Mass spectrometric identification of a naturally processed melanoma peptide recognized by CD8+ cytotoxic T lymphocytes.

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.

Isolation and characterization of a resistant core peptide of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF); confirmation of the GM-CSF amino acid sequence by mass spectrometry.

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.

The role of mass spectrometry in the drug discovery process.

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.

Antiulcer agents. 2. Gastric antisecretory, cytoprotective, and metabolic properties of substituted imidazo[1,2-a]pyridines and analogues.

The search for a successor to 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine, Sch 28080 (27), a compound that exhibits gastric antisecretory and cytoprotective properties and has undergone clinical evaluation as an antiulcer agent, has culminated in the identification of four related compounds that exhibit pharmacologic profiles similar to that of 27. In three of these potential successors an amino group functions as a surrogate for the 3-cyanomethyl substituent of the prototype. The present work concerns, in addition to an evaluation of the structure-activity relationships of a series of analogues of 27, preliminary studies of the pharmacodynamics and metabolism of 27, performed with the aid of cyano carbon labeled versions of the drug (13C labeled; 28; 14C labeled, 29). These studies have shown that 27 is well-absorbed and extensively metabolized and that the major metabolite of 27 is the thiocyanate anion. A similar study performed on 3-amino-2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine, labeled at the 3-position with carbon-13 (41) or carbon-14 (42), revealed that this compound, which has an antisecretory/cytoprotective profile comparable to that of 27, is also metabolized to thiocyanate anion, although this must occur via a different mechanism. The chemistry section includes a discussion of the potential sites of protonation of the pharmacologically similar 3-amino analogue 40 and the structurally related imidazo[1,2-a]pyrazine 67. Predictions based on charge density and protonation product stabilities are presented. That N1 is the site of protonation in these analogues has been definitively demonstrated by X-ray crystal structure analysis, which also unequivocally established the assigned imidazo[1,2-a]pyrazine ring structure.

Fast-atom bombardment and tandem mass spectrometry of macrolide antibiotics.

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.

Extraction and characterization of adenovirus proteins from sodium dodecylsulfate polyacrylamide gel electrophoresis by matrix-assisted laser desorption/ionization mass spectrometry.

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.

Application of electrospray mass spectrometry in probing protein-protein and protein-ligand noncovalent interactions.

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.

Electrospray ionization mass spectrometry for the study of non-covalent complexes: an emerging technology.

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.

High-resolution LC/MS for analysis of minor components in complex mixtures: negative ion ESI for identification of impurities and degradation products of a novel oligosaccharide antibiotic.

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.

Fast atom bombardment mass spectrometric characterization of peptides.

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.

Urinary metabolites of (R),(R)-labetalol.

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.

Comparative mapping of recombinant proteins and glycoproteins by plasma desorption and matrix-assisted laser desorption/ionization mass spectrometry.

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.

Structural analysis of biologically active peptides and recombinant proteins and their modified counterparts by mass spectrometry.

The structural characterization of the Escherichia coli-expressed human interferon alpha-2b (rh-IFN alpha-2b) was carried out by employing the fast atom bombardment (FAB) and plasma desorption (PD) mapping methods. The mass spectral data of the rh-IFN alpha-2b and the trypsin-generated peptide mixture allowed rapid and facile confirmation of the cDNA-derived sequence and determination of the existing disulfide pattern in the protein molecule. The same PD/FAB mapping approach was successfully employed in the structural determination of the iodination reaction product of rh-IFN alpha-2b and the potent vasoconstrictor peptide endothelin.

Bacterial phospholipid analysis by fast atom bombardment mass spectrometry.

The structural analysis of naturally occurring bacterial phospholipids in mixtures by fast atom bombardment (FAB) mass spectrometry are reported. The bacterial strains examined included several genera of actinomycetes, two strains of Escherichia coli, and one strain each of Proteus mirabilis and Pseudomonas aeruginosa. FAB mass spectrometry proved to be a useful tool for the structural identification of phospholipids in mixtures and provided stable pseudo-molecular ions and characteristic fragment ions which permitted the identification of phosphatidylethanolamine and phosphatidyl choline. Information regarding the chain length of the fatty acids, their degree of unsaturation in the chains and the presence of hydroxyl groups was also obtained. The results obtained by FAB mass spectrometry were supported by high-resolution mass spectral data, tandem mass spectrometric studies and FAB mass spectrometry of components which had been separated and partially purified by thin-layer chromatography. Each organism displayed a highly characteristic phospholipid profile suggesting the possible use of FAB mass spectrometry as a method for rapid bacterial detection and identification.