A constant-momentum/energy-selector time-of-flight mass spectrometer.

A matrix assisted laser desorption/ionization time-of-flight mass spectrometer has been built with an ion source that can be operated in either constant-energy or constant-momentum acceleration modes. A decreasing electric field distribution in the ion-accelerating region makes it possible to direct ions onto a space-focal plane in either modes of operation. Ions produced in the constant-momentum mode have velocities and, thus, flight times that are linearly dependent on mass and kinetic energies that are inversely dependent on mass. The linear mass dispersion doubles mass resolving power of ions accelerated with space-focusing conditions in constant-momentum mode. The mass-dependent kinetic energy is exploited to disperse ions according to mass in a simple kinetic energy filter constructed from two closely spaced, oblique ion reflectors. Focusing velocity of ions of the same mass can substantially improve ion selection for subsequent post source decay or tandem time-of-flight analyses.

A gas chromatograph/resonant electron capture-TOF mass spectrometer for four dimensions of negative ion analytical information.

A prototype gas chromatograph (GC) electron monochromator (EM) reflectron time-of-flight (TOF) mass spectrometer has been constructed and demonstrated to simultaneously record four-dimensional resonant electron capture (REC) mass spectra (m/z, ion-intensity, electron-energy, and retention time) of electron-capturing compounds in real time. Specifically, complete REC mass spectra of all of the components in a mixture of perfluorocarboxylic acids and in a sample of pentafluorobenzyl alcohol were recorded in the GC mode. For each compound, the data enable one to distinguish different electronic states of the molecular ion and different possible decomposition pathways for each state. This new instrument can be used to obtain analytical information unrecognizable by any other mass spectrometric technique from the isomeric species of a variety of electron-capturing structures.

Selective production of rat mutant selenoprotein W with and without bound glutathione.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and electrospray ionization mass spectrometry (ESI MS) analysis of a 6x His-tagged recombinant form of rat mutant selenoprotein W (RMSW) reveals that aerobic growth conditions primarily produce a form of RMSW without bound glutathione (10,305 Da) whereas anaerobic conditions produce a glutathione-bound (305 Da) form (10,610 Da). Purification of RMSW was achieved with a procedure employing acetone precipitation and DEAE-cellulose chromatography, in addition to Ni-NTA agarose chromatography. Additional steps, including polyvalent metal ion binding (PMIB) resin chromatography and CM-cellulose chromatography, were necessary after elution from the Ni-NTA agarose column, in order to maintain solubility of the purified protein.

An exponential dilution gradient system for nanoscale liquid chromatography in combination with MALDI or nano-ESI mass spectrometry for proteolytic digests.

A simple and inexpensive nano high performance liquid chromatography system (nano-LC) employing the exponential dilution method for gradient separations was built. The system was used to analyze a tryptic digest of Escherichia coli uracil DNA glycosylase (Ung; Mr = 25,563), a DNA-binding protein that initiates the uracil-excision DNA repair process by catalyzing the release of uracil from the deoxyribose phosphate backbone of DNA. Both on-line and off-line approaches to analyzing peptides produced by in-gel digestion of Ung are demonstrated. The on-line approach uses nano-high performance liquid chromatography (HPLC)/micro-electrospray MS to assign peptide masses. The off-line approach uses matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and nano-electrospray/collision-induced dissociation (CID) tandem mass spectrometry, to analyze fractions (2-3 microL) collected manually from the nano-LC system. The nano-electrospray technique allows detailed fragmentation information to be obtained at different collision energies with only a marginal increase in sample handling due to the nano-LC step.

A high resolving power ion selector for post-source decay measurements in a reflecting time-of-flight mass spectrometer.

An electrostatic deflector has been designed and constructed that can be used in a reflecting time-of-flight mass spectrometer for either single-deflector or dual-deflector velocity selection in post-source decay measurements. The deflector consists of an interleaved set of parallel deflection electrodes as in a Loeb/Cravath/Bradbury device, but thin metal ribbon instead of wire is used for the deflection electrodes. The time for reversing the electric field, which depends on various factors such as the electronics for pulsing the voltage and the time constant of a particular electrode geometry, is about 19 ns for the deflectors used in this study. By properly timing the reversal of the electric field, the time-window for ion transmission can be made substantially less than the switching time of each individual deflector. In conjunction with matrix-assisted laser desorption/ionization, the single-deflector's resolving power and transmission are robust with respect to laser fluence, i.e. they remain high even when the fluence is raised well above threshold. By contrast the operational features of the dual-deflector gate offer more versatility in locating and sizing the selection window. Operating the ion selector in a single-deflector mode, we have achieved a resolving power of approximately 710 full width at half maximum (FWHM) for different isotopes of protonated, sodiated, and potassiated substance-P (m/z 1348.6, 1370.6 and 1386.6 respectively; 10.073 keV). Operating it in the dual-deflector mode under two different sets of conditions, we have succeeded in obtaining resolving powers of approximately 1100 (FWHM) for protonated substance-P (m/z 1348.6; 10.8 keV) and approximately 5200 (FWHM) for an isotopomer of PEG 6000 (approximately m/z 6000; 10.04 keV). This accomplishment implies that high-resolution ion selection can be coupled to post-source decay analyses.

Intraphagosomal chlorination dynamics and yields determined using unique fluorescent bacterial mimics.

Fluorescein was covalently attached through a cystamine linker group to carboxy-derivatized polyacrylamide microspheres to generate phagocytosable particles containing fluorescent reporter groups. A unique feature of these beads is that the dye was recoverable in near-quantitative yield from intracellular environments by thiol reduction of the cystamine disulfide bond. Fluorescence microscopy indicated that individual neutrophils could bind as many as approximately 20 serum-opsonized beads, although no appreciable cellular association was observed for unopsonized beads. By using methyl viologen to quench external fluorescence, it was demonstrated that 70-90% of the neutrophil-associated fluorescein on opsonized beads was inaccessible to the medium. The particle-bound fluorescein underwent near-stoichiometric conversion to chlorinated derivatives when reacted with HOCl or the cell-free myeloperoxidase (MPO)-H2O2-Cl- system; products were identified by HPLC separation and electrospray ionization mass spectrometry of the recovered dye. Fluorescence changes accompanying phagocytosis were consistent with chlorination of the dye; fluorescence spectrometric and chemical trapping measurements indicated that intraphagosomal chlorination was far more extensive than extracellular chlorination. Yields of recovered chlorofluoresceins determined by HPLC indicated that sufficient HOCl had been produced intracellularly to kill entrapped bacteria. Fluorescein chlorination coincided approximately with phagocytosis and stimulated uptake of O2 by the cells. Demonstration that HOCl is produced within phagosomes in sufficient concentrations to kill bacteria on a time scale associated with death constitutes strong evidence in support of a primary role for HOCl in the microbicidal action of neutrophils.

Characterization of peptide-oligonucleotide heteroconjugates by mass spectrometry.

Two peptide-oligothymidylic acids, prepared by joining an 11 residue synthetic peptide containing one internal carboxyl group (Asp side chain) to amino-linker-5'pdT6 and amino-linker-5'pdT10 oligonucleotides, were analyzed by matrix-assisted laser desorption/ionization (MALDI) on a linear time-of-flight mass spectrometer and by electrospray ionization (ESI) on a triple-quadrupole system. These synthetic compounds model peptide-nucleic acid heteroconjugates encountered in antisense research and in studies that use photochemical crosslinking to investigate molecular aspects of protein-nucleic acid interactions. MALDI and ESI sensitivities for the two hybrid compounds were found to be similar respectively to their sensitivities for the pure oligonucleotide parts. In general, MALDI proved to be less affected by sample impurities and more sensitive than ESI, while ESI on the quadrupole produced greater mass accuracy and resolution than MALDI on the time-of-flight instrument. A hybrid's behavior in a MALDI-matrix or an ESI-spray-solvent was found to be governed mainly by the oligonucleotide. A single positive ESI tandem mass spectrum of the peptide-dT6 accounted for the heteroconjugate's entire primary structure including the point of the oligonucleotide's covalent attachment to the peptide.

Resistance of morpholino phosphorodiamidate oligomers to enzymatic degradation.

Oligomers possessing the Morpholino phosphorodiamidate backbone were evaluated for resistance to a variety of enzymes and biologic fluids. A 25-mer was incubated with nucleases, proteases, esterases, and serum, and the reaction mixtures were directly analyzed by MALDI-TOF mass spectrometry. The 25-mer was completely resistant to 13 different hydrolases and serum and plasma. The excellent resistance of Morpholino phosphorodiamidates to enzymatic attack indicates their suitability for in vivo use.

Alkylation of Escherichia coli thioredoxin by S-(2-chloroethyl)glutathione and identification of the adduct on the active site cysteine-32 by mass spectrometry.

Alkylation of reduced Escherichia coli thioredoxin by the episulfonium ion derived from S-(2-chloroethyl)glutathione (CEG) at physiologic pH resulted in at least three different alkylation products. These adducts were separated by reverse phase chromatography, digested with trypsin, and peptide-mapped. The peptide containing the active site cysteines was collected and sequenced by tandem mass spectrometry. Results indicate that the site of alkylation was at Cys-32 exclusively with no alkylation at Cys-35. Raising the pH above the pKa of Cys-35 to ionize the thiol before reacting with the episulfonium ion of CEG did not lead to alkylation at Cys-35, suggesting that a steric factor prevents the alkylating moiety of CEG from accessing this cysteine. A tryptic digest of a minor bis-adduct yielded an alkylated peptide which contained tyrosine, an amino acid known to be alkylated at its hydroxyl group by CEG. Sequencing by tandem mass spectrometry, however, was unsuccessful due to fragmentation of the alkylating moiety from the peptide. Results of this study confirm that the episulfonium ion of CEG can adduct thioredoxin at the active site and may have important toxicologic significance regarding the mechanism of 1,2-dichloroethane toxicity.

Thioredoxin alkylation by a dihaloethane-glutathione conjugate.

Glutathione is a thiol-containing tripeptide which functions to protect cellular constituents from endogenous and xenobiotic electrophiles via conjugation and eventual excretion. In the case of compounds such as 1,2-dihaloethanes, however, conjugate formation results in bioactivation of the species rather than detoxification. The conjugate can then act as an alkylating agent toward cellular constituents including DNA, proteins, or lipids. Alkylation of protein thiols in cells exposed to dihaloethane may contribute substantially to the toxicity produced by these compounds. We examined the reactivity of the conjugate S-(2-chloroethyl)-glutathione (CEG) toward the model protein Escherichia coli thioredoxin. At physiological pH, treatment of thioredoxin by CEG resulted in the production of several bands visible on isoelectric focusing, which were determined by matrix-assisted laser desorption ionization (MALDI) mass spectrometry to be mono-, di-, tri-, and tetra-alkylated forms of thioredoxin. A concomitant loss of in vitro enzymatic activity was observed. These products were also observed when reaction was allowed to take place at pH 11.4. Treatment at pH 4.4 resulted in lesser alkylation of thioredoxin, with only the mono- and di-alkylated forms detected. Iodoacetic acid treatment of CEG-alkylated thioredoxin revealed that the iodoacetic acid-susceptible Cys32 was not carboxymethylated, suggesting that this is one of the sites alkylated by CEG.

UV-catalyzed cross-linking of Escherichia coli uracil-DNA glycosylase to DNA. Identification of amino acid residues in the single-stranded DNA binding site.

Photochemical cross-linking of Escherichia coli uracil-DNA glycosylase (Ung) to oligonucleotide dT20 was performed to identify amino acid residues that reside in or near the DNA-binding site. UV-catalyzed cross-linking reactions produced a covalent Ung x dT20 complex which was resolved from uncross-linked enzyme by SDS-polyacrylamide gel electrophoresis. Cross-link formation required native Ung and was inhibited by increasing concentrations of NaCl in a manner characteristics of NaCl inhibition of Ung catalytic activity. The Ung x dT20 complex was purified to apparent homogeneity, and mass spectrometry revealed that Ung was cross-linked to dT20 in 1:1 stoichiometry as a 31,477 dalton complex. Purified Ung x dT20 lacked detectable uracil-DNA glycosylase activity and failed to bind single-stranded DNA. Recently, we demonstrated that the bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) binds Ung and prevents further interaction with DNA (Bennett, S. E., Schimerlik, M. I., and Mosbaugh, D. W. (1993) J. Biol. Chem. 268, 26879-26885). Addition of the Ugi protein to the cross-linking reaction blocked formation of the Ung x dT20 cross-link. Conversely, the Ung x dT20 cross-link was refractory to Ugi binding. Upon trypsin digestion of Ung x dT20, four distinct products were identified as peptide x dT20 cross-links. A combination of amino acid sequence and mass spectrometric analysis revealed that four tryptic peptides (T6, T18, T19, and T18/19) were adducted to dT20. These observations suggest that dT20 is cross-linked to the Ung DNA-binding site.

Direct observation of UV-crosslinked protein-nucleic acid complexes by matrix-assisted laser desorption ionization mass spectrometry.

Interactions between proteins and nucleic acids are important in the fundamental cellular processes that drive replication, recombination, dynamic alteration and repair of DNA, transcription and processing of RNA, synthesis of proteins, and regulation of enzyme activities. As part of an effort to develop a general, sensitive mass spectrometric strategy for the characterization of protein-nucleic acid interactions, we have used matrix-assisted laser desorption-ionization (MALDI) time-of-flight mass spectrometry to analyze protein-nucleic acid complexes that have been covalently crosslinked by ultraviolet (UV) light. In general, the application of MALDI mass spectrometric techniques to studies of UV-induced crosslinking of nucleoprotein complexes is demonstrated to be feasible. Specifically, MALDI mass analysis was used to determine the molecular weights of the phage T4 gene 32 protein (gp32) crosslinked to the oligonucleotide (dT)20, and the Escherichia coli transcription termination factor rho, photoaffinity labeled with 4-thio-uridine-diphosphate (4sUDP). The covalent gp32:(dT)20 complex is readily detected at a concentration of 1-2 microM in 1 microL of an unpurified solution of reactants that has been exposed to a single, 266 nm UV laser pulse. Mass spectrometric molecular weight determinations of the covalent rho:4sUDP complex add directness and specificity to the ATPase inactivation assay normally used to monitor the formation of 4sUDP photoaffinity labeled rho. It is found that successful MALDI mass spectrometry of protein-nucleic acid complexes is as critically dependent on the choice of solvents and additives as it is on the primary matrix compound.

Reversed phase high performance liquid chromatography of glycopeptides.

A practical procedure for isolating and purifying glycopeptides is described, viz. enzymatic hydrolysis-gel permeation chromatography-ion exchange chromatography-reversed phase HPLC. Using this procedure 28 glycopeptides from hen ovalbumin have been isolated some of which hitherto have not been identified. Water was a suitable mobile phase for preparing pure glycopeptides, and control of column temperature was important for good separations and reproducible retention times. Structural confirmation was by fast atom bombardment mass spectrometry.

Mass spectrometric charting of bovine posterior/intermediate pituitary peptides.

The feasibility for charting neuropeptides in neuroendocrine tissues on the basis of the universal property and inherent specificity of their molecular weights was explored. As a model, a comprehensive MS analysis of extractable peptides from bovine posterior/intermediate pituitary was performed. Two suitable MS techniques--namely, plasma-desorption time-of-flight and fast atom bombardment MS--were evaluated, and each method could identify more than 20 peptides, including N-terminally acetylated and C-terminally amidated species. In toto these peptides account for almost the entire lengths of propressophysin, prooxyphysin, and proopiomelanocortin. Some of the experimentally determined molecular weights did not match any known peptides. Three of these species were identified as acidic joining peptide (4-24) [proopiomelanocortin(83-103)], C-terminal glycopeptide(22-39) [propressophysin(130-147)], and glycosylated C-terminal glycopeptide(1-19) [propressophysin(109-127)] by conventional sequence analysis.

Mass spectrometry of L-beta-aspartamido carbohydrates isolated from ovalbumin.

Using ovalbumin as a starting material, we have isolated and purified relatively large stocks of known glycopeptides for use as models with which to investigate and develop mass spectrometric procedures for the analysis of this class of compounds. In the process, we have discovered six new components in the L-beta-aspartamido carbohydrate fraction of ovalbumin. Positive and negative fast atom bombardment mass spectra, produced on a KRATOS MS50-TC, confirm that the newly found chromatographic peaks correspond to as yet unidentified compounds in the molecular weight range of 1000-3000. Fragment ion peaks present in the mass spectra of some of the unknown compounds can be rationalized in terms of the fragment ion patterns established from the structures of the known glycoasparagines.

Stable glucopyranosylpalladium complexes with cis-beta-hydrogen. A six-membered ring metallocycle with an oxygen donor ligand.

Two stable glucopyranosylpalladium complexes, chloro[1,3-dimethyl-5-(3,4,6-tri-O-acetyl-2-deoxy-alpha-D -arabinohexopyranosyl)-2,4(1H,3H)-pyrimidinedionnato] (triphenylphosphine)-palladium and the corresponding triphenylarsine analog, were studied using fast atom bombardment mass spectrometry, 1H, 13C and 31P nuclear magnetic resonance, UV and IR spectroscopy to establish structures for these complexes. The data obtained indicate that the pyranosyl ring is in a chair conformation in which palladium (C2'), acetoxy (C3' C4') and acetoxymethyl (C5') are equatorial and 1,3-dimethyl-2,4(1H,3H) pyrimidinedion-5-yl (C1') is axial. The palladium(II) ion is encompassed in a six-membered ring metallocycle in which C2' of the glucopyranosyl ring and the oxygen of the C4 carbonyl of the pyrimidinedionyl group occupy adjacent ligand sites. The other two ligand sites on square planar palladium are occupied by triphenylphosphine (or triphenylarsine) cis to C2' and trans to carbonyl oxygen, and chloride trans to C2' and cis to oxygen. This stable metallocycle has three unusual features, a cis-beta-hydrogen, a six-membered Pd-containing ring and an oxygen donor ligand. Its surprising stability is due to conformational barriers to the proper alignment of Pd with pyranosyl ring substituents required for elimination reactions.

Sample derivatization and structure analysis by field desorption mass spectrometry. Peptide methylation-methanolysis.

Analysis by field desorption mass spectrometry of reaction product mixtures produced by treatment of peptides with methanol and hydrogen chloride augments significantly structural data derived by direct field desorption mass spectrometry analysis of the peptides. Analyses of data obtained by three peptides, beta-Ala-HisOH, p-Glu-Ser-GlyOH and Glu-Ser-Gly-AspOH are used for illustration. Data from field desorption mass spectrometry of peptide methylation-methanolysis product mixtures are used to (a) distinguish [M]+- from [M + H]+ in peptide field desorption mass spectrometry spectra, (b) ascertain the number of carboxyl groups present in a peptide, (c) identify a pyro-glutamyl N-terminus and (d) derive peptide sequence information. Definitive assignments of ion relationships are facilitated by use of methanol and deuteromethanol in paired experiments.