The Electrospray Ionization (ESI) is a soft ionization technique extensively used for production of gas phase ions (without fragmentation) of thermally labile large supramolecules. In the present review we have described the development of Electrospray Ionization mass spectrometry (ESI-MS) during the last 25 years in the study of various properties of different types of biological molecules. There have been extensive studies on the mechanism of formation of charged gaseous species by the ESI. Several groups have investigated the origin and implications of the multiple charge states of proteins observed in the ESI-mass spectra of the proteins. The charged analytes produced by ESI can be fragmented by activating them in the gas-phase, and thus tandem mass spectrometry has been developed, which provides very important insights on the structural properties of the molecule. The review will highlight recent developments and emerging directions in this fascinating area of research.

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Electrospray Ionization Mass Spectrometry: A Technique to Access the Information beyond the Molecular Weight of the Analyte

The present article is a survey of ESI and MALDI mass spectrometric analysis of nucleic acid oligomers and polymers. In order to limit the extent of the review, mass spectrometry of mononucleotides is generally not considered, except where such data are important for an understanding of the analysis of larger nucleic acids. The first part of the review is a condensed description of the structure and the acid-base properties of nucleic acids. The remaining part is divided into three main sections, dealing with the practical aspects of the two ionization techniques, fragmentation, and applications, respectively. The first section includes an extensive discussion of experimental parameters and problems, which are important for the analysis of different types of nucleic acid samples, including noncovalent complexes and mixtures. At the end of this section, as well as the following one, a comparison between MALDI and ESI as ionization techniques for nucleic acid is given. In addition to a detailed discussion of ion fragmentation, the fragmentation section includes an overview of the direct mass spectrometric sequencing of nucleic acids performed with either technique. The fragmentation reactions occurring upon MALDI and ESI are compared. The last section describes the life science applications of ESI-MS and MALDI-MS of nucleic acids; an account of experiments demonstrating the potential of a method, and of the bona fide solving of problems by ESI and MALDI is given. © 1997 John Wiley & Sons, Inc.

Mass spectrometry of nucleic acids.

The rifamycin antibacterials, rifampicin (rifampin), rifabutin and rifapentine, are uniquely potent in the treatment of patients with tuberculosis and chronic staphylococcal infections. Absorption is variably affected by food; the maximal concentration of rifampicin is decreased by food, whereas rifapentine absorption is increased in the presence of food. The rifamycins are well-known inducers of enzyme systems involved in the metabolism of many drugs, most notably those metabolised by cytochrome P450 (CYP) 3A. The relative potency of the rifamycins as CYP3A inducers is rifampin > rifapentine > rifabutin; rifabutin is also a CYP3A substrate. The antituberculosis activity of rifampicin is decreased by a modest dose reduction from 600 to 450mg. This somewhat surprising finding may be due to the binding of rifampicin to serum proteins, limiting free, active concentrations of the drug. However, increasing the administration interval (after the first 2 to 8 weeks of therapy) has little effect on the sterilising activity of rifampicin, suggesting that relatively brief exposures to a critical concentration of rifampicin are sufficient to kill intermittently metabolising mycobacterial populations. The high protein binding of rifapentine (97%) may explain the suboptimal efficacy of the currently recommended dose of this drug. The toxicity of rifampicin is related to dose and administration interval, with increasing rates of presumed hypersensitivity with higher doses combined with administration frequency of once weekly or less. Rifabutin toxicity is related to dose and concomitant use of CYP3A inhibitors. The rifamycins illustrate the complexity of predicting the pharmacodynamics of treatment of an intracellular pathogen with the capacity for dormancy.

Comparative Pharmacokinetics and Pharmacodynamics of the Rifamycin Antibacterials

Since its introduction in 1987, capillary electrophoresis-mass spectrometry (CE-MS) has developed to a well accepted multidimensional analytical approach complementary and/or competitive to classical MS-hyphenated separation techniques. The threefold combination of rapid developments of an exceptional separation technique, of selective mass detection possibilities, and of very mild ionization modes first allowed these progresses. This article shows the CE specificities that need to be well controlled/known, compared to classical and more routinely used liquid chromatography in the light of its coupling to MS. The major trends and developments over the last 15 years and most of the reviews and applications found in ISI Web of science and publisher databases are presented in a tabulated way. The reader can thus rapidly find existing CE-MS analysis techniques in his field of research and application (forensics, environment, bioanalytics, pharmaceutics, and metabolites).

Capillary electrophoresis-mass spectrometry: 15 years of developments and applications.

Procedures are described for interpretation of mass spectra from collision-induced dissociation of polycharged oligonucleotides produced by electrospray ionization. The method is intended for rapid sequencing of oligonucleotides of completely unknown structure at approximately the 15-mer level and below, from DNA or RNA. Identification of sequence-relevant ions that are produced from extensive fragmentation in the quadrupole collision cell are based primarily on (1) recognition of 3‘- and 5‘-terminal residues as initial steps in mass ladder propagation, (2) alignment of overlapping nucleotide chains that have been constructed independently from each terminus, and (3) use of experimentally measured molecular mass in rejection of incorrect sequence candidates. Algorithms for sequence derivation are embodied in a computer program that requires <2 s for execution. The interpretation procedures are demonstrated for sequence location of simple forms of modification in the base and sugar. The potential for direc...

Interpretation of oligonucleotide mass spectra for determination of sequence using electrospray ionization and tandem mass spectrometry.

Electrospray coupled with tandem mass spectrometry is shown to have utility in determining the molecular mass and the sequence of short modified oligonucleotides. The product ion spectrum of a triply deprotonated molecule precursor is dominated by ions formed via two fragmentation pathways: first, fragmentation across the 5′PO phosphate bond, and second, loss of a non-terminal base with concomitant cleavage of the ribose 3′CO bond. If either of these two pathways is complete with fragmentation occurring at each equivalent position along the phosphodiester backbone, then the oligomer may be sequenced. Alternatively, if both these pathways are only partially complete, their complementarity may be used for more extensive sequencing. The inductive effect of the modification on the nucleobase is shown to influence the latter fragmentation process. The greater the electron affinity of the base modification, the more facile is the loss of that base when the molecule is collisionally activated. In fact, this latter fragmentation dominates the product ion spectrum when a nucleobase contains a substituent that is highly inductively withdrawing. This domination can be such that few other first-generation fragment ions are present in the product ion spectrum. Sequence information may then require utilization of second-generation product ions.

Mass and sequence verification of modified oligonucleotides using electrospray tandem mass spectrometry

Capillary zone electrophoresis (CZE) coupled with negative ion electrospray mass spectrometry (ES-MS) is used for the detection and identification of adducts formed from the reaction of DNA with (±)-anti-7,8,9,10-tetrahydrobenzo[a]pyrene-7,8-diol 9,10-epoxide (BPDE), an active metabolite of benzo[a]pyrene (BaP). Results presented in this paper demonstrate low nanogram detection limits (<10 ng or <15 pmol) for normal scan spectra and collision-induced dissociation spectra of the main nucleotide adduct formed from this reaction. (BPDE reacts predominantly with the exocyclic amino group of guanine.) Exploitation of selective reaction monitoring (SRM) produces detection limits in the low picogram range (<85 pg or <130 fmol). The application of sample stacking significantly increases the concentration detection limit (to approximately 10-8 M). Nucleotide adducts are negatively charged at most pHs and are therefore ideally suited to the stacking process used in this research. These techniques have been applied ...

Detection and identification of benzo[a]pyrene diol epoxide adducts to DNA utilizing capillary electrophoresis-electrospray mass spectrometry.

Analysis of modified oligonucleotides by capillary electrophoresis in a polyvinylpyrrolidone matrix coupled with electrospray mass spectrometry

This paper describes a novel method to map guanine bases in short oligonucleotides using a simple chemical modification reaction and subsequent analysis by electrospray ionization ion trap mass spectrometry (ITMS). In situ guanine-specific methylation followed by gas-phase fragmentation permits the determination of the positions of all guanine residues. Collision-induced dissociation (CID) of the monomethylated oligonucleotide strand promotes rapid depurination and further collision (MS3) of the apurinic oligonucleotide leads to preferential cleavage of the backbone at the site of depurination. The mass of the resulting complementary product ions verifies the position of each guanine base in the sequence. The utility of this methodology is demonstrated for oligonucleotide sequences up to 10 bases in length. In addition, this technique successfully illustrates the use of selective fragmentation for sequencing oligonucleotides by ITMS. Copyright © 1999 John Wiley & Sons, Ltd.

Oligonucleotide sequencing using guanine-specific methylation and electrospray ionization ion trap mass spectrometry.

The antimycobacterial drug rifabutin is extensively metabolized in humans and laboratory animals. About 40% of the dose is excreted in urine as unchanged drug, and lipophilic (extractable with 1-chlorobutane) and polar metabolites. Polar metabolites accounted for 59.1 +/- 2.5% and 88.8 +/- 4.4% of radioactivity in urine collected over 96 hr after intravenous administration of 25 and 1 mg/kg of [14C]rifabutin to Sprague-Dawley rats, respectively. After 48 hr, all urinary radioactivity consisted of polar metabolites. The most abundant polar metabolite, identified by electrospray ionization-MS, collision-induced dissociation-MS, and comparison of HPLC retention times with the synthetic standard, was N-isobutyl-4-hydroxy-piperidine. Lipophilic metabolites accounted for <20% of urinary radioactivity. Major lipophilic metabolites, 25-O-deacetyl-rifabutin, 27-O-demethyl-rifabutin, 31-hydroxy-rifabutin, 32-hydroxy-rifabutin, and 20-hydroxy-rifabutin were isolated from both human and rat urine by HPLC and identified by electrospray ionization-MS, collision-induced dissociation-MS, and NMR spectrometry. In addition, two metabolites formed by the oxidation of the N-isobutyl-piperidyl group of rifabutin were found in the urine of rats, but not humans.

John P. Barry

Papers

Mass and sequence verification of modified oligonucleotides using electrospray tandem mass spectrometry

Detection and identification of benzo[a]pyrene diol epoxide adducts to DNA utilizing capillary electrophoresis-electrospray mass spectrometry.

Analysis of modified oligonucleotides by capillary electrophoresis in a polyvinylpyrrolidone matrix coupled with electrospray mass spectrometry

Oligonucleotide sequencing using guanine-specific methylation and electrospray ionization ion trap mass spectrometry.

Isolation and identification of major urinary metabolites of rifabutin in rats and humans