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J. Mass Spectrom. Soc. Jpn., 46(1), 11-16, 1998
In the electron impact mass spectra of a series of cycloalkylacetones with ring sizes varying from cyclopropane to cycloheptane, the intensity of the McLafferty rearrangement ion at m/z 58 increases with the larger, less strained rings. The larger ring ketones give surprisingly intense "McLafferty plus one" ions at m/z 59, which is the base peak in the mass spectrum of cyclohexylacetone. This double hydrogen McLafferty rearrangement is favored over the single hydrogen rearrangement at both high (70 eV) and low (14 eV) ionizing voltages. Deuterium labeling confirms the expected considerable hydrogen migration in the cycloalkyl ring, occurring following hydrogen transfer to the carbonyl oxygen and prior to C-C bond cleavage.
J. Mass Spectrom. Soc. Jpn., 46(1), 17-24, 1998
The EI mass spectra of 1,4-disubstituted-1,2,3,6-tetrahydropyridinyl derivs. were studied using ten regioselectively labeled isomers of the neurotoxin MPTP. Anal. of the isotopically enriched ions obtained from each labeled analog allowed us to identify the origins of the fragments. Fragmentation pathways are proposed that account for the formation of most of the major peaks. Based on the fragment ions detected and the calcd. HOMO energies, it is proposed that electron bombardment results in a resonance delocalized radical cation that could be derived from initial electron loss from the nitrogen atom or the olefinic double bond.
J. Mass Spectrom. Soc. Jpn., 46(1), 25-31, 1998
A sensitive and specific method involving gas chromatog.-mass spectrometry (GC-MS) using selected ion monitoring has been developed and validated for the detn. of bucillamine (SA96) and its metabolites (SA679, SA981, and SA672) in rat blood by derivatizing free sulfhydryl groups with Me acrylate (MA). The collected blood sample was immediately mixed with MA in 0.05 M tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 9.1) to stabilize free sulfhydryl groups of SA96 and SA679. The reaction of MA with a sulfhydryl group was successfully completed under the conditions tested and derivs. produced were sufficiently stable to allow the development of a simultaneous detn. method of SA96 and its metabolites. Derivatized analytes were purified using a combination of deproteinization and liq.-liq. extn. Analytes in exts. were converted to methylesters, and finally subjected to GC-MS. The limit of quantitation of SA96, SA679, and SA672 was 2 ng/mL and that of SA981 was 10 ng/mL in rat blood. All std. curves for analytes were linear, and the repeatability and reproducibility of this method were satisfactory. It has been successfully applied to pharmacokinetic studies of bucillamine after oral administration to rats.
J. Mass Spectrom. Soc. Jpn., 46(1), 33-36, 1998
The ionization efficiency under fast atom bombardment conditions with liq. matrix have been studied. The dependence of fast atoms, Xe, Ar, and He, for the ionization efficiency from the surface of matrix soln. bombarded with 6 keV atom beams has been rationalized from the standpoint of elastic/inelastic collisions between a fast atom and a matrix mol. in the surface layer. A relationship between ionization efficiency and at. mass, which was formulated by a collision theory, predicted a tendency that the efficiency approaches asymptotically to a finite value with increasing at. mass. The curves for the ionization efficiency vs. at. mass, obtained by using a few compds., were in agreement with the theor. prediction in the tendency.
J. Mass Spectrom. Soc. Jpn., 46(1), 37-43, 1998
Positive and negative ion fast atom bombardment tandem mass spectrometry 8FAB-CID MS/MS) has established the fragmentation pattern of the 18 imidazolone derivatives which are related to the structure of the GFP (green fluorescent protein) chromophore in the jelly fish Aequorea bioluminescence. Ring cleavage of the methylidene group as well as the C4-C5 and C2-N3 bonds also occurred in the positive ion mode. On the basis of the CID-MS/MS (collision induced dissociation tandem mass spectrometry) the structures of protonated molecules produced upon FAB are discussed. When the structure of the fragment ion has a p-quinonoid moiety, distinct peaks due to radical anion species such as (M-H-CH3)- ? are observed in their CID-MS/MS of parent anions (M-H)-, which seems to be characteristics of the conjugated p-hydroxyphenyl structures.
J. Mass Spectrom. Soc. Jpn., 46(1), 45-52, 1998
Here we report that FAB CID-MS/MS, choosing an ion originating from the in-source degrdn. prior to the full acceleration of the mol.-related ion as the precursor, is an excellent means to differentiate linkage isomers. FAB CID-MS/MS performed in this way results in a product ion spectrum, which is enriched with the fragmentation information, and which may thus indicate the position of the newly formed OH group already produced in the ion source. Linkage knowledge may be classified into one of three categories as described later. This method was applied to acidic Lewis-type glycoconjugates-putative ligands for selectins [L. L. W. Cooling, D.-S. Zhang, and T. A. W. Koerner, Trends in Glycosci. Glycotech., 9, 191 (1997)]- and their neutral analogs to differentiate linkage isomers by mass spectrometry. With these glycoconjugates FAB CID-MS/MS having [M+H]+s as the precursor ions is a good means to distinguish the linkage isomers only in simple cases, but not in general cases. By using ions originating from in-source degrdn. prior to the full acceleration of the mol.-related ion as the precursor, we were able to differentiate between Lea and Lex.
J. Mass Spectrom. Soc. Jpn., 46(1), 53-56, 1998
Pyoverdins are chromopeptides produced by the so-called fluorescent group of the bacterial genus Pseudomonas. Species or even strain specific they differ in the compn. of their peptide chains. For classification purposes it is of importance to get fast information regarding the nature of the latter. For two new representatives from hydrolysis results and esp. from an anal. of the data obtained from electrospray ionization (ESI) and fast atom bombardement (FAB) mass spectrometry measurements the amino acid sequences can be suggested as Asp-BuOHOrn-Dab-aThr-Gly-Ser-Ser-OHAsp-aThr (Pseudomonas putida BTP16) and Ser-Ser-FoOHOrn-Ser-Ser-Ser-FoOHOrn-Lys-Lys (Pseudomonas fluorescens BTP7).
J. Mass Spectrom. Soc. Jpn., 46(1), 57-62 (in Japanese), 1998
Polar derivatization method for electrospray ionization (ESI) was developed and applied to the sensitive anal. of nonpolar ketosteroid in plasma. 4-Androstene-3,17-dione (androstenedione) in human plasma was purified by solid-phase extn. with Bond Elut C18 and derivatized to the carboxymethyloxime form (androstenedioxime) as a polar deriv. Androstenedione-d6 as an internal std. was synthesized and its deuterium contents were satisfactory for the sensitive anal. of androstenedione. The LC/ESI-MS condition for androstenedioxime was performed and single peak was obtained without sepn. of four steric isomers. Sensitivity of androstenedioxime in this mehtod was enhanced 5 times higher than androstenedione. Calibration graphs were linear (r=0.999) from 2.5 to 100 ng/mL for androstenedione extd. from human plasma and inter-assay precision (RSD) was within 6%.
J. Mass Spectrom. Soc. Jpn., 46(1), 63-68, 1998
A reliable identification method for laccaic acids which are the main components in lac dye has been established using ESI LC/MS/MS with a volatile mobile phase contg. acetylacetone. Addn. of acetylacetone to the mobile phase enables us to sep. laccaic acids without redn. of peak resoln. The volatile mobile phase, acetonitrile-0.05% aq. TFA soln. (1-4) contg. 0.005 M acetylacetone, is applicable to directly interfaced ESI LC/MS/MS without clogging problems. In all ES tandem mass spectra of laccaic acids, [M-H-CO2]- and [M-H-2CO2]- appeared in considerable abundance, together with the weak ions of [M-H]-, [M-H-H2O]-, [M-H-CO2-H2O]-, and [M-H-2CO2-H2O]- when [M-H]- was selected as the precursor ion. Laccaic acids can be easily identified by these product ions. The method has been successfully applied to identification of laccaic acids in a jelly and in "Shi-kou," which is a stick lac imported 1,200 yr ago.
J. Mass Spectrom. Soc. Jpn., 46(1), 69-73, 1998
Non-covalent complexes between the glycopeptide antibiotic vancomycin and the cell-wall precursor analog N-acetyl-D-Ala-D-Ala, as well as its deuterated enantiomer N-acetyl-L-Ala-L-Ala-d3 were probed using electrospray ionization mass spectrometry. In soln., the D,D-peptide interacts strongly with the antibiotic, whereas the L,L-peptide shows no observable complexation. Using a competition strategy, mass spectra were obtained from solns. contg. vancomycin, N-acetyl-D-Ala-D-Ala, and N-acetyl-L-Ala-L-Ala-d3. With acetonitrile-water as the solvent, the ratio of free vancomycin to vancomycin complex was much higher than expected from soln. interactions and the specific D,D-peptide complex and the non-specific L,L-peptide complex were detected in a 1.7:1 ratio. In contrast, sprayed from an ammonium acetate buffer soln. both the amt. and specificity of obsd. complex reflected soln. interactions (only D,D-peptide complex was found). Thus, our results demonstrate that in electrospray mass spectrometry the conditions must carefully be chosen and control expts. must be carried out in order to exclude non-specific aggregation as a possible cause of complex formation.
J. Mass Spectrom. Soc. Jpn., 46(1), 75-82, 1998
Hydrogen/deuterium exchange (H/D) was performed on ubiquitin, cytochrome c and myoglobin, and deuterium incorporation was investigated by electrospray ionization mass spectrometry (ESIMS) under several conditions, with different pH and different content of org. solvent. CD (CD) was employed to observe conformational changes in proteins under the conditions for H/D exchange and ESIMS measurements. Comparing the results of H/D exchange expts., ubiquitin was found to be the most stable among the three proteins against acid and org. solvent. Cytochrome c seemed to have interconvertible conformers under acidic condition with the addn. of acetonitrile, though it was not indicated in CD spectra. Myoglobin appeared to be folded very loosely in mildly acidic condition and it was difficult to observe its H/D exchange rate by ESIMS, though its CD spectra showed little change under the conditions used for ESIMS analyses. It was possible to investigate slight conformational changes in proteins by utilizing H/D exchange and ESIMS analyses complementary to CD spectra.
J. Mass Spectrom. Soc. Jpn., 46(1), 83-89, 1998
A new methodol. using 2H-labeled leucine (Leu(D10)) was developed for peptide mapping of a large protein monoclonal IgG2b (IgG2b). This method enabled easy assignment of mol. masses of digested peptides to an amino acid sequence of the original protein. It also enabled leucine and isoleucine residues to be distinguished from each other. Mouse 2H-labeled IgG2b (IgG2b(d)) was produced in the cells grown in a serum-free medium that contained Leu(D10) instead of Leu. The mol. mass of a peptide contg. one leucine residue produced by the enzymic digestion of RCM (reduced and carboxymethylated)-IgG2b(d) was 9 u larger than that of an unlabeled peptide. It was revealed that the no. of leucine residues in the digested peptide was estd. easily by the mass difference between labeled and unlabeled peptides. Peptide mappings of RCM-IgG2b, RCM-IgG2b(d), and their equiv. mixt., RCM-IgG2b(mix), were obtained by digestion with lysyl-endopeptidase and consecutive MALDI-TOFMS analyses. It was easy to attribute the mass values to the peptides by the knowledge of the no. of leucine residues, mol. masses of peptides and specificity of an enzyme. Leucine and isoleucine residues were easily distinguished in a protein when the protein contained 2H-labeled leucine residue.
J. Mass Spectrom. Soc. Jpn., 46(1), 91-96, 1998
Post-source decay (PSD) mass spectra of several peptides were obtained on a time-of-flight mass spectrometer with a curved-field reflectron to det. their amino acid sequences using very small quantities of sample. Peptides included angiotensin II, modified angiotensin II, and the tryptic fragments from somatostatin. Samples were prepd. on the mass spectrometer probe using a piezo-elec. micropipetting app. that enabled deposition of nanoliter vols. The curved-field reflectron then enables recording of PSD mass spectra of mass-selected precursors without the need for stepping the reflectron voltage to bring different mass regions in focus. Using this instrument, sequence-specific ions could be obsd. with as little as a few atomoles of peptide.
J. Mass Spectrom. Soc. Jpn., 46(1), 97-102, 1998
A study of the induction of metallothionein isoforms in human T24 bladder tumor cells revealed the presence of coeluting unknown proteins that were also heat stable and had mol. masses in the same range. In order to det. any relationship to metallothionein isoforms, these unknown proteins were identified, using database searching based on mass spectrometric detn. of mol. wts., tryptic peptide mass maps and partial sequences. This study demonstrates that heat stable thymosin b-4 and b-10 are expressed in human bladder tumor cells.
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