Identification of the site of phosphorylation of the chemotaxis response regulator protein, CheY.

The protein (Escherichia coli CheY) that controls the direction of flagellar rotation during bacterial chemotaxis has been shown to be phosphorylated on the aspartate 57 residue. The residue phosphorylated is present within a conserved sequence in every member of a family of bacterial regulatory proteins. The phosphorylation is transient, with a much shorter half-life than that expected of a simple acyl phosphate intermediate, indicating that the sequence and conformation of the protein is designed to achieve a rapid hydrolysis.

Characterization of structural xenobiotic modifications in proteins by high sensitivity tandem mass spectrometry. Human hemoglobin treated in vitro with styrene 7,8-oxide.

Alkylation of DNA by xenobiotic agents, or their electrophilic metabolites, is believed to be the major initiating process that may result ultimately in carcinogenesis. The study of hemoglobin alkylated in vivo by chemical carcinogens has previously been proposed as an indicator for DNA alkylation. Xenobiotically modified proteins, however, are not readily amenable to conventional methods for amino acid sequencing.

Asparagine-linked glycosylation of the scrapie and cellular prion proteins.

Post-translational modification of the scrapie prion protein (PrP) is thought to account for the unusual features of this protein. Molecular cloning of a PrP cDNA identified two potential Asn-linked glycosylation sites. Both the scrapie (PrPSc) and cellular (PrPC) isoforms were susceptible to digestion by peptide N-glycosidase F (PNGase F) but resistant to endoglycosidase H as measured by migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Structure, oligosaccharide structures, and posttranslationally modified sites of the nicotinic acetylcholine receptor.

Using mass spectrometry, we have examined the transmembrane topography of the nicotinic acetylcholine receptor, a five-subunit glycosylated protein complex that forms a gated ion channel in the neuromuscular junction. The primary sequences of the four polypeptide chains making up the acetylcholine receptor from Torpedo californica contain many possible sites for glycosylation or phosphorylation. We have used liquid secondary ion mass spectrometry to identify posttranslationally modified residues and to determine the intact oligosaccharide structures of the carbohydrate present on the acetylcholine receptor.

A new Saccharomyces cerevisiae mnn mutant N-linked oligosaccharide structure.

We find that the N-linked Man8GlcNAc2- core oligosaccharide of Saccharomyces cerevisiae mnn mutant mannoproteins is enlarged by the addition of the outer chain to the alpha 1----3-linked mannose in the side chain that is attached to the beta 1----4-linked mannose rather than by addition to the terminal alpha 1----6-linked mannose. This conclusion is derived from structural studies on a phosphorylated oligosaccharide fraction and from mass spectral fragment analysis of neutral core oligosaccharides.

O6-substituted-2'-deoxyguanosine-3'-phosphate adducts detected by 32P post-labeling of styrene oxide treated DNA.

32P post-labeling of DNA reacted with styrene oxide resulted in the detection of six adducts. In order to determine which of these corresponded to modification at the O6 position of guanine, O6-substituted styrene oxide-deoxyguanosine-3'-monophosphate derivatives were synthesized. The two synthetic isomers were purified by HPLC and the structures were confirmed by mass spectrometry and 1H NMR.

Structure and heterogeneity of the oligosaccharides from the lipopolysaccharides of a pyocin-resistant Neisseria gonorrhoeae.

The compositions and partial structures of the oligosaccharides from the lipopolysaccharides (LPS) of a pyocin-resistant Neisseria gonorrhoeae (strain JW31R) have been determined by liquid secondary ion mass spectrometry (LSIMS), tandem mass spectrometry, and methylation analysis. Four major structures were identified with Mr 2123, 2000, 1961, and 1838, as well as seven species of lower abundance of Mr 1758-1272. The largest of the major oligosaccharides (Mr, 2122) consists of 3-deoxymanno-2-ketooctulosonic acid (KDO)-Hep2GalNAcGlcNAcGal4Glc2 (Hep, heptose) and phosphoethanolamine (PEA).

Cesium ion liquid secondary ion mass spectrometry of membrane-bound glycoproteins: structural and topological considerations of acetylcholine receptor from Torpedo californica.

We report mass mapping of a large (270 kD) multisubunit membrane bound glycoprotein, nicotinic acetylcholine receptor from Torpedo californica, using enzymic digests of the affinity purified whole receptor and cesium ion liquid secondary ion mass spectrometry. Peptides, glycopeptides and derivatized N-linked oligosaccharides were isolated by HPLC and identified by LSIMS. We have shown that mass spectrometric sensitivity is improved a hundred-fold through use of computer-controlled mass window stepping of an electro-optical multichannel array detection system on a LSIMS double focusing mass spectrometer.

Environmental applications of organic mass spectrometry in China.

A review of more than 30 papers on the environmental applications of organic mass spectrometry in China in studies of environmental carcinogens, such as nitrosamines, polycyclic aromatic hydrocarbons (PAHs), and nitro-PAH, and trace organics in water, polychlorinated biphenyls, and reaction products and mechanism is presented.

Revision of the blocked N terminus of rat heart fatty acid-binding protein by liquid secondary ion mass spectrometry.

The primary structure of rat heart muscle fatty acid-binding protein was investigated by liquid secondary ion mass spectrometry. The protein was digested with trypsin, chymotrypsin, and Staphylococcus aureus V8 protease and the resulting peptides were separated by reverse phase high performance liquid chromatography. The masses of the protonated molecular ions (MH+) of the tryptic, chymotryptic, and S.

Structural characterization of intact, branched oligosaccharides by high performance liquid chromatography and liquid secondary ion mass spectrometry.

We report results of a mass-spectrometric-based strategy for determining the detailed structural features of N-linked oligosaccharides from glycoproteins. The method was used to characterize a series of intact, high mannose oligosaccharides isolated from human immunoglobulin M (IgM). The IgM was purified from a patient with Waldenstrom's macroglobulinemia.

Structure of the oligosaccharide portion of human hepatitis B surface antigen.

The hepatitis B surface antigen, which constitutes the currently available vaccine, is the empty envelope of the hepatitis B virus. We investigated the carbohydrate structures of the envelope glycoproteins. The intact oligosaccharides were enzymatically released from the coat glycoproteins using peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F and isolated by gel permeation chromatography.

Positive and negative liquid secondary ion mass spectrometry of chloro- and nitrophenylglucuronides.

Mass spectra of a series of chloro- and nitrophenylglucuronides by liquid secondary ion (LSI) mass spectrometry were obtained. In the positive ion mode class characteristic fragmentations and adduct ions are observed only in the presence of alkali salt additives. No additives were necessary in the negative ion mode to see abundant class characteristic [M-H]- and aglycone fragment ions.

High-performance liquid chromatographic isolation and fast atom bombardment mass spectrometric identification of Di-N-desethylamiodarone, a new metabolite of amiodarone in the dog.

Amiodarone is an antiarrhythmic drug which has received considerable attention in recent years. It has been suggested that the unusual pharmacodynamic characteristics of this drug may be due in part to the influence of active metabolites. Using fast atom bombardment (FAB) mass spectrometry we have identified a new metabolite of amiodarone, the di-N-desethyl analog (DDEA).

Human stratum corneum lipids: characterization and regional variations.

The lipids of mammalian stratum corneum are known to be important regulators of skin permeability. Since the human stratum corneum displays remarkable regional variations in skin permeability, we assessed the total lipid concentration, the distribution of all major lipid species, and the fatty acid composition in Bligh-Dyer extracts from four skin sites (abdomen, leg, face, and sole) that are known to display widely disparate permeability. Statistically significant differences in lipid weight were found at the four sites that were inversely proportional to their known permeability.

Carcinogen binding to DNA.

A key initiating event in the induction of neoplasia by a chemical carcinogen is believed to be the covalent reaction of the carcinogen with the DNA of the target cell. Most carcinogens are not biologically active as such, but require metabolic conversion to a chemically reactive form (ultimate carcinogen). Chemical carcinogens undergo an extremely complex set of metabolic reactions, leading for the most part to inactive detoxification products as well as reactive electrophilic species.

Identification of 2-nitrofluorene in diesel exhaust particulates.

Diesel exhaust particulates contain mutagens which are direct-acting in the Ames test. A dichloromethane extract of these particulates was separated by liquid chromatography and analyzed by high resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS). 2-Nitrofluorene, a known carcinogen and direct mutagen, was positively identified by comaxima of ion fragments and retention time on the gas chromatograph.

Structural identification of autoinducer of Photobacterium fischeri luciferase.

Synthesis of bacterial luciferase in some strains of luminous bacteria requires a threshold concentration of an autoinducer synthesized by the bacteria and excreted into the medium. Autoinducer excreted by Photobacterium fischeri strain MJ-1 was isolated from the cell-free medium by extraction with ethyl acetate, evaporation of solvent, workup with ethanol-water mixtures, and silica gel chromatography, followed by normal-phase and then reverse-phase high-performance liquid chromatography. The final product was greater than 99% pure.

Field desorption mass spectrometry of oligosaccharides.

Field desorption mass spectrometry has been used to analyze carbohydrate polymers with 5 to 14 hexose units without prior derivatization. In all examples, the molecular weight of the oligosaccharide could be determined by means of the abundant quasimolecular ions of the type MNa(+), MH(+), MNa(2) (2+), and MNa(3) (3+). Fragmentation at glycosidic linkages was observed in varying extents.