Reactions of O*- with methyl benzoate: a negative ion chemical ionization and Fourier transform ion cyclotron resonance study.

The reactions of O*- with methyl benzoate have been examined by the measurement of negative ion chemical ionization (NICI) mass spectra using a CI source, with confirmatory studies carried out on a Fourier transform ion cyclotron resonance mass spectrometer. Reaction mechanisms have been elucidated using isotopically labeled esters. Nucleophilic attack at the carbonyl carbon and the aromatic ring were important reaction pathways. Nucleophilic attack at the carbonyl carbon was followed by the production of products (C6HsCO2- and CH3OCO2-) characteristic of radical, beta-fragmentation. Using 18O-labeled methyl benzoate, the SN2 reaction was found to account for a smaller percentage, 21(+/-1)%, of the benzoate product. Aromatic ring attack resulted in formation of [M + O - H]- and [M - 2H]*- ions. Although aryl hydrogens accounted for most H2*+ abstracted by O*-, evidence for abstraction of HarylH*+alkyl and HalkylH*+alkyl was also found. Although present at much lower abundance, dehydrobenzoate, dehydrophenoxy, and C7H6*- ([M - 2H - CO2]*-) radical anions were also observed. An Haryl/Halkyl exchange associated with formation of the benzoate anion was attributed to an Halkyl abstraction that occurred within the methanol/dehydrobenzoate ion-dipole complex. The [M - 2H]*-, dehydrobenzoate, dehydrophenoxy, and [M - 2H - CO2]*- ion signals were quenched by reaction with O2. Conditions required for production of O*- spectra under NICI conditions were also examined.

Investigation of oligonucleotide fragmentation with matrix-assisted laser desorption/ionization Fourier-transform mass spectrometry and sustained off-resonance irradiation.

Matrix-assisted laser desorption/ionization (MALDI) can be combined with Fourier-transform ion cyclotron resonance mass spectrometry (FTMS) for the detailed structural examination of biomolecules such as peptides and oligonucleotides. We have been able to detect molecular ions for bovine heart cytochrome c (MW = 12,327) by MALDI-FTMS (355 nm laser desorption, 2,5-dihydroxybenzoic acid matrix). Although the mass resolution of these molecular ions is poor, the experiments verify that the MALDI-FTMS mass range for our 3-tesla instrument is in excess of m/z 12,000. Accurate mass measurements and selective dissociation experiments were used to examine the fragmentation pathways of small oligonucleotides in detail. Sustained off-resonance irradiation (SORI) was found to be superior to conventional on-resonance collisionally activated dissociation (CAD) for the efficient dissociation and detection of fragment ions for oligonucleotides. These experiments indicated that oligonucleotide fragmentation is a complex process and results not only from simple elimination of nucleic bases and cleavages of phosphate ester bonds, but also by rearrangement processes in which a terminal phosphate moiety can be transferred to an internal phosphate group.

Analysis of modified oligonucleotides by matrix-assisted laser desorption/ionization Fourier transform mass spectrometry.

Matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance mass spectrometry (FTMS) has been applied to the structural characterization of modified oligodeoxyribonucleotide 4-, 6-, and 11-mers. Each oligonucleotide contained one modified base, either an O6-methyl-substituted guanine, an N6-(10R)-trans-opened benzo[a]pyrenediol epoxide adduct of adenine, or an N2-(R)-styrene oxide adduct of guanine. 3-Hydroxypicolinic acid was used as the MALDI matrix for molecular weight and purity determinations, while either 2,5-dihydroxybenzoic acid (DHBA) or an anthranilic/nicotinic acid (AA/NA) mixture was used to induce fragmentation for the production of structurally significant fragment ions. For the 4- and 6-mers, the oligonucleotide sequence could be obtained from the direct AA/NA or DHBA spectra. Sequence information was also obtained by inserting a time delay between the laser desorption event and ion detection to permit metastable decomposition. For the 11-mers, high-mass sequence ions were not detected. Although similar sequence ions were observed in both the positive and the negative ion mass spectra, more fragmentation was generally observed in the positive ion mode. In the positive ion mode, modified base fragment ions were observed when DHBA was used, and these fragments were examined using accurate mass measurements, collisionally induced dissociations, and ion-molecule reactions to characterize the modified base. MALDI-FTMS signals from one sample application can be used for the measurement of hundreds of spectra. The direct MALDI-FT mass spectra show matrix-dependent, structurally informative fragments, and CID experiments can be implemented using low-picomole sample quantities.

Structural characterization of polycyclic aromatic hydrocarbon dihydrodiol epoxide DNA adducts using matrix-assisted laser desorption/ionization Fourier transform mass spectrometry.

Matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance mass spectrometry (FTMS) has been applied for the structural characterization of four polycyclic aromatic hydrocarbon dihydrodiol epoxide (PAHDE) adducts, including the 5,6-dimethylchrysene DE adduct of 2'-deoxyadenosine, the 5-methyl- and 5,6-dimethylchrysene DE adducts of 2'-deoxyguanosine, and the benzo[a]pyrene-DE adduct of 2'-deoxyguanosyl 3'-phosphate. Measurement of positive and negative ion mass spectra, accurate mass determinations, and CID experiments were carried out using 10-40 ng (20-70 pmol) of sample. An evaluation of five MALDI matrices showed that matrix selection can be used to control the degree of analyte fragmentation. Three MALDI matrices commonly used for the analysis of proteins (sinapinic acid, ferulic acid, 2,5-dihydroxybenzoic acid) gave positive ion adduct mass spectra showing protonated or sodiated molecular ions accompanied by abundant, structurally informative fragment ions. Fragmentation was significantly reduced when working with two matrices used for oligonucleotide analysis (an anthranilic-nicotinic acid mixture and 3-hydroxypicolinic acid). Using the CID capabilities of FTMS, isolation and activation of the MALDI-produced ions was used to provide additional structural information. While characteristic negative ions were not detected for the adenosyladduct, the guanosyl and guanosyl 3'-phosphate adducts gave [M-H]- ions when the anthranilic-nicotinic acid matrix mixture was used. The guanosyl adducts also showed [M-H-2H2O]- fragments. Compared with FAB or FAB-MS/MS for the analysis of underivatized PAH-DE adducts, MALDI-FTMS signals are long-lived, the direct MALDI-FT mass spectra show more structurally informative fragments, and accurate mass and CID experiments require lower sample quantities.

Gas-Phase reactions of O 2 (-.) with alkyl and aryl esters of benzenedicarboxylic acids.

The reactions of O 2 (-.) with alkyl and aryl esters of benzenedicarboxylic acids have been studied under negative-ion chemical ionization (NICI) conditions via a conventional chemical ionization source. Reaction mechanisms have been elucidated by using ion isolation techniques on a Fourier transform ion cyclotron resonance mass spectrometer. In addition, (18)O 2 (-.) has been used as the reagent and the products of competitive reactions that involve the mixed esters of benzenedicarboxylic acids have been studied. O 2 (-.) reactions with the alkyl esters of 1,2- and l,3-benzenedicarboxylic acids are attributed to SN2 displacement at the O-alkyl carbon. The spectra of mixed alkyl esters show that O 2 (-.) attack is reduced at sterically hindered alkyl groups. In contrast with the spectra of 1,2- and l,3-benzenedicarboxylic acids, the spectra of 1,4-benzenedicarboxylic acids are dominated by M(-.) production. Reactions of O 2 (-.) with phenyl benzoates and the aryl esters of benzenedicarboxylic acids proceed via addition-elimination pathways. Experiments with mixed alkyl-aryl benzenedicarboxylic acid esters show that the addition-elimination reaction pathway is preferred over O-alkyl SN2 displacement. The O2/ Ar-NICI mass spectra show features that can be used to distinguish 1,2-, 1,3-, and 1/4-benzenedicarboxylic acid esters. Molecular and fragment ions provide structural information complementary to that generated under electron ionization and chemical ionization conditions.

Matrix-assisted laser desorption/ionization Fourier-transform mass spectrometry of oligodeoxyribonucleotides.

Conditions for the matrix-assisted laser desorption/ionization (MALDI) of oligodeoxyribonucleotides at 355 nm, developed using a 3-Tesla Fourier-transform ion cyclotron resonance mass spectrometer (FTMS), are reported. Efficient ion trapping and matrix selection are critical to the desorption and detection of oligonucleotides by FTMS. The achievable upper mass limit for the MALDI-FTMS of biomolecules on our 3-Tesla system has been extended from approximately 2 kDa to 6 kDa through the use of pulsed-trapping-plate ion deceleration techniques. By implementing the deceleration techniques, molecular ions for bovine insulin (MW = 5733.5), an oligodeoxythymidylic acid, pd[T]10 (MW = 3060.0), and a mixed-base 12-mer (MW = 3611.5) have been measured. For the analysis of oligonucleotides by FTMS, selection of an appropriate MALDI matrix is essential for the generation of [M-H]- ions. 3-Hydroxypicolinic acid provides a significant improvement over 2,5-dihydroxybenzoic acid for production of deprotonated molecules particularly for mixed-base oligomers. MALDI studies using FTMS have been duplicated using a newly constructed time-of-flight mass spectrometer (TOFMS) and oligonucleotide fragmentation on the TOFMS is reduced relative to that observed by FTMS. This may be a consequence of the longer times (milliseconds) required for FTMS detection.