Application of electrospray ionization mass spectrometry and tandem mass spectrometry in combination with capillary electrophoresis for biochemical investigations.
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Biochemistry↗
Biomedical subjects
C G Edmonds
Publications and source records attributed to C G Edmonds.
Posttranscriptional modification of tRNA in thermophilic archaea (Archaebacteria).
Nucleoside modification has been studied in unfractionated tRNA from 11 thermophilic archaea (archaebacteria), including phylogenetically diverse representatives of thermophilic methanogens and sulfur-metabolizing hyperthermophiles which grow optimally in the temperature range of 56 (Thermoplasma acidophilum) to 105 degrees C (Pyrodictium occultum), and for comparison from the most thermophilic bacterium (eubacterium) known, Thermotoga maritima (80 degrees C). Nine nucleosides are found to be unique to the archaea, six of which are structurally novel in being modified both in the base and by methylation in ribose and occur primarily in tRNA from the extreme thermophiles in the Crenarchaeota of the archaeal phylogenetic tree. 2-Thiothymine occurs in tRNA from Thermococcus sp., and constitutes the only known occurrence of the thymine moiety in archaeal RNA, in contrast to its near-ubiquitous presence in tRNA from bacteria and eukarya. A total of 33 modified nucleosides are rigorously characterized in archaeal tRNA in the present study, demonstrating that the structural range of posttranscriptional modifications in archaeal tRNA is more extensive than previously known. From a phylogenetic standpoint, certain tRNA modifications occur in the archaea which are otherwise unique to either the bacterial or eukaryal domain, although the overall patterns of modification are more typical of eukaryotes than bacteria.
Adenosine↗
Sensitivity considerations for large molecule detection by capillary electrophoresis-electrospray ionization mass spectrometry.
The use of the electrospray ionization (ESI) method for interfacing capillary electrophoresis with mass spectrometry (CE-MS) is particularly well suited for the analysis of large molecules due to the multiple charging phenomenon. While ionization efficiency is very high, the available ion current is dispersed over more peaks so that the maximum peak intensity obtainable declines significantly for large molecules. Sensitivity with ESI can be improved by operation at very low flow-rates, an ideal situation for CE-MS. These and other considerations related to sensitivity are illustrated using ESI-MS measurements for cytochrome c.
Animals↗
New developments in biochemical mass spectrometry: electrospray ionization.
The principles, development, and recent application of electrospray ionization-mass spectrometry (ESI-MS) to biological compounds are reviewed. ESI-MS methods now allow determination of accurate molecular weights for proteins extending to over 50,000, and in some cases well over 100,000. Similar capabilities are being developed for oligonucleotides. The instrumentation used for ESI-MS is briefly described and it is shown that, although ionization efficiency appears to be uniformly high, detector sensitivity may be directly correlated with molecular weight. The use of tandem mass spectrometry (e.g., MS/MS) for extending collision-induced dissociation (CID) methods to the structural studies of large molecules is described. For example, effective CID of various albumin species (molecular weight approximately 66,000) can be obtained, far larger than obtainable for singly charged molecular ions. The combination of capillary electrophoresis, in both free solution zone electrophoresis and isotachophoresis formats, as well as microcolumn liquid chromatography with ESI-MS, provides the capability for on-line separation and analysis of subpicomole quantities of proteins. These and other new developments related to ESI-MS are illustrated by a range of examples. Fundamental considerations suggest even more impressive developments may be anticipated related to detection sensitivity and methods for obtaining structural information.
Biochemistry↗
Primary sequence information from intact proteins by electrospray ionization tandem mass spectrometry.
Tandem mass spectrometry has been used to obtain information related to portions of the primary sequence for an intact protein, bovine ribonuclease A. Multiply charged molecular ions, generated by electrospray ionization, were collisionally dissociated at low energies in a triple quadrupole mass spectrometer to yield singly and multiply charged fragment ions that can be assigned to the known sequence of the protein. Dissociation of the highly charged molecular ions resulted in pairs of complementary product ions. The higher order (gas-phase) protein structure affects the dissociation processes, as observed in comparisons of tandem mass spectra of the native and disulfide-reduced forms of ribonuclease A.
Amino Acid Sequence↗
Effect of reducing disulfide-containing proteins on electrospray ionization mass spectra.
Electrospray ionization produces multiply charged molecular ions for biomolecules with molecular weights in excess of 100,000. This allows mass spectrometers with limited mass-to-charge range to extend their molecular weight range by a factor equal to the number of charges. The maximum number of observed charges for peptides and smaller proteins correlates well with the number of basic amino acid residues (Arg, Lys, His), except for disulfide-containing molecules, such as lysozyme and bovine albumin. However, reduction of disulfide linkages with 1,4-dithiothreitol (Cleland's reagent) may allow the protein to be in an extended conformation and make "buried" basic residues available for protonation to yield higher charged molecular ions by the electrospray ionization process. For larger proteins reduction of disulfide bridges greatly increases the maximum charge state, but charging of basic amino acid residues remains less efficient than for smaller proteins.
Disulfides↗
5-[[(carboxymethyl)amino]methyl]uridine is found in the anticodon of yeast mitochondrial tRNAs recognizing two-codon families ending in a purine.
The modified nucleoside (U*) present in the wobble position of Saccharomyces cerevisiae mitochondrial tRNA(Leu) and tRNA(Trp) was isolated by thin-layer chromatography and HPLC. Its chromatographic, UV spectral, and mass spectrometric properties were shown to be identical with those of 5- [[(carboxymethyl)amino]methyl]uridine (cmnm5U). This nucleoside found in yeast mitochondrial tRNAs reading two-codon families ending in a purine permits the selective recognition of A and G in the third codon position.
Anticodon↗
Electrospray ionization mass spectrometry.
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Amino Acid Sequence↗
Chorismic acid, a key metabolite in modification of tRNA.
Chorismic acid is the common precursor for the biosynthesis of the three aromatic amino acids as well as for four vitamins. Mutants of Escherichia coli defective in any of the genes involved in the synthesis of chorismic acid are also unable to synthesize uridine 5-oxyacetic acid (cmo5U) and its methyl ester (mcmo5U). Both modified nucleosides are normally present in the wobble position of some tRNA species. Mutants defective in any of the specific pathways leading to phenylalanine, tyrosine, tryptophan, folate, enterochelin, ubiquinone, and menaquinone have normal levels of cmo5U and mcmo5U in their tRNA. The presence of shikimic acid in the growth medium restores the ability of an aroD mutant to synthesize cmo5U, while O-succinylbenzoate, which is an early intermediate in the synthesis of menaquinone, does not. Thus, chorismic acid is a key metabolite in the synthesis of these two modified nucleosides in tRNA. The absence of chorismic acid blocks the formation of cmo5U and mcmo5U at the first step, which might be the formation of 5-hydroxyuridine. This results in an unmodified U in the wobble position of tRNA(1Val) and in most of the tRNAs normally containing cmo5U and mcmo5U. Since cmo5U and mcmo5U are synthesized under anaerobic conditions, the formation of these nucleosides does not require molecular oxygen. One of the carbon atoms of the side chain, --O--CH2--COOH, originates from the methyl group of methionine. The other carbon atom does not originate directly from the C-1 pool, from the carboxyl group methionine, or from bicarbonate. This metabolic link between intermediary metabolism and translation also exists for another member of the family Enterobacteriaceae, Salmonella typhimurium, as well as for the distantly related gram-positive organism Bacillus subtilis.
Bacillus subtilis↗
Capillary zone electrophoresis and isotachophoresis-mass spectrometry of polypeptides and proteins based upon an electrospray ionization interface.
The special capabilities of the capillary electrophoresis electrospray ionization-mass spectrometer interface for the analysis of peptides and proteins with molecular weights extending to in excess of 100,000 are reviewed. The dynamic combinations of both capillary zone electrophoresis and capillary isotachophoresis with electrospray ionization are illustrated for mixtures of peptides and proteins. Myoglobin and cytochrome c detection limits were ca. 100 fmol. The potential extension of these methods for determination of the primary structure (sequence) of polypeptides using tandem mass spectrometry is shown to be facilitated by the high charge state of ions produced by the electrospray interface. The relevance of these results for advances in analytical biochemistry are discussed.
Animals↗
Thermospray liquid chromatography/mass spectrometry in deuterium oxide.
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Chromatography, Liquid↗
Novel salvage of queuine from queuosine and absence of queuine synthesis in Chlorella pyrenoidosa and Chlamydomonas reinhardtii.
Partially purified extracts from Chlorella pyrenoidosa and Chlamydomonas reinhardtii catalyze the cleavage of queuosine (Q), a modified 7-deazaguanine nucleoside found exclusively in the first position of the anticodon of certain tRNAs, to queuine, the base of Q. This is the first report of an enzyme that specifically cleaves a 7-deazapurine riboside. Guanosine is not a substrate for this activity, nor is the epoxide a derivative of Q. We also establish that both algae can incorporate exogenously supplied queuine into their tRNA but lack Q-containing tRNA when cultivated in the absence of queuine, indicating that they are unable to synthesize Q de novo. Although no physiological function for Q has been identified in these algae, Q cleavage to queuine would enable algae to generate queuine from exogenous Q in the wild and also to salvage (and recycle) queuine from intracellular tRNA degraded during the normal turnover process. In mammalian cells, queuine salvage occurs by the specific cleavage of queuine from Q-5'-phosphate. The present data also support the hypothesis that plants, like animals, cannot synthesize Q de novo.
Chlamydomonas↗
Transfer RNA(5-methylaminomethyl-2-thiouridine)-methyltransferase from Escherichia coli K-12 has two enzymatic activities.
The tRNA(5-methylaminomethyl-2-thiouridine)-methyltransferase, which is involved in the biosynthesis of the modified nucleoside 5-methylaminomethyl-2-thiouridine (mnm5s2U) present in the wobble position of some tRNAs, was purified close to homogeneity (95% purity). The molecular mass of the enzyme is 79,000 daltons. The enzyme activity has a pH optimum of 8.0-8.5, is inhibited by magnesium ions, and stimulated by ammonium ions. Two different intermediates in the biosynthesis of mnm5s2U34 are present in tRNA from the mutants trmC1 and trmC2. Unexpectedly, the product present in tRNA from trmC1 cells was identified by mass spectrometric and chromatographic analyses as 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), i.e. a more complex derivative than the final product mnm5s2U. The product present in tRNA from trmC2 cells was identified as 5-aminomethyl-2-thiouridine (nm5s2U). In the presence of S-adenosylmethionine the most purified enzyme fraction converts both cmnm5s2U34 and nm5s2U34 into mnm5s2U34. In the absence of S-adenosylmethionine, however, cmnm5s2U34 is converted into nm5s2U by this enzyme fraction. We conclude that the purified polypeptide has two enzymatic activities; one actually demodifies cmnm5s2U to nm5s2U and the other catalyzes the transfer of a methyl group from S-adenosylmethionine to nm5s2U, thus forming mnm5s2U. The sequential order of the biosynthesis of mnm5s2U34 is suggested to be: (Formula: see text). The molecular activity of the methyltransferase activity (nm5s2U34----mnm5s2U34) is 74 min-1, and the steady state concentration of the enzyme is only 78 molecules/genome equivalent in cells growing at a specific growth rate of 1.0/h.
Escherichia coli↗
Identification of 12-keto-5,8,10-heptadecatrienoic acid as an arachidonic acid metabolite produced by human HL-60 leukemia cells.
An unusual cyclooxygenase-derived metabolite of arachidonic acid has been shown to be produced by N,N-dimethylformamide (DMF)-induced, terminally differentiated human HL-60 promyelocytic leukemia cells and to a much lesser extent by untreated cells. Biochemical evidence in conjunction with gas chromatography/mass spectrometry and liquid chromatography/thermospray mass spectrometry analyses indicates that the product is 12-keto-5,8,10-heptadecatrienoic acid (KHT). Both KHT and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) were produced when arachidonic acid was incubated with cell lysates obtained from differentiated HL-60 granulocytes. Indomethacin and the thromboxane synthetase inhibitor UK-38485 inhibited the production of both metabolites, whereas ethacrynic acid inhibited only the production of KHT. In 100,000 g supernatant fractions, obtained from either untreated or differentiated cells, KHT was produced when HHT was used as substrate. The addition of exogenous NAD, but not NADP, to incubations caused a significant increase in the production of KHT coincident with a decrease in the level of HHT. These data suggest that, in both differentiated and undifferentiated HL-60 cells, an NAD-dependent enzyme, apparently 15-prostaglandin dehydrogenase (15-PGDH), is expressed and catalyzes the conversion of HHT to KHT. In differentiated HL-60 cells, this metabolite is produced from arachidonic acid through a multi-enzymatic process involving the activities of cyclooxygenase, thromboxane synthetase and 15-PGDH. The production of KHT from arachidonic acid in undifferentiated HL-60 cells is probably limited, therefore, by the virtual absence of cyclooxygenase activity in these cells.
Arachidonic Acid↗
Isolation and structure elucidation of an epoxide derivative of the hypermodified nucleoside queuosine from Escherichia coli transfer RNA.
A new nucleoside has been identified in tRNATyr from Escherichia coli MRE 600, where it replaces the highly modified nucleoside queuosine. The nucleoside is also present in a large amount relative to queuosine in mixed tRNA from E. coli strains MRE 600 and W (from which it was isolated for characterization). The new nucleoside has been characterized as an epoxy derivative of queuosine: 7-(5-[(2,3-epoxy-4,5-dihydroxycyclopent-1-yl)amino]methyl)-7-de azaguanosine, oQ, based on data from directly combined liquid chromatography/mass spectrometry, high resolution mass spectrometry, and proton NMR spectroscopy. Nucleoside oQ is also present in small amounts in mixed tRNA from E. coli B. Isomerization of oQ occurs readily under alkaline conditions to give a rearranged product, oQ', characterized as 7-(5-[(3,4-epoxy-2,5-dihydroxycyclopent-1-yl)amino]methyl)-7-deaza guanosine. The present finding constitutes the first report of epoxide formation during post-transcriptional processing of RNA.
Chromatography, High Pressure Liquid↗
Structure determination of a new fluorescent tricyclic nucleoside from archaebacterial tRNA.
A highly fluorescent nucleoside was detected in enzymatic digests of the extremely thermophilic archaebacterium Sulfolobus solfataricus by combined liquid chromatography-mass spectrometry (LC/MS). Following isolation, the structure was determined primarily by mass spectrometry, to be 3-(beta-D-ribofuranosyl)-4,9-dihydro-4,6,7-trimethyl-9-oxoimidazo[ 1, 2-a]purine (mimG), a new derivative of the Y (wye) nucleoside. The structural assignment was verified by comparison of the base released by acid hydrolysis with the corresponding synthetic base, using mass spectrometry, chromatography, and UV absorption and fluorescence properties. Nucleoside mimG was also detected by LC/MS in hydrolysates of the thermophiles Thermoproteus neutrophilus and Pyrodictium occultum. These results constitute the first finding of a member of the hypermodified Y family of nucleosides in archaebacteria.
Archaea↗
Stable isotope dilution quantification of mutagens in cooked foods by combined liquid chromatography-thermospray mass spectrometry.
A method of general applicability for the detection and quantification of mutagens in cooked foods at the ppb level is presented. A minimal sample prefractionation is employed and [Me-2H3]-labeled analogs of the compounds of interest are added for identification and quantification of mutagens by accurate measurement of chromatographic retention (K') in reverse-phase high-performance liquid chromatography (HPLC), and by measurement of the ratio of response of the protonated molecular ions of analyte and internal standard by directly coupled liquid chromatography-mass spectrometry (LC/MS). Initial application is demonstrated in the analysis of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) in broiled salmon. Measured levels of IQ and MeIQ in broiled salmon flesh were 0.3-1.8 ppb and 0.6-2.8 ppb, respectively, and for the skin of broiled salmon 1.1-1.7 ppb and 1.5-3.1 ppb, respectively. Results on cooked beef and sardine are also reported.
Animals↗
Analysis of mutagens from cooked foods by directly combined liquid chromatography-mass spectrometry.
Directly combined high performance liquid chromatography-mass spectrometry (LC/MS) has been studied as a method of analysis of heterocyclic aromatic mutagens in cooked foods, in the parts per billion concentration range. Identification and semiquantitative estimation of mutagens is based on accurate measurement of chromatographic retention (k') and molecular weight-selective detection of mutagens, which are protonated during passage of the chromatographic eluant into a thermospray interface of a quadrupole mass spectrometer. Standard chromatographic retention (k') values in two reversed-phase systems and data from thermospray mass spectra from nine mutagens are reported. An isolation scheme employing CH3OH extraction, acid-base partition, cellulose-trisulfo-Cu-phthalocyanine adsorption, and normal-phase HPLC was used prior to LC/MS analysis. Initial applications have been demonstrated in the analysis of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) in broiled salmon flesh. Levels measured were estimated to be in the range 0.2 to 0.4 microgram/kg IQ and 0.4 to 0.9 microgram/kg MeIQ. The method is judged to be generally applicable with minimal sample prefractionation to detection of mutagens at the parts per billion level in cooked foods.
Animals↗