Determination of binding positions in oligosaccharides and glycosphingolipids by fast-atom-bombardment mass spectrometry

Application of f.a.b.-m.s. to the products obtained from glycoconjugates upon periodate oxidation followed by borohydride reduction and methylation gives the positions of binding of the monosaccharide residues on the basis of the sequences of the primary and secondary ions.     

Modulation by nutrients and drugs of liver acyl-CoAs analyzed by mass spectrometry

The profile of liver acyl-CoAs induced by dietary fats of variable compositions or by xenobiotic hypolipidemic amphipathic carboxylates was evaluated in vivo using a novel electrospray ionization tandem mass spectrometry methodology of high resolution, sensitivity, and reliability. The composition of liver fatty acyl-CoAs was found to reflect the composition of dietary fat. Treatment with hypolipidemic carboxylates resulted in liver dominant abundance of their respective acyl-CoAs accompanied by an increase in liver fatty acyl-CoAs. Cellular effects exerted by dietary fatty acids and/or xenobiotic carboxylic drugs may be transduced in vivo by their respective acyl-CoAs.     

Chemometric discrimination of unfractionated plant extracts analyzed by electrospray mass spectrometry

Metabolic fingerprints were obtained from unfractionated Pharbitis nil leaf sap samples by direct infusion into an electrospray ionization mass spectrometer. Analyses took less than 30 s per sample and yielded complex mass spectra. Various chemometric methods, including discriminant function analysis and the machine-learning methods of artificial neural networks and genetic programming, could discriminate the metabolic fingerprints of plants subjected to different photoperiod treatments. This rapid automated analytical procedure could find use in a variety of phytochemical applications requiring high sample throughput.     

Analysis of the in vivo phosphorylation state of rabbit skeletal muscle glycogen synthase by fast-atom-bombardment mass spectrometry

The in vivo phosphorylation state of glycogen synthase was re-examined by fast-atom-bombardment mass spectrometry and a procedure in which phosphoserine residues are first converted to S-ethylcysteine. In animals injected with the beta-adrenergic antagonist propranolol, the phosphorylation sites in the N-terminal (N) and C-terminal (C) cyanogen bromide peptides were identified as the serine residues at N7, the region C28-C39, C42, C46 and C100. In animals injected with adrenalin, the phosphorylation of N7 increased from 0.6 to 0.8 mol/mol, the region C28-C39 from 0.7 to 1.2 mol/mol and C100 from 0.3 to 0.6 mol/mol. The phosphorylation states of C42 (0.7 mol/mol) and C46 (0.9 mol/mol) were unchanged. In addition, two further serine residues became phosphorylated at positions N10 (0.5 mol/mol) and C87 (0.5 mol/mol), which were not phosphorylated in the absence of adrenalin. Residues N10 and C42 have not been recognized as in vivo sites of phosphorylation previously. The results suggest that N10 is phosphorylated by a novel protein kinase which may be activated by cyclic-AMP-dependent protein kinase. The phosphorylation of C42 is likely to be catalysed by glycogen synthase kinase 3. The protein kinases responsible for phosphorylating N7, the region C28-C39, C46, C87 and C100 in vivo and the molecular mechanisms by which adrenalin inactivates glycogen synthase in vivo are discussed. Residue N3, a major site phosphorylated by casein kinase-I in vitro is not phosphorylated in vivo. This and other evidence indicates that casein kinase-I is not a glycogen synthase kinase in vivo.     

Doping control for metandienone using hair analyzed by gas chromatography-tandem mass spectrometry

A sensitive, specific and reproducible method for the quantitative determination of the anabolic metandienone in human hair has been developed. The preparation involved a decontamination step with methylene chloride. The hair sample (about 50 mg) was solubilised in 1 ml 1 M NaOH, 10 min at 95 degrees C, in presence of 2 ng of nandrolone-d(3) used as internal standard. The homogenate was neutralized and extracted using consecutively a solid-phase extraction (Isolute C(18) eluted with methanol) and a liquid-liquid extraction with pentane. The residue was derivatized by adding 5 microl MSTFA/NH(4)I/2-mercaptoethanol (250 microl; 5 mg; 15 microl) and 45 microl MSTFA, then incubated for 20 min at 60 degrees C. A 1 microl aliquot of derivatized extract was injected into the column (HP5-MS capillary column, 5% phenyl-95% methylsiloxane, 30 m x 0.32 mm i.d., 0.25 microm film thickness) of a Hewlett Packard (Palo Alto, CA, USA) gas chromatograph (6890 Series). Metandienone was identified using three transitions (its daughter ions at m/z 339 and 206 for the parent 444 and 191 for 206) using a Waters Quattro Micro MS-MS system. The transition m/z 444 to 206 has been used as quantification transition and the others as identification transitions. The assay was capable of detecting 2 pg/mg of metandienone when approximately 50 mg of hair material was processed. Linearity was observed for metandienone concentrations ranging from 2 to 500 pg/mg with a correlation coefficient of 0.9997. Intra-day and between-day precisions at 50 pg/mg were 13.4-16.5% and 22.0%, respectively, with an extraction recovery of 48%. The analysis of hair, cut into four segments, obtained from an athlete, revealed the presence of metandienone at the concentrations of 78, 7, 10 and 108 pg/mg in each segment of hair (0-1, 1-2, 2-3 and 3 cm to the tip).     

Ligand-induced conformational changes in the acetylcholine-binding protein analyzed by hydrogen-deuterium exchange mass spectrometry

Recent x-ray crystallographic studies of the acetylcholine-binding protein (AChBP) suggest that loop C, found at the circumference of the pentameric molecule, shows distinctive conformational changes upon antagonist and agonist occupation. We have employed hydrogen-deuterium exchange mass spectrometry to examine the influence of bound ligands on solvent exposure of AChBP. Quantitative measurements of deuterium incorporation are possible for approximately 56% of the Lymnaea AChBP sequence, covering primarily the outer surface of AChBP. In the apoprotein, two regions flanking the ligand occupation site at the subunit interface, loop C (residues 175-193) and loop F (residues 164-171), show greater extents of solvent exchange than other regions of the protein including the N- and C-terminal regions. Occupation by nicotinic agonists, epibatidine and lobeline, and nicotinic antagonists, methyllycaconitine, alpha-bungarotoxin, and alpha-cobratoxin, markedly restricts the exchange of loop C amide protons, influencing both the rates and degrees of exchange. Solvent exposure of loop C and its protection by ligand suggest that in the apoprotein, loop C exhibits rapid fluctuations in an open conformation. Bound agonists restrict solvent exposure through loop closure, whereas the larger antagonists restrict solvent exposure largely through occlusion of solvent. Loop F, found on the complementary subunit surface at the interface, also reveals ligand selective changes in amide proton exchange rates. Agonists do not affect solvent accessibility of loop F, whereas certain antagonists cause subtle accessibility changes. These results reveal dynamic states and fluctuating movements in the vicinity of the binding site for unligated AChBP that can be influenced selectively by ligands.     

Vibrio cholerae toxin-coregulated pilus structure analyzed by hydrogen/deuterium exchange mass spectrometry

The bacterial pathogen Vibrio cholerae uses toxin-coregulated pili (TCP) to colonize the human intestine, causing the severe diarrheal disease cholera. TCP are long, thin, flexible homopolymers of the TcpA subunit that self-associate to hold cells together in microcolonies and serve as the receptor for the cholera toxin phage. To better understand TCP's roles in pathogenesis, we characterized its structure using hydrogen/deuterium exchange mass spectrometry and computational modeling. We show that the pilin subunits are held together by tight packing of the N-terminal alpha helices, but loose packing of the C-terminal globular domains leaves substantial gaps on the filament surface. These gaps expose a glycine-rich, amphipathic segment of the N-terminal alpha-helix, contradicting the consensus view that this region is buried in the filament core. Our results explain extreme filament flexibility, suggest a molecular basis for pilus-pilus interactions, and reveal a previously unrecognized therapeutic target for V. cholerae and other enteric pathogens.     

Contaminant inclusion into protein crystals analyzed by electrospray mass spectrometry and X-ray crystallography.

The inclusion of protein contaminants into crystals of turkey egg white lysozyme (TEWL) was investigated by electrospray mass spectrometry of the dissolved crystals. The results show that significant amounts of the structurally related contaminant hen egg white lysozyme (HEWL) are included in the crystals of TEWL. The structurally unrelated contaminant RNAse A, on the other hand, is not included. The X-ray diffraction data statistics of a hybrid TEWL/HEWL crystal and an uncontaminated TEWL crystal were of similar quality. This indicates that, even though the crystals contain much higher levels of the contaminant than one would have expected after a recrystallization experiment, they are still suitable for X-ray diffraction experiments. However, attempts to detect the presence of the contaminant in the crystal by crystallographic structure refinement did not yield conclusive results.     

Assignment of the five disulfide bridges in an alpha-amylase inhibitor from wheat kernel by fast-atom-bombardment mass spectrometry and Edman degradation.

The assignment of the five disulfide bridges in an alpha-amylase monomeric inhibitor from wheat kernel (coded 0.28) was achieved by combining fast-atom-bombardment mass spectrometry (FAB-MS) and automatic sequencing based on Edman degradation. Direct FAB-MS analysis of the native and reduced enzymatic digests of the protein allowed the assignment of three disulfide bridges out of five, including those involving two adjacent cysteine residues. The remaining two disulfide bridges were assigned by sequencing automatically the peptide clusters purified from the tryptic digest of the native protein.     

Unique molecular signatures of glycerophospholipid species in different rat tissues analyzed by tandem mass spectrometry

Glycerophospholipids (GPL) in animal tissues are composed of a large array of molecular species that mainly differ in the fatty acyl composition. In order to further understand the roles of GPL at the molecular level, it is necessary to have comprehensive, accurate accounts of the molecular makeup for these molecules in animal tissues. However, this task was difficult simply because the conventional technologies of profiling GPL species depended heavily on technical skill for accuracy and reliability and were extremely labor-intensive. In recent years, tandem mass spectrometry (MS/MS) proved to be a highly reliable and sensitive technology for profiling small molecules, including GPL, in biological samples. In this study, we used this technology to perform simultaneous comparative analyses for phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI) in the same lipid preparations of liver, lung, kidney, heart, pancreas, stomach, small intestine, spleen, skeleton muscle and brain of an adult rat. We produced molecular profiles of these 4 GPL classes in these 10 different tissues that are highly reproducible between different scans of the same sample and between samples from different animals. It is intriguing that each tissue was found to possess a unique signature of GPL profile that may be used to identify unknown tissues. More importantly, these profiles may also set reference points for studying changes of GPL metabolism in different physiological and pathological conditions.     

Formaldehyde adducts of glutathione. Structure elucidation by two-dimensional n.m.r. spectroscopy and fast-atom-bombardment tandem mass spectrometry.

Aqueous mixtures of formaldehyde and glutathione react to form a variety of cyclized adducts in addition to S-hydroxymethylglutathione. The adducts are in labile equilibrium with each other and are not readily separated. The structures of two of the other major adducts were determined by concerted application of 13C-1H two-dimensional chemical-shift correlation, fast-atom-bombardment mass spectrometry and tandem mass spectrometry to the adduct mixtures in aqueous solution.     

Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry

The quadroma antibody represents the first attempt to produce a bispecific heterodimeric IgG antibody by somatic fusion of 2 hybridoma cells each expressing monoclonal antibodies with distinctive specificities. However, because of random heavy and light chain pairing, the desired functional bispecific antibody represents only a small fraction of the protein produced. Subsequently, the knobs-into-holes (KiH) approach was developed to enforce correct heavy chain heterodimerization. Assuming equimolar expression of 4 unmodified chains comprising 2 heavy and 2 light chains, the statistical distribution of all paired combinations can be calculated. With equimolar expression as the goal, we transfected HEK cells with 1:1:1:1 plasmid ratios and analyzed the protein A affinity-purified antibodies from the quadroma and KiH approaches qualitatively and quantitatively with regard to the estimated relative amounts of the products using electrospray quadrupole time-of-flight mass spectrometry. Our results show that all expected species are formed, and that, within the methodological limits, the species distribution in the mixtures corresponds approximately to the statistical distribution.     

Formation of prostamides from anandamide in FAAH knockout mice analyzed by HPLC with tandem mass spectrometry

We investigated the formation of PGF(2alpha) 1-ethanolamide, PGE(2) 1-ethanolamide, and PGD(2) 1-ethanolamide (prostamides F(2alpha), E(2), and D(2), respectively) in liver, lung, kidney, and small intestine after a single intravenous bolus administration of 50 mg/kg of anandamide to normal and fatty acid amide hydrolase knockout (FAAH -/-) male mice. One group of three normal mice was not dosed (na?ve) while another group of three normal mice received a bolus intravenous injection of 50 mg/kg of anandamide. Three FAAH -/- mice also received an intravenous injection of 50 mg/kg of anandamide. After 30 min, the lung, liver, kidney, and small intestine were harvested and processed by liquid-liquid extraction. The concentrations of prostamide F(2alpha), prostamide E(2), prostamide D(2), and anandamide were determined by HPLC-tandem mass spectrometry. Prostamide F(2alpha) was detected in tissues in FAAH -/- mice after administration of anandamide. Concentrations of anandamide, prostamide E(2), and prostamide D(2) in liver, kidney, lung, and small intestine were much higher in the anandamide-treated FAAH -/- mice than those of the anandamide-treated control mice. This report demonstrates that prostamides, including prostamide F(2alpha), were formed in vivo from anandamide, potentially by the cyclooxygenase-2 pathway when the competing FAAH pathway is lacking.     

Protein complexes in the archaeon Methanothermobacter thermautotrophicus analyzed by blue native/SDS-PAGE and mass spectrometry

Methanothermobacter thermautotrophicus is a thermophilic archaeon that produces methane as the end product of its primary metabolism. The biochemistry of methane formation has been extensively studied and is catalyzed by individual enzymes and proteins that are organized in protein complexes. Although much is known of the protein complexes involved in methanogenesis, only limited information is available on the associations of proteins involved in other cell processes of M. thermautotrophicus. To visualize and identify interacting and individual proteins of M. thermautotrophicus on a proteome-wide scale, protein preparations were separated using blue native electrophoresis followed by SDS-PAGE. A total of 361 proteins, corresponding to almost 20% of the predicted proteome, was identified using peptide mass fingerprinting after MALDI-TOF MS. All previously characterized complexes involved in energy generation could be visualized. Furthermore the expression and association of the heterodisulfide reductase and methylviologen-reducing hydrogenase complexes depended on culture conditions. Also homomeric supercomplexes of the ATP synthase stalk subcomplex and the N5-methyl-5,6,7,8-tetrahydromethanopterin:coenzyme M methyltransferase complex were separated. Chemical cross-linking experiments confirmed that the multimerization of both complexes was not experimentally induced. A considerable number of previously uncharacterized protein complexes were reproducibly visualized. These included an exosome-like complex consisting of four exosome core subunits, which associated with a tRNA-intron endonuclease, thereby expanding the constituency of archaeal exosomes. The results presented show the presence of novel complexes and demonstrate the added value of including blue native gel electrophoresis followed by SDS-PAGE in discovering protein complexes that are involved in catabolic, anabolic, and general cell processes.     

Subproteomes of soluble and structure-bound Helicobacter pylori proteins analyzed by two-dimensional gel electrophoresis and mass spectrometry

Helicobacter pylori is one of the most common bacterial pathogens and causes a variety of diseases, such as peptic ulcer or gastric cancer. Despite intensive study of this human pathogen in the last decades, knowledge about its membrane proteins and, in particular, those which are putative components of the type IV secretion system encoded by the cag pathogenicity island (PAI) remains limited. Our aim is to establish a dynamic two-dimensional electrophoresis-polyacrylamide gel electrophoresis (2-DE-PAGE) database with multiple subproteomes of H. pylori (http://www.mpiib-berlin.mpg.de/2D-PAGE) which facilitates identification of bacterial proteins important in pathogen-host interactions. Using a proteomic approach, we investigated the protein composition of two H. pylori fractions: soluble proteins and structure-bound proteins (including membrane proteins). Both fractions differed markedly in the overall protein composition as determined by 2-DE. The 50 most abundant protein spots in each fraction were identified by peptide mass fingerprinting. We detected four cag PAI proteins, numerous outer membrane proteins (OMPs), the vacuolating cytotoxin VacA, other potential virulence factors, and few ribosomal proteins in the structure-bound fraction. In contrast, catalase (KatA), gamma-glutamyltranspeptidase (Ggt), and the neutrophil-activating protein NapA were found almost exclusively in the soluble protein fraction. The results presented here are an important complement to genome sequence data, and the established 2-D PAGE maps provide a basis for comparative studies of the H. pylori proteome. Such subproteomes in the public domain will be effective instruments for identifying new virulence factors and antigens of potential diagnostic and/or curative value against infections with this important pathogen.     

Enzymatic methyl esterification of synthetic tripeptides: structural requirements of the peptide substrate. Detection of the reaction products by fast-atom-bombardment mass spectrometry

Eukaryotic protein carboxyl methyltransferase catalyzes a two-substrates reaction in which the methyl group of S-adenosylmethionine is transferred to the free carboxyl group of D-aspartyl and L-isoaspartyl-containing peptide or protein substrates. It has been previously shown that at least three binding sites are required for the interaction of adenosylmethionine with the enzyme and/or the protein substrate [Oliva A., Galletti P., Zappia V., Paik W. K. & Kim S. (1980) Eur. J. Biochem. 104, 595-602], while very little is known concerning the structural requirements of the protein substrate. In this study several synthetic tripeptides were selected in order to elucidate the structural requirements of the methyl-accepting substrates. The results obtained with this series of peptides suggested that: (1) three residues appear to be the minimal length, so far identified, required for a productive enzyme-substrate interaction, several dipeptides being ineffective as substrates [McFadden P. N. & Clarke S. (1986) J. Biol. Chem. 261, 11,503-11,511]; (2) the isoaspartyl residue is not recognized unless its alpha-amino group is involved in a carboamide bond; (3) an hydrogen atom on the amide linkage following the isoaspartyl residue is essential for both recognition and catalysis; (4) oligopeptides containing both D-aspartyl and D-isoaspartyl residues are not recognized by this methyltransferase. On the basis of these results, interaction sites between the peptide substrate and the enzyme molecule have been proposed. This paper also reports the first application of fast-atom-bombardment mass spectrometry to the detection of the products of the enzymatic methyl esterification reaction. By this soft ionization technique, the methyl-esterified peptides as well as the corresponding cyclic imides generated during the spontaneous demethylation process have been identified.     

The proteomic characterization of ram sperm during cryopreservation analyzed by the two-dimensional electrophoresis coupled with mass spectrometry

The aim of this study was to analyze the effects of the cryopreservation process on the protein profile of ram sperm using two-dimensional electrophoresis (2-DE) coupled with mass spectroscopy. Semen was collected from five rams and cryopreserved in a Tris-based extender supplemented with glycerol and egg yolk as the main cryoprotectants. The fresh and post-thaw sperm total proteins were extracted and purified, followed by the 2-DE. The differential proteins in the stained gel were determined by mass spectrometry. The results indicated that there were 39 differential proteins between fresh sperm and frozen-thawed sperm. Among these proteins, the abundance of 28 proteins in fresh sperm was higher than those in post-thaw sperm (P < 0.05). However, 11 proteins in post-thaw sperm were up-regulated instead. The gene ontology (GO) analysis showed that most of differential proteins were implicated in cellular process, metabolism and regulation of the biological process. The networks of protein-protein interaction indicated a strong interaction among these differential proteins, which may be involved in sperm metabolism, acrosomal function, sperm motility, and reducing ROS level. In conclusion, the cryopreservation process modifies the proteome of ram sperm, which may be directly associated with ram sperm cryodamage, consequently influencing their fertility. Additionally, these differential proteins can be used as biomarkers for evaluation of frozen ram semen quality.