ABSTRACT
High-resolution mass spectrometry has been routinely used for structural confirmation and identification; however, it has mostly been applied to relatively pure samples. Exact mass measurement of minor components such as impurities, degradation products or metabolites in complex mixtures has been difficult without prior separation and isolation. Here we report the utilization of on-line liquid chromatography in combination with high-resolution mass spectrometry for the identification of impurities and base degradation products of Sch 27899, a member of the everninomicin class of antibiotics. Nine Sch 27899-related impurities and degradation products were detected by negative ion electrospray ionization using a magnetic sector mass spectrometer. Exact mass measurements were obtained at a resolution of 5000 using polyethylene glycol (PEG) sulfates as internal standards. Corresponding elemental compositions were determined within a 2 ppm error tolerance and structures were proposed for all components.
Subject(s)
Aminoglycosides , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Chromatography, Liquid/methods , Oligosaccharides/analysis , Spectrometry, Mass, Electrospray Ionization/methods , Anti-Bacterial Agents/analysis , Calibration , Carbohydrate Sequence , Micromonospora/chemistry , Molecular Sequence Data , Molecular Structure , Oligosaccharides/chemistry , Oligosaccharides/metabolism , Sensitivity and SpecificityABSTRACT
A new methodology for the extraction and characterization of proteins from Coomassie-stained sodium dodecylsulfate polyacrylamide gel electrophoresis using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been described. The utility of this methodology was demonstrated in the characterization of adenovirus proteins. The key steps in the extraction and destaining process involve washing the excised band with a combination of solvents that include 10% acetic acid, acetonitrile, methanol, and formic acid:water:isopropanol mixture. By using this procedure, we determined adenovirus proteins with molecular weights ranging from 10,000 to 110,000 Da by MALDI-MS, obtaining a detection limit of approximately 6 pmol. Parallel experiments were successfully carried out to analyze adenovirus proteins from Cu-stained gels. It was observed that increase in laser intensity resulted in significant improvements in the quality of MALDI mass spectra for the analysis of inefficiently destained proteins from Cu-stained gels.
Subject(s)
Adenoviridae/chemistry , Viral Proteins/chemistry , Coloring Agents , Copper , Electrophoresis, Polyacrylamide Gel , Molecular Weight , Rosaniline Dyes , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Viral Proteins/isolation & purificationABSTRACT
The structure characterization of biologically-active organic compounds, developed from synthetic and natural sources, is an integral part of the drug discovery effort to identify novel therapeutic agents. Mass spectrometric methods (electrospray ionization, matrix-assisted laser desorption/ionization, fast atom bombardment, electron ionization and chemical ionization) are uniquely qualified to solve a wide variety of structural identification problems with high speed and accuracy. This report provides an overview of the recent developments in mass spectrometry (MS) and discusses their contribution to several areas of pharmaceutical research: the automation of MS for high-throughput analysis to support new entity research, the use of liquid chromatography (LC)-MS for mixture analysis of degradation products and drug metabolites, the expanded role of highly sensitive MS for the structure elucidation of unknown organic compounds (especially natural products), the study of peptides and proteins, and the detection of non-covalent complexes.
ABSTRACT
Mutated, tumorigenic Ras is present in a variety of human tumors. Compounds that inhibit tumorigenic Ras function may be useful in the treatment of Ras-related tumors. The interaction of a novel GDP exchange inhibitor (SCH-54292) with the Ras-GDP protein was studied by NMR spectroscopy. The binding of the inhibitor to the Ras protein was enhanced at low Mg2+ concentrations, which enabled the preparation of a stable complex for NMR study. To understand the enhanced inhibitor binding and the increased GDP dissociation rates of the Ras protein, the conformational changes of the Ras protein at low Mg2+ concentrations was investigated using two-dimensional 1H-15N HSQC experiments. The Ras protein existed in two conformations in slow exchange on the NMR time scale under such conditions. The conformational changes mainly occurred in the GDP binding pocket, in the switch I and the switch II regions, and were reversible. The Ras protein resumed its regular conformation after an excess amount of Mg2+ was added. A model of the inhibitor in complex with the Ras-GDP protein was derived from intra- and intermolecular NOE distance constraints, and revealed that the inhibitor bound to the critical switch II region of the Ras protein.
Subject(s)
Glucosides/metabolism , Guanosine Diphosphate/metabolism , Proteins/antagonists & inhibitors , Sulfonamides/metabolism , ras Proteins/metabolism , Amino Acid Sequence , Binding Sites , Computer Simulation , Glucosides/chemistry , Guanine Nucleotide Exchange Factors , Humans , Macromolecular Substances , Magnesium/metabolism , Models, Molecular , Molecular Sequence Data , Monte Carlo Method , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Conformation , Proteins/chemistry , Sulfonamides/chemistry , ras Guanine Nucleotide Exchange FactorsABSTRACT
The detection of non-covalent complexes in the mass range 19,000-34,000 Da, using electrospray ionization mass spectrometry (ESI-MS), is reviewed. The examples discussed include (1) a protein-ligand interaction (ras-GDP), (2) an inhibitor-protein-ligand interaction (SCH 54292/SCH 54341-ras-GDP), (3) a protein-protein interaction (gamma-IFN homodimer) and (4) a protein-metal complex [HCV (1-181)-Zn]. In each case, the ESI-MS method is capable of releasing the intact non-covalent complex from its native solution state into the gas phase in the form of multiply-charge ions. The molecular masses of these complexes were determined with a mass accuracy of better than 0.01%, which is far superior to the traditional methods of sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel permeation chromatography. The method provides the researcher with a quick, reliable and reproducible method for probing difficult biological problems. The key to success in the study of non-covalent complexes depends on careful understanding and manipulation of ESI source parameters and sample solution conditions; special care must be taken with the source orifice potential and the solution pH and organic co-solvents must be avoided. This paper also illustrates the usefulness of ESI-MS for addressing biological problems leading to the discovery of new therapeutics; the approach involves the rapid screening of potential drug candidates, such as weakly bound inhibitors.
Subject(s)
Mass Spectrometry/methods , Proteins/chemistry , Chelating Agents/chemistry , Genes, ras , Glucosides/analysis , Guanosine Diphosphate/analysis , Hepacivirus/chemistry , Humans , Interferon-gamma/chemistry , Ligands , Mass Spectrometry/instrumentation , Metals/chemistry , Molecular Weight , Proteins/analysis , Sulfonamides/analysisABSTRACT
Structural characterization of peptides in the range of 500-5000 Da, using fast atom bombardment (FAB) and Cs+ ion liquid secondary ion mass spectrometry (SIMS), is reviewed. These include synthetic peptides Kemptamide (mol wt 1516); GIF-C15 (mol wt 1875), an isolated natural product as an acylated pentapeptide; and polypeptides generated from enzymatic digests of proteins. MS data is shown to reveal molecular weight and sequence information as well as determine disulfide bonds between cysteine residues and glycosylation sites in the case of a glycopeptide. The complementarity of MS technique to classical biochemical methods for peptide characterization is highlighted. The reader is briefly acquainted with two newer ionization techniques namely, electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). Synthetic chemists and biochemists can refer to the in-depth review articles that are cited throughout this article.
Subject(s)
Peptides/chemistry , Spectrometry, Mass, Fast Atom Bombardment/methods , Spectrometry, Mass, Secondary Ion/methods , Amino Acid Sequence , Glycosylation , Molecular Sequence Data , Molecular Weight , Peptide Mapping , Sequence Analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methodsABSTRACT
MS based methodology employing electrospray ionization (ESI) is described for the detection of ternary complexes in which SCH 54292 or SCH 54341 and GDP are noncovalently bound to oncogenic ras protein. The observed molecular weights of 19,816 and 19,570 Da confirmed the presence of noncovalent complexes of ras-GDP-SCH 54292 and ras-GDP-SCH 54341, respectively. We have also performed selective chemical modification of lysine residues of the ras protein complex followed by enzymatic digestion and on-line LC-ESI MS peptide mapping to determine protein-drug binding topography. There was a good correlation between nucleotide exchange inhibition as determined by the enzyme assay and evidence of complex formation as determined by MS.
Subject(s)
ras Proteins/antagonists & inhibitors , ras Proteins/chemistry , Amino Acid Sequence , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Binding Sites , Drug Evaluation, Preclinical , Glucosides/chemistry , Glucosides/pharmacology , Guanine Nucleotide Exchange Factors , In Vitro Techniques , Macromolecular Substances , Mass Spectrometry/methods , Molecular Sequence Data , Molecular Weight , Peptide Mapping , Proteins/antagonists & inhibitors , Proteins/chemistry , Sulfonamides/chemistry , Sulfonamides/pharmacology , ras Guanine Nucleotide Exchange Factors , ras Proteins/geneticsABSTRACT
The nucleotide exchange process is one of the key activation steps regulating the ras protein. This report describes the development of potent, non-nucleotide, small organic inhibitors of the ras nucleotide exchange process. These inhibitors bind to the ras protein in a previously unidentified binding pocket, without displacing bound nucleotide. This report also describes the development and use of mass spectrometry, NMR spectroscopy and molecular modeling techniques to elucidate the structure of a drug-protein complex, and aid in designing new ras inhibitor targets.
Subject(s)
Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Crystallography, X-Ray , Drug Design , Magnetic Resonance Spectroscopy , Proto-Oncogene Proteins p21(ras)/chemistry , Spectrometry, Mass, Fast Atom BombardmentABSTRACT
N-nitrosoamides of 7 beta-hydroxylated bile acid conjugates, particularly of the ursodeoxycholic acid family have been synthesized. The products and synthetic intermediates were fully characterized by the results of high-resolution 1H NMR, FT-IR, FABMS and ESI-MS studies. The compounds, N-nitrosoglycoursodeoxycholic acid (NOGUDCA), N-nitrosoglycoursocholic acid (NOGUCA) and N-nitrosoglycodeoxycholic acid (NOGDCA) decomposed between pH 6 and 9 in aqueous buffer solutions, indicating a t1/2 of 5-7 h while N-nitrosotauroursodeoxycholic acid (NOTUDCA) indicated a much longer t1/2 of 15-17 h. These results suggest that the compounds are relatively stable and may enter the enterohepatic circulation. Their decomposition is similar to that of other N-nitrosamides, which generate alkylating agents and thereby act as DNA mutagens.
Subject(s)
Bile Acids and Salts/chemical synthesis , Deoxycholic Acid/chemical synthesis , Nitroso Compounds/chemistry , Bile Acids and Salts/chemistry , Deoxycholic Acid/analogs & derivatives , Half-Life , Magnetic Resonance Spectroscopy , Spectrometry, Mass, Fast Atom BombardmentABSTRACT
PURPOSE: To isolate and characterize a monomethioninesulfoxide variant of the commercially available therapeutic protein interferon alpha-2b. METHODS: The methionine (Met)-oxidized variant was isolated by reverse-phase high performance liquid chromatography and characterized by SDS-PAGE, peptide mapping and mass spectrometric analysis of the trypsin/V8-generated peptide fragments. The biological and immunological activities of the isolated variant were also evaluated. RESULTS: The rHuIFN alpha-2b variant was found to contain a Met sulfoxide residue at position 111 of the rHuIFN alpha-2b molecule. The far-UV CD spectra showed a slight loss of alpha-helical content and an increase in the beta-sheet contribution. The CD spectra indicate that both chromatographic conditions and Met oxidation contribute to the observed secondary structure changes. Both interferon alpha-2b main component and its methionine-oxidized variant showed different reactivity to monoclonal antibodies employed in immunoassays for the protein. CONCLUSIONS: A monomethioninesulfoxide rHuIFN alpha-2b variant was found to be present in the rHuIFN alpha-2b bulk drug substance in solution. The Met(111) residue was identified as Met sulfoxide by comparative tryptic/V8 mapping and mass spectrometric analysis. Nevertheless, the oxidation of the Met(111) residue did not seem to have a detectable effect on the biological activity of the molecule.
Subject(s)
Antiviral Agents/isolation & purification , Interferon-alpha/isolation & purification , Methionine/analogs & derivatives , Amino Acid Sequence , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Cells, Cultured , Chromatography, High Pressure Liquid , Circular Dichroism , Electrophoresis, Polyacrylamide Gel , Humans , Interferon alpha-2 , Interferon-alpha/chemistry , Interferon-alpha/pharmacology , Mass Spectrometry , Methionine/chemistry , Molecular Sequence Data , Peptide Mapping , Recombinant Proteins , Spectrophotometry, UltravioletABSTRACT
We and others have previously reported that melanoma-specific, cytotoxic T lymphocytes (CTL) define a minimum of six class I-presented peptide epitopes common to most HLA-A2+ melanomas. Here we show that three of these peptide epitopes are coordinately recognized by a CTL clone obtained by limiting dilution from the peripheral blood of an HLA-A2+ melanoma patient. Tandem mass spectrometry was used to characterize and sequence one of these three naturally processed melanoma peptides. One of the potential forms of the deduced peptide sequence (XXTVXXGVX, X = I or L) matches positions 32-40 of the recently identified melanoma gene MART-1/Melan-A. This peptide (p939; ILTVILGVL) binds to HLA-A2 with an intermediate-to-low affinity and is capable of sensitizing the HLA-A2+ T2 cell line to lysis by CTL lines and clones derived from five different melanoma patients. A relative high frequency of anti-p939-specific effector cells appear to be present in situ in HLA-A2+ melanoma patients, since p939 is also recognized by freshly isolated tumor infiltrating lymphocytes. p939 represents a good candidate for the development of peptide-based immunotherapies for the treatment of patients with melanoma.
Subject(s)
Antigens, Neoplasm/immunology , CD8-Positive T-Lymphocytes/immunology , Melanoma/immunology , T-Lymphocytes, Cytotoxic/immunology , Amino Acid Sequence , Clone Cells , Epitope Mapping , HLA-A2 Antigen/immunology , Humans , Mass Spectrometry , Molecular Sequence Data , Peptides/chemistry , Peptides/immunologyABSTRACT
Dilevalol, the (R),(R)-isomer of labetalol, is a novel antihypertensive agent with both beta-adrenoreceptor activity and direct vasodilatory action. The biotransformation of dilevalol was studied in the rat, dog, monkey, and human. Nine metabolites were isolated and characterized by NMR spectroscopy and MS. The simple benzylic glucuronide is the major metabolite in the dog, monkey, and human, whereas the phenolic glucuronide is the major metabolite in the rat. Seven other metabolites that arise from phase 1 oxidation were also isolated, including a family of catechol-like metabolites formed by hydroxylation at the C3 position of the benzamide ring. This catechol also undergoes ring cyclization forming two novel indolic metabolites.
Subject(s)
Labetalol/metabolism , Animals , Biotransformation , Dogs , Glucuronates/urine , Humans , Macaca fascicularis , Male , Rats , Rats, Sprague-Dawley , Species SpecificityABSTRACT
The mass spectrometric (MS) techniques of 252Cf-plasma desorption (PD) and matrix-assisted laser desorption/ionization (MALDI) are compared in the molecular weight determination and the mapping analysis of several recombinant proteins and glycoproteins. MALDI MS analysis exhibited better sensitivity and mass measurement accuracy and a remarkably short analysis time compared with PD MS analysis. The latter was not successful in the analysis of rhIFN-gamma and the higher mass mammalian cell-derived IL-5 glycoproteins. Mapping of the Escherichia coli-derived rhIFN alpha-2b and rhIL-4 proteins, by direct PD or MALDI MS analysis of the trypsin-generated peptide mixtures provided signals for ca. 95% and 88% of the expected tryptic peptides, respectively. Peptide signals below m/z 1500 were generally more intense in the PD mass spectra, while higher mass signals were more intense in the MALDI mass spectra. Both PD and MALDI MS analyses provided a rapid confirmation of the existing two and three disulfide bonds in the rhIFN alpha-2b and rhIL-4 proteins, respectively. In the mapping of the CHO IL-4 glycoprotein, detection of the trypsin-generated glycopeptides was only possible by MALDI, where their detection was greatly improved by using the super-DHB (sDHB) matrix, a 9:1 mixture of 2,5-dihydroxybenzoic acid (DHB) with 2-hydroxy-5-methoxybenzoic acid. This sDHB matrix also generated significantly enhanced and better resolved MALDI peptide signals, which in turn resulted in a much improved mass measurement accuracy.
Subject(s)
Glycoproteins/analysis , Peptide Mapping , Recombinant Proteins/analysis , Animals , Humans , Lasers , Mass Spectrometry , MiceABSTRACT
Molecular weights of macrolide antibiotics can be determined from either (M + H)(+) or (M + Met)(+), the latter desorbed from alkali metal salt-saturated matrices. The ion chemistry of macrolides, as determined by tandem mass spectrometry (MS/MS), is different for ions produced as metallated than those formed as (M + H)(+) species. An explanation for these differences is the location of the charge. For protonated species, the charge is most likely situated on a functional group with high proton affinity, such as the dimethylamino group of the ammo sugar. The alkali metal ion, however, is bonded to the highly oxygenated aglycone. As a result, the collision-activated dissociation spectra of protonated macrolides are simple with readily identifiable fragment ions in both the high and low mass regions but no fragments in the middle mass range. In contrast, the cationized species give complex spectra with many abundant ions, most of which are located in the high mass range. The complementary nature of the fragmentation of these two species recommends the study of both by MS/MS when determining the structure or confirming the identity of these biomaterials.
ABSTRACT
A trypsin-resistant core peptide of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) was isolated and analyzed by high-energy Cs+ liquid secondary-ion (LSI) mass spectrometric analysis. This analysis provided successful detection of the high-mass disulfide-linked core peptide as well as information confirming the existence of disulfide pairing. Similarly, LSI mass spectrometric analysis of the peptide fragments isolated chromatographically from a Staphylococcus aureus V8 protease digest of rhGM-CSF provided rapid confirmation of the cDNA-derived sequence and determination of the existing disulfide bonds between cysteine residues 54-96 and 88-121. Electrospray ionization mass spectrometry was employed to measure the molecular weight of the intact protein and to determine the number of the disulfide bonds in the protein molecule by comparative analysis of the protein before and after reduction with beta-mercaptoethanol.
Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor/chemistry , Peptide Fragments/chemistry , Amino Acid Sequence , Chromatography, High Pressure Liquid , Escherichia coli/genetics , Gas Chromatography-Mass Spectrometry , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Humans , Molecular Sequence Data , Peptide Fragments/genetics , Recombinant Proteins/chemistry , Sequence Analysis , Serine Endopeptidases/metabolism , Trypsin/metabolismABSTRACT
A novel mass spectrometry-based methodology using electrospray ionization (ESI) is described for the detection of protein-protein [interferon (IFN)-γ dimer] and protein-ligand [ras-guanosine diphosphate (GDP)] noncovalent interactions. The method utilizes ESI from aqueous solution at appropriate pH. The presence of the noncovalent complex of the IFN-γ dimer was confirmed by the observed average molecular weight of 33,819 Da. The key to the detection of the IFN-γ dimer is the use of an alkaline solution (pH ≈ 9) for sample preparation and for mass spectrornetry analysis. The effect of the declustering energy in the region of the ion sampling orifice and focusing quadrupole on the preservation of the gas-phase noncovalent complex (IFN-γ dimer) was also studied. The effect of the declustering energy on complex dissociation was further extended to probe the noncovalent protein-ligand association of ras-GDP. It was found that little energy is required to dissociate the IFN-γ dimer, whereas a substantial amount of energy is required to dissociate the gas-phase ras-GDP complex.