of stoichiometry were not detected. Identical results were obtained by chemically cross-linking the HIV-gp120/sCD4 complex with subsequent analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and MALDI-MS. This study confirms the earlier suggestions of the stoichiometry of the sCD4/HIV-gp120 complex in solution and also demonstrates the potential of MALDI-MS in investigations of specific noncovalent complexes of glycoproteins.
CD4 Antigens↗
Preliminary comparison of precursor scans and liquid chromatography-tandem mass spectrometry on a hybrid quadrupole time-of-flight mass spectrometer.
Recent mass spectrometry instrumentation developments include the appearance of novel hybrid tandem instrumentation, Q-TOF, consisting of a quadrupole mass analyzer (MS1) and a time-of-flight (TOF) analyzer. The TOF analyzer is not scanned, but collects all fragment ions entering the analyzer at a given time. Thus, the typical precursor scan experiment cannot be performed. Instead, a full MS-MS spectrum can be acquired for each mass passed by MS1. Appropriate data manipulation, i.e. extracted ion current chromatograms, can correlate specific fragment ion formation to the parent ion. Precursor scanning and LC-MS-MS are compared on a Q-TOF instrument for the determination of protein modifications, including acetylation and phosphorylation. Model peptides used for phosphopeptide detection were generated from a mixture of beta-casein. Model acetylated peptides were generated from a mixture of acetylated substance P1-9 and substance P1-11. The results were then applied to a more complex mixture, a digest of HIV-p24. Results indicate that precursor scanning is useful for screening, but that LC-MS-MS has a sensitivity advantage and is less susceptible to suppression effects. LC-MS-MS, therefore, appears to be better for the detection of trace components in complex mixtures.
Acetylation↗
Effect of nonenzymatic glycation of albumin and superoxide dismutase by glucuronic acid and suprofen acyl glucuronide on their functions in vitro.
Acyl glucuronides bind irreversibly to plasma proteins, and one mechanism proposed for this covalent binding is similar to that for glycation of protein by reducing sugars. Because glycation of protein by glucose and other reducing sugars can alter protein function, this lead to the hypothesis that the glycation of proteins by acyl glucuronides may cause similar effects. When human serum albumin (HSA) was incubated with 0.5 M glucose for 5 days, the unbound fractions of diazepam and warfarin were increased by 41 and 35%, respectively, less than that caused by glucuronic acid which increased the unbound fractions by 90% for diazepam and 420% for warfarin. When HSA was incubated with suprofen glucuronide (SG) at a much lower concentration of 0.005 M for only 24 h, the effects on the unbound fractions of diazepam and warfarin to HSA were altered dramatically with increases of 340 and 230%, respectively. After incubation of superoxide dismutase (SOD) with 0.5 or 1 M reducing sugars for 14 days, the enzyme activity decreased to 82 and 61% of initial levels at day 14, respectively, whereas glucuronic acid almost completely inactivated the enzyme activity over the same period. Even at a very low concentration (0.005 M) of SG, SOD activity was reduced significantly to 11% of initial levels by day 14, which was comparable to the effect by 0.5 and 1.0 M concentrations of glucuronic acid. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and matrix associated laser desorption/ionization time of flight mass spectrometry indicated that several equivalents of reducing sugars or SG became attached to albumin after incubation. These results suggest that acyl glucuronides may affect the function of proteins by the formation of glycated protein in vivo and may be associated with the toxicity of xenobiotics metabolized to labile acyl glucuronides.
Animals↗
Molecular cloning, enzymatic characterization, developmental expression, and cellular localization of a mouse cytochrome P450 highly expressed in kidney.
A cDNA encoding a new cytochrome P450 was isolated from a mouse liver library. Sequence analysis reveals that this 1,886-base pair cDNA encodes a 501-amino acid polypeptide that is 69-74% identical to CYP2J subfamily P450s and is designated CYP2J5. Recombinant CYP2J5 was co-expressed with NADPH-cytochrome P450 oxidoreductase in Sf9 cells using a baculovirus system. Microsomal fractions of CYP2J5/NADPH-cytochrome P450 oxidoreductase-transfected cells metabolize arachidonic acid to 14,15-, 11,12-, and 8, 9-epoxyeicosatrienoic acids and 11- and 15-hydroxyeicosatetraenoic acids (catalytic turnover, 4.5 nmol of product/nmol of cytochrome P450/min at 37 degrees C); thus CYP2J5 is enzymologically distinct. Northern analysis reveals that CYP2J5 transcripts are most abundant in mouse kidney and present at lower levels in liver. Immunoblotting using a polyclonal antibody against a CYP2J5-specific peptide detects a protein with the same electrophoretic mobility as recombinant CYP2J5 most abundantly in mouse kidney microsomes. CYP2J5 is regulated during development in a tissue-specific fashion. In the kidney, CYP2J5 is present before birth and reaches maximal levels at 2-4 weeks of age. In the liver, CYP2J5 is absent prenatally and during the early postnatal period, first appears at 1 week, and then remains relatively constant. Immunohistochemical staining of kidney sections with anti-human CYP2J2 IgG reveals that CYP2J protein(s) are present primarily in the proximal tubules and collecting ducts, sites where the epoxyeicosatrienoic acids are known to modulate fluid/electrolyte transport and mediate hormonal action. In situ hybridization confirms abundant CYP2J5 mRNA within tubules of the renal cortex and outer medulla. Epoxyeicosatrienoic acids are endogenous constituents of mouse kidney thus providing direct evidence for the in vivo metabolism of arachidonic acid by the mouse renal epoxygenase(s). Based on these data, we conclude that CYP2J5 is an enzymologically distinct, developmentally regulated, protein that is localized to specific nephron segments and contributes to the oxidation of endogenous renal arachidonic acid pools. In light of the well documented effects of epoxyeicosatrienoic acids in modulating renal tubular transport processes, we postulate that CYP2J5 products play important functional roles in the kidney.
Amino Acid Sequence↗
Characterization of cytochrome c free radical reactions with peptides by mass spectrometry.
The reactions of horse heart cytochrome c, hydrogen peroxide, and the spin trap 3,5-dibromo-4-nitrosobenzenesulfonic acid with a series of polypeptides were investigated using mass spectrometry. The mass spectra obtained from these reactions revealed that after a free radical has been generated on the heme-containing protein horse heart cytochrome c, it can be transferred to other biomolecules. In addition, the number of free radicals transferred to the target molecule could be determined. Recipient peptides/proteins that contained a tyrosine and/or tryptophan amino acid residue were most susceptible to free radical transfer. Using tandem mass spectrometry, the location of the 3,5-dibromo-4-nitrosobenzenesulfonic acid radical adduct on the nonapeptide RWIILGLNK was unequivocally determined to be at the tryptophan residue. We also demonstrated that the presence of an antioxidant in the reaction mixture not only inhibits free radical formation on horse heart cytochrome c, but also interferes with the transfer of the free radical, once it has been formed on cytochrome c.
Amino Acid Sequence↗
Affinity capillary electrophoresis investigation of an epitope on human immunodeficiency virus recognized by a monoclonal antibody.
Affinity capillary electrophoresis (ACE) has been used to investigate the epitope on the human immunodeficiency virus (HIV) core protein p24 recognized by the monoclonal antibody (mAb) 13-102-100. The affinity of a series of peptides with N- and C-terminal truncations of the epitope sequence determined by mass spectrometry was studied. The peak area change assay was used for the study of the interactions of the mAb with those peptides, exhibiting tight binding to the mAb, and the migration time shift assay was used to probe the relative affinities of peptides showing weak binding to the mAb. The experimental results show that the monoclonal antibody 13-102-100 recognizes the peptide VHPVHAGPIAP with highest affinity. Smaller peptides incorporating only part of the epitope, however, are recognized to some extent in the ACE experiments.
Amino Acid Sequence↗
Matrix-assisted laser desorption ionization/mass spectrometry mapping of human immunodeficiency virus-gp120 epitopes recognized by a limited polyclonal antibody.
In this study we have applied epitope excision and epitope extraction strategies, combined with matrix assisted laser desorption/ionization mass spectrometry, to determine the fine structure of epitopes recognized by a polyclonal antibody to human immunodeficiency virus envelope glycoprotein gp120. This is the first application of this approach to epitope mapping on a large, heavily glycosylated protein. In the epitope excision method, gp120 in the native form is first bound to the antibody immobilized on sepharose beads and cleaved with endoproteinase enzymes. In the epitope extraction method, the gp120 was first proteolytically cleaved and then allowed to react with the immobilized antibody. The fragments that remain bound to the antibody, after repeated washing to remove the unbound peptides, contain the antigenic region that is recognized by the antibody, and the bound peptides in both methods can be characterized by direct analysis of the immobilized antibody by matrix assisted laser desorption ionization/mass spectrometry. In this study we have carried out epitope excision and extraction experiments with three different enzymes and have identified residues 472-478 as a major epitope. In addition, antigenic regions containing minor epitopes have also been identified.
Amino Acid Sequence↗
Molecular cloning, expression, and functional significance of a cytochrome P450 highly expressed in rat heart myocytes.
A cDNA encoding a P450 monooxygenase was amplified from reverse transcribed rat heart and liver total RNA by polymerase chain reaction using primers based on the 5'- and 3'-end sequences of two rat pseudogenes, CYP2J3P1 and CYP2J3P2. Sequence analysis revealed that this 1,778-base pair cDNA contained an open reading frame and encoded a new 502 amino acid protein designated CYP2J3. Based on the deduced amino acid sequence, CYP2J3 was approximately 70% homologous to both human CYP2J2 and rabbit CYP2J1. Recombinant CYP2J3 protein was co-expressed with NADPH-cytochrome P450 oxidoreductase in Sf9 insect cells using a baculovirus expression system. Microsomal fractions of CYP2J3/NADPH-cytochrome P450 oxidoreductase-transfected cells metabolized arachidonic acid to 14,15-, 11,12-, and 8, 9-epoxyeicosatrienoic acids and 19-hydroxyeicosatetraenoic acid as the principal reaction products (catalytic turnover, 0.2 nmol of product/nmol of cytochrome P450/min at 37 degrees C). Immunoblotting of microsomal fractions prepared from rat tissues using a polyclonal antibody raised against recombinant CYP2J2 that cross-reacted with CYP2J3 but not with other known rat P450s demonstrated abundant expression of CYP2J3 protein in heart and liver. Immunohistochemical staining of formalin-fixed paraffin-embedded rat heart tissue sections using the anti-CYP2J2 IgG and avidin-biotin-peroxidase detection localized expression of CYP2J3 primarily to atrial and ventricular myocytes. In an isolated-perfused rat heart model, 20 min of global ischemia followed by 40 min of reflow resulted in recovery of only 44 +/- 6% of base-line contractile function. The addition of 5 microM 11, 12-epoxyeicosatrienoic acid to the perfusate prior to global ischemia resulted in a significant 1.6-fold improvement in recovery of cardiac contractility (69 +/- 5% of base line, p = 0.01 versus vehicle alone). Importantly, neither 14,15-epoxyeicosatrienoic acid nor 19-hydroxyeicosatetraenoic acid significantly improved functional recovery following global ischemia, demonstrating the specificity of the biological effect for the 11, 12-epoxyeicosatrienoic acid regioisomer. Based on these data, we conclude that (a) CYP2J3 is one of the predominant enzymes responsible for the oxidation of endogenous arachidonic acid pools in rat heart myocytes and (b) 11,12-epoxyeicosatrienoic acid may play an important functional role in the response of the heart to ischemia.
8,11,14-Eicosatrienoic Acid↗
CYP2J subfamily cytochrome P450s in the gastrointestinal tract: expression, localization, and potential functional significance.
Our laboratory recently described a new human cytochrome P450 arachidonic acid epoxygenase (CYP2J2) and the corresponding rat homologue (CYP2J3), both of which were expressed in extrahepatic tissues. Northern analysis of RNA prepared from the human and rat intestine demonstrated that CYP2J2 and CYP2J3 mRNAs were expressed primarily in the small intestine and colon. In contrast, immunoblotting studies using a polyclonal antibody raised against recombinant CYP2J2 showed that CYP2J proteins were expressed throughout the gastrointestinal tract. Immunohistochemical staining of formalin-fixed, paraffin-embedded intestinal sections using anti-CYP2J2 IgG and avidin-biotin-peroxidase detection revealed that CYP2J proteins were present at high levels in nerve cells of autonomic ganglia, epithelial cells, intestinal smooth muscle cells, and vascular endothelium. The distribution of this immunoreactivity was confirmed by in situ hybridization using a CYP2J2-specific antisense RNA probe. Microsomal fractions prepared from human jejunum catalyzed the NADPH-dependent metabolism of arachidonic acid to epoxyeicosatrienoic acids as the principal reaction products. Direct evidence for the in vivo epoxidation of arachidonic acid by intestinal cytochrome P450 was provided by documenting, for the first time, the presence of epoxyeicosatrienoic acids in human jejunum by gas chromatography/mass spectrometry. We conclude that human and rat intestine contain an arachidonic acid epoxygenase belonging to the CYP2J subfamily that is localized to autonomic ganglion cells, epithelial cells, smooth muscle cells, and vascular endothelium. In addition to the known effects on intestinal vascular tone, we speculate that CYP2J products may be involved in the release of intestinal neuropeptides, control of intestinal motility, and/or modulation of intestinal fluid/electrolyte transport.
8,11,14-Eicosatrienoic Acid↗
Predominant expression of an arachidonate epoxygenase in islets of Langerhans cells in human and rat pancreas.
Our laboratory recently described a new human cytochrome P450 arachidonic acid epoxygenase (CYP2J2) and the corresponding rat homolog (CYP2J3). Immunoblotting studies using a polyclonal antibody raised against recombinant human CYP2J2 confirmed CYP2J protein expression in human and rat pancreatic tissues. Immunohistochemical staining of formalin-fixed paraffin-embedded rat and human pancreas using the anti-CYP2J2 IgG and avidin-biotin-peroxidase detection revealed that CYP2J2 protein expression was highly localized to cells in the islets of Langerhans, with minimal staining in pancreatic exocrine cells. Colocalization studies using antibodies to the glucagon, insulin, somatostatin, and pancreatic polypeptide as markers for alpha-, beta-, delta-, and PP cells, respectively, showed that CYP2J protein expression was abundantly present in all four cell types, but was highest in the glucagon-producing alpha-cells. Direct evidence for the epoxidation of arachidonic acid by pancreatic cytochrome P450 was provided by documenting, for the first time, the presence of epoxyeicosatrienoic acids in vivo in human and rat pancreas by gas chromatography/mass spectrometry. Importantly, the levels of immunoreactive CYP2J2 in different human pancreatic tissues were highly correlated with endogenous epoxyeicosatrienoic acid concentrations. We conclude that human and rat pancreas contain an arachidonic acid epoxygenase belonging to the CYP2J subfamily that is highly localized to islet cells. These data together with previous work showing effects of epoxyeicosatrienoic acids in stimulating insulin and glucagon secretion from isolated rat pancreatic islets support the hypothesis that epoxygenase products may be involved in stimulus-secretion coupling in the pancreas.
8,11,14-Eicosatrienoic Acid↗
Monitoring cleavage of fusion proteins by matrix-assisted laser desorption ionization/mass spectrometry: recombinant HIV-1IIIB p26.
Matrix-associated laser desorption ionization/mass spectrometry (MALDI/MS) has been used to examine whole bacteria for the presence of a recombinant HIV p26 fusion protein. MALDI/MS, combined with affinity-purification techniques, is also shown to be very useful in monitoring the enzymatic cleavage of both affinity-bound fusion protein and fusion protein in solution. The combination of mass resolution, sensitivity, and speed of analysis makes MALDI/MS an attractive alternative to SDS-PAGE.
Chromatography, Affinity↗
Epitope mapping by mass spectrometry: determination of an epitope on HIV-1 IIIB p26 recognized by a monoclonal antibody.
Matrix-assisted laser desorption mass spectrometry in combination with proteolytic protection assays has been used to identify the functional epitope on HIV-1 IIIB p26 recognized by a mAb. In this procedure, the intact protein is affinity bound to an immobilized mAb under physiologic conditions. A combination of proteolytic enzymatic cleavages was then performed to remove unprotected residues. Protected residues were identified by matrix-assisted laser desorption mass spectrometry based on their m.w. With this approach, an 11-residue sequence was identified as the most tightly affinity-bound fragment. in addition, two less tightly bound segments were observed. These latter two residues may contain elements of a discontinuous epitope or may be residues involved in a wider contact area. The combination of matrix-assisted laser desorption and proteolytic epitope footprinting has been applied to the determination of the epitope on a recombinant protein recognized by a mAb but should be equally applicable to the definition of an epitope on a native protein in its natural folded conformation.
Amino Acid Sequence↗
ESR spin-trapping of a protein-derived tyrosyl radical from the reaction of cytochrome c with hydrogen peroxide.
The reaction of horse heart cytochrome c with hydrogen peroxide was investigated using the ESR spin-trapping technique and the nitroso spin traps 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS) and 2-methyl-2-nitrosopropane (MNP). The ESR spectra obtained using both spin traps were typical of an immobilized nitroxide and indicated that the adduct was a macromolecule. The intensity of the ESR spectrum corresponding to the DBNBS/*cytochrome c radical adduct was greatly enhanced by performing the reaction under anaerobic conditions, which suggested that the spin trap was competing with O2 for reaction with the radical site(s). Nonspecific proteolysis of either the DBNBS or the MNP adducts revealed isotropic three-line spectra. In addition, a high resolution ESR spectrum for the protease-treated MNP cytochrome c-derived protein radical adduct was obtained. The superhyperfine couplings detected in this spectra were identical to those detected from an authentic MNP/tyrosyl adduct. Carbon-13 labeling of the aromatic ring positions of tyrosine yielded additional hyperfine coupling, demonstrating that the radical site was definitely located on the ring of tyrosine. Mass spectrometry detected as many as four DBNBS/.cytochrome c-derived adducts from the reaction of cytochrome with H2O2. Thus, it would appear four radical sites are formed during the reaction, at least one of which is tyrosine.
Animals↗
Biochemical characterization of the human liver cytochrome P450 arachidonic acid epoxygenase pathway.
Human liver microsomal fractions metabolized arachidonic acid in the presence of NADPH yielding epoxyeicosatrienoic acids and their hydration products, dihydroxyeicosatrienoic acids, as the principal reaction products. Inhibition studies using polyclonal antibodies prepared against recombinant CYP2C8, an abundant human liver cytochrome P450 epoxygenase, demonstrated 85-90% inhibition of arachidonic acid epoxide formation. Both epoxyeicosatrienoic acids and dihydroxyeicosatrienoic acids were detected in large amounts in human liver using gas chromatography/mass spectrometry. Chiral analysis of the endogenous liver epoxides demonstrated a preference for the 14(R),15(S)-, 11(R),12(S)-, and 8(S),9(R)-epoxyeicosatrienoic acid enantiomers. Importantly, the chirality of liver epoxyeicosatrienoic acids matched that previously reported for recombinant CYP2C8 (Zeldin et al. (1995) Arch. Biochem. Biophys. 322, 76-86). Incubations of both human liver cytosolic and microsomal fractions with synthetic epoxyeicosatrienoic acids revealed a 10-fold higher rate of dihydroxyeicosatrienoic acid formation with cytosolic relative to microsomal fractions. Recombinant human liver cytosolic epoxide hydrolase rapidly and regioselectively hydrated epoxyeicosatrienoic acids. We conclude, based on these data, that CYP2C8 is one of the primary, constitutive hepatic arachidonic acid epoxygenases responsible for formation of epoxyeicosatrienoic acids and that cytosolic epoxide hydrolase is the principal liver enzyme which forms the dihydroxyeicosatrienoic acids. We speculate that these biologically active eicosanoids may be important in maintaining homeostasis in the liver.
Arachidonic Acid↗
Molecular cloning and expression of CYP2J2, a human cytochrome P450 arachidonic acid epoxygenase highly expressed in heart.
A cDNA encoding a human cytochrome P450 arachidonic acid epoxygenase was isolated from a human liver cDNA library. Sequence analysis revealed that this 1,876-base pair cDNA contained an open reading frame and encoded a new 502-amino acid protein designated CYP2J2. Blot hybridization analysis of RNA prepared from human tissues revealed that CYP2J2 was highly expressed in the heart. Recombinant CYP2J2 protein was prepared using the baculovirus expression system and purified to near electrophoretic homogeneity. The enzyme metabolized arachidonic acid predominantly via olefin epoxidation to all four regioisomeric cis-epoxyeicosatrienoic acids (catalytic turnover 65 pmol of product formed/nmol of cytochrome P450/min at 30 degrees C). Epoxidation of arachidonic acid by CYP2J2 at the 14,15-olefin was highly enantioselective for (14R, 15S)-epoxyeicosatrienoic acid (76% optical purity). Immunoblotting of microsomal fractions prepared from human tissues using a polyclonal antibody raised against the recombinant hemoprotein confirmed primary expression of CYP2J2 protein in human heart. The in vivo significance of CYP2J2 was suggested by documenting the presence of epoxyeicosatrienoic acids in the human heart using gas chromatography/mass spectroscopy. Importantly, the chirality of CYP2J2 products matched that of the epoxyeicosatrienoic acid enantiomers present, in vivo, in human heart. We propose that CYP2J2 is one of the enzymes responsible for epoxidation of endogenous arachidonic acid pools in human heart and that epoxyeicosatrienoic acids may, therefore, play important functional roles in cardiac physiology.
Amino Acid Sequence↗
Coaxial continuous-flow fast-atom bombardment vs electrospray ionization: a sensitivity comparison on a magnetic sector instrument.
The performance of an electrospray (ESI) source was evaluated and compared to that of a coaxial continuous-flow fast-atom bombardment source on a magnetic mass spectrometer using ten different peptides over the mass range of 400 to 3500 Da and using a variety of scanning modes. Results show that sensitivities using the two ionization techniques are similar, with limits-of-detection in the attomole to low femtomole range. In addition, proteins can be routinely detected using ESI at femtomole levels. The observance of the noncovalent complex of RNase A and cytidine 2'-monophosphate yields evidence that these complexes can be studied using instruments that operate at high accelerating voltages.
Cytidine Monophosphate↗