Dual polarity accurate mass calibration for electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry using maltooligosaccharides

In view of the fact that memory effects associated with instrument calibration hinder the use of many mass-to-charge (m/z) ratios and tuning standards, identification of robust, comprehensive, inexpensive, and memory-free calibration standards is of particular interest to the mass spectrometry community. Glucose and its isomers are known to have a residue mass of 162.05282 Da; therefore, both linear and branched forms of polyhexose oligosaccharides possess well-defined masses, making them ideal candidates for mass calibration. Using a wide range of maltooligosaccharides (MOSs) derived from commercially available beers, ions with m/z ratios from approximately 500 to 2500 Da or more have been observed using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) and time-of-flight mass spectrometry (TOF-MS). The MOS mixtures were further characterized using infrared multiphoton dissociation (IRMPD) and nano-liquid chromatography/mass spectrometry (nano-LC/MS). In addition to providing well-defined series of positive and negative calibrant ions using either electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI), the MOSs are not encumbered by memory effects and, thus, are well-suited mass calibration and instrument tuning standards for carbohydrate analysis.     

Specialized natural product analysis and chemophenetics of some Turkish endemic Centaurea L. (Asteraceae) taxa by electrospray ionization mass spectrometry fingerprinting and liquid chromatography-tandem mass spectrometry

Specialized natural product analysis of six Turkish endemic and two narrowly distributed Centaurea L. taxa was performed via electrospray ionization mass spectrometry (ESI-MS) fingerprinting and liquid chromatography-tandem mass spectrometry (LC-MS/MS), which is an effective methodology that is widely used for fast screening of complex natural mixtures such as food extracts, but not has not been used as commonly for plant chemophenetics. This method is preferable when it is aimed to compare a large number of plant extracts for chemophenetic purposes and when it is difficult to provide equally good chromatographic separation in all of the extracts. ESI-MS shows the major compounds in fingerprinting extracts. LC-MS/MS provides identification according to fragmentation with the advantage of MS/MS, and validation can be performed in selected reaction monitoring (SRM) mode with simultaneous precursor and product ion scans. Herein, sixteen flavones, four flavonols, four flavanones, two lignans, three sesquiterpene lactones, and four phenolic acids, a total of thirty three substances, were identified tentatively or unambiguously from the extracts. It was concluded that ESI-MS fingerprinting is a suitable method for plant chemophenetics when coupled and validated with LC-MS/MS. Moreover, it was concluded that sesquiterpene lactones, lignans, and flavonoids are suitable for taxonomic purposes in Centaurea owing to species-specific metabolite profiles.     

Use of electrospray ionization mass spectrometry to study binding interactions between a replication terminator protein and DNA

Tus protein binds tightly to specific DNA sequences (Ter) on the Escherichia coli chromosome halting replication. We report here conditions for detecting the 1 : 1 Tus-Ter complex by electrospray ionization mass spectrometry (ESI-MS). ESI mass spectra of a mixture of Tus and nonspecific DNA showed ions predominantly from uncomplexed Tus protein, indicating that the Tus-Ter complex observed in the gas phase was the result of a specific interaction rather than nonspecific associations in the ionization source. The Tus-Ter complex was very stable using a spray solvent of 10 mM ammonium acetate at pH 8.0, and initial attempts to distinguish binding affinities of Tus and mutant Tus proteins for Ter DNA were unsuccessful. Increasing the ammonium acetate concentration in the electrospray solvent (800 mM at pH 8.0) increased the dissociation constants sufficiently such that relative orders of binding affinity for Tus and various mutant Tus proteins for various DNA sequences could be determined. These were in agreement with the dissociation constants determined in solution studies. A dissociation constant of 700 x 10(-9) M for the binding of the mutant Tus protein A173T (where residue 173 is changed from alanine to threonine) to Ter DNA was estimated, compared with a value of 相似文献   

Base composition analysis of human mitochondrial DNA using electrospray ionization mass spectrometry: a novel tool for the identification and differentiation of humans

In traditional approaches, mitochondrial DNA (mtDNA) variation is exploited for forensic identity testing by sequencing the two hypervariable regions of the human mtDNA control region. To reduce time and labor, single nucleotide polymorphism (SNP) assays are being sought to possibly replace sequencing. However, most SNP assays capture only a portion of the total variation within the desired regions, require a priori knowledge of the position of the SNP in the genome, and are generally not quantitative. Furthermore, with mtDNA, the clustering of SNPs complicates the design of SNP extension primers or hybridization probes. This article describes an automated electrospray ionization mass spectrometry method that can detect a number of clustered SNPs within an amplicon without a priori knowledge of specific SNP positions and can do so quantitatively. With this technique, the base composition of a PCR amplicon, less than 140 nucleotides in length, can be calculated. The difference in base composition between two samples indicates the presence of an SNP. Therefore, no post-PCR analytical construct needs to be developed to assess variation within a fragment. Of the 2754 different mtDNA sequences in the public forensic mtDNA database, nearly 90% could be resolved by the assay. The mass spectrometer is well suited to characterize and quantitate heteroplasmic samples or those containing mixtures. This makes possible the interpretation of mtDNA mixtures (as well as mixtures when assaying other SNPs). This assay can be expanded to assess genetic variation in the coding region of the mtDNA genome and can be automated to facilitate analysis of a large number of samples such as those encountered after a mass disaster.     

A study of immunoglobulin G glycosylation in monoclonal and polyclonal species by electrospray and matrix-assisted laser desorption/ionization mass spectrometry

N-linked oligosaccharides were released from human and bovine polyclonal immunoglobulin G (IgG) obtained from commercial sources and also from a monoclonal IgG(1) secreted by murine B-lymphocyte hybridoma cells (CC9C10) grown under different serum-free conditions. These conditions differed according to their steady-state dissolved oxygen concentrations. This work is based on a previous quantitative study where released glycans were characterized by fluorophore-assisted carbohydrate electrophoresis (FACE) and high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (J. P. Kunkel, D. C. H. Jan, J. C. Jamieson, and M. Butler, 1998, J. Biotechnol. 62, 55-71). In the present article, peptide-N-glycosidase F-released glycans from different species of polyclonal IgG and murine monoclonal IgG were characterized qualitatively by high-performance liquid chromatography (HPLC) coupled to electrospray ionization mass spectrometry (ESI-MS). The glycans were also analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The MALDI mass spectrometer used allowed acquisition of MS and tandem MS data, which were useful in structural investigations at a more detailed level than allowed by FACE and HPAEC-PAD. Predominant N-linked structures, as determined by all techniques, were core-fucosyl asialyl biantennary chains with varying galactosylation. Minor amounts of afucosyl, bisected, and monosialyl oligosaccharides were also detected. In contrast to FACE and HPAEC-PAD, MALDI-double quadrupole/time-of-flight MS and HPLC/ESI-MS also detected low-abundance high-mannose and hybrid structures in some of the species under investigation.     

Identification of cardiac glycosides in fractions from Periploca forrestii by high-performance liquid chromatography/diode-array detection/electrospray ionization multi-stage tandem mass spectrometry and liquid chromatography/nuclear magnetic resonance

Cardiac glycosides are a class of naturally occurring compounds that are characterized by some interesting biological activities and are widely distributed in the plant kingdom and can also be found in some animals. There is an interest in the chemical characterization of these molecules due to their toxicity and their use in medicines. In the study reported here, a combination of electrospray ionization tandem mass spectrometry with high-performance liquid chromatography equipped with diode-array detector (HPLC-DAD/ESI-MSⁿ), and hyphenation to both liquid chromatography and nuclear magnetic resonance spectroscopy (HPLC/NMR) were utilized for the on-line analyses of cardiac glycosides from Periploca forrestii. The fragmentation patterns and ¹H NMR spectra of nine isolated cardiac glycosides were investigated; their fragmentation rules and ¹H NMR spectral characteristics were summarized and applied to the structural identification of similar constituents in fractions from P. forrestii. As a result, a total of nine trace cardiac glycosides were tentatively determined by analyses of accurate molecular masses, representative fragment ions and characteristic ¹H NMR signals provided by HPLC/high-resolution mass spectrometry (HRMS), HPLC-DAD/ESI-MSⁿ and HPLC/¹H NMR experiments, respectively. Of these, eight (2–9) are new compounds and one (1) is reported from P. forrestii for the first time. Results of the present study can benefit the rapid identification and targeted isolation of new cardiac glycosides from crude plant extracts.     

Assessment of alterations in X-ray irradiation-induced DNA damage of glioma cells by using proton nuclear magnetic resonance spectroscopy

Glioma is one of the most common types of brain tumors. DNA damage is closely associated with glioma cell apoptosis induced by X-ray irradiation. Alterations of metabolites in glioma can be detected noninvasively by proton nuclear magnetic resonance (1H NMR) spectroscopy. To noninvasively explore the micro mechanism in X-ray irradiation-induced apoptosis, the relationship between metabolites and DNA damage in glioma cells was investigated. Three glioma cell lines (C6, U87 and U251) were randomly designated as control (0 Gy) and treatment groups (1, 5, 10, 15 Gy). After X-ray exposure, each group was separated into four parts: (i) to detect metabolites by 1H NMR spectroscopy; (ii) to make cell colonies; (iii) to detect cell cycle distribution and apoptosis rate by flow cytometry; and (iv) to measure DNA damage by comet assay. The metabolite ratios of lactate/creatine and succinate/creatine decreased (lactate/creatine: C6, 22.17–66.27%; U87, 15.93–44.56%; U251, 26.27–74.48%. succinate/creatine: C6, 14.41–48.35%; U87, 22.03–70.62%; U251, 17.33–60.06%) and choline/creatine increased (C6, 52.22–389.68%; U87, 56.15–82.36%; U251, 31.87–278.62%) in the treatment groups compared with the control group (each P < 0.05), which linearly depended on DNA damage. An increasing dose of X-ray irradiation increased numbers of apoptotic cells (P < 0.01), and the DNA damage parameters were dose-dependent (P < 0.05). The colony-forming rate declined (P < 0.01) and the percentage of cells at G1 stage increased when exposed to 1 Gy X-ray (three cell lines, P < 0.05). Metabolite alterations detected by 1H NMR spectroscopy can be used to determine DNA damage induced by X-ray irradiation. 1H NMR spectroscopy is a noninvasive method to predict DNA damage of glioma cell at the micro level.     

Characterization of the interface structure of enzyme-inhibitor complex by using hydrogen-deuterium exchange and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

We investigated the interaction between a thiol protease inhibitor, cystatin, and its target enzyme, papain, by hydrogen-deuterium (H/D) exchange in conjunction with successive analysis by collision-induced dissociation (CID) in an rf-only hexapole ion guide with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS). The deuterium incorporation into backbone amide hydrogens of cystatin was analyzed at different time points in the presence or absence of papain, examining the mass of each fragment produced by hexapole-CID. In the absence of papain, amide hydrogens in short amino-terminal fragments, such as b10(2+) and b12(2+), were highly deuterated within 1 min. Although fewer fragments were observed for the cystatin-papain complex in the hexapole-CID spectra, significant reductions in initial deuterium content were recognized throughout the sequence of cystatin. This suggests that complex formation restricted the flexibility of the whole cystatin molecule. Detailed analyses revealed that a marked reduction in deuterium content in the region of residues 1-10 persisted for hours, suggesting that the flexible N-terminal region was tightly fixed in the binding pocket with hydrogen bonds. Our results are consistent with those of previous studies on the structure and inhibition mechanism of cystatin. We demonstrated here that enzyme-inhibitor interactions can be characterized by H/D exchange in combination with CID in a hexapole ion guide using ESI-FTICR MS rapidly and using only a small amount of sample.     

26 kDa endochitinase from barley seeds: Real-time monitoring of the enzymatic reaction and substrate binding experiments using electrospray ionization mass spectrometry

A 26 kDa endochitinase from barley seeds was enzymatically characterized exclusively by electrospray ionization mass spectrometry (ESI-MS). At first, oligosaccharide hydrolysis catalyzed by the barley chitinase was monitored in real-time by ESI-MS. The reaction time-course obtained by ESI-MS monitoring was found to be consistent with the data obtained earlier by HPLC, and the quantitative profile was successfully simulated by kinetic modeling of the enzymatic hydrolysis. It is obvious that the real-time monitoring method by ESI-MS allows a faster and cheaper determination of the chitinase activity with unlabeled substrate. Further, the enzymatic activity of the E67Q mutant of the barley chitinase was analyzed and the role of Glu67 was discussed comparing the mass spectra of enzyme protein obtained in native and in denatured conditions. Then it was determined that the observed loss of the enzymatic activity in E67Q is definitely caused by a point mutation of Glu67 but not due to partial unfolding of the mutated enzyme. Finally, association constants of enzyme–oligosaccharide complexes were calculated from Scatchard plots obtained by mass spectra. The binding free energy values obtained for E67Q were found to be comparable to those previously obtained in liquid phase, but less dependent upon the chain length of the oligosaccharides. To our knowledge, this study is the first enzymatic characterization of chitinase exclusively by such an innovative ESI-MS system.     

Mechanism of enzymatic degradation of the azo dye Orange II determined by ex situ 1H nuclear magnetic resonance and electrospray ionization-ion trap mass spectrometry

Removal of azo dye effluents generated by textile photography industries is a main issue in wastewater treatment. Enzymatic treatment of dyes appears to be one of the most efficient processes for their degradation. The elucidation of degradation pathways is of special interest considering health and environmental priorities. Ex situ nuclear magnetic resonance (NMR) spectroscopy and electrospray ionization (ESI)-ion trap mass spectrometry performed directly on incubation medium have been used for the first time to follow kinetics of sulfonated azo dye Orange II enzymatic degradation. Nine transformation products were identified using these complementary analyses performed ex situ without any prior treatment. Three types of cleavage are proposed for the degradation pathway: (i) a symmetrical splitting of the azo linkage that leads to the formation of 4-aminobenzenesulfonate (and 1-amino-2-naphthol, not detected); (ii) an asymmetrical cleavage on the naphthalene side that generates 1,2-naphthoquinone and 4-diazoniumbenzenesulfonate as products, with the latter one being transformed into 4-hydroxybenzensulfonate; and (iii) a third degradation pathway that leads to 2-naphthol and 4-hydroxybenzenesulfonate. Moreover, three other intermediates have been identified. This study, which constitutes the first concomitant use of (1)H NMR spectroscopy and ESI-ion trap mass spectrometry in this field, illustrates the indubitable interest of the ex situ approach.     

Metabolomic analysis of biofluids from rats treated with Aconitum alkaloids using nuclear magnetic resonance and gas chromatography/time-of-flight mass spectrometry

The Aconitum alkaloids aconitine, mesaconitine, and hypaconitine are the main toxic components in a commonly used traditional Chinese herbal medicine Fu Zi. To provide guidelines for the safe use of this medicine, metabolic changes in Wistar rats caused by these compounds were investigated by means of integrated analysis of two metabonomic approaches: ¹H nuclear magnetic resonance (NMR) and gas chromatography/time-of-flight mass spectrometry (GC/TOF–MS). Rats were given a single dose of aconitine, mesaconitine, hypaconitine, or vehicle. The largest metabolic changes were observed 6 h after treatment. Every group receiving a dose had higher urine concentrations of glucose, acetate, dimethylglycine, succinate, and alanine and had lower concentrations of creatinine, citrate, 2-oxoglutarate, N-acetylated metabolites, and trimethylamine-N-oxide (TMAO) than did the control group. These results may reflect the perturbation of renal tubular function within the first 24 h after treatment. The results also revealed a larger perturbation of metabolic profiles in the aconitine group than in the mesaconitine and hypaconitine groups, illustrating how these alkaloids exhibit different toxicities. An analysis of plasma samples collected 7 days postdose showed that there were higher levels of lactate, alanine, and lipids along with lower levels of glucose, β-hydroxybutyrate, and creatine in the plasma of the aconitine and mesaconitine groups than there were in the control and hypaconitine groups. The GC/TOF–MS data from the plasma samples showed that the number of metabolites, with significant changes or with a tendency to change, in the aconitine and mesaconitine groups were dissimilar, suggesting a possible difference in the acute toxicity mechanisms of these alkaloids.     

Quantitative profiling of endocannabinoids and related compounds in rat brain using liquid chromatography-tandem electrospray ionization mass spectrometry

A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described for the simultaneous identification and quantification of eight endocannabinoid (EC) or related "entourage" compounds in rat brain tissue. Analytes were extracted and purified from rat brain tissue using an ethyl acetate/hexane solvent extraction, followed by a solid phase extraction (SPE) protocol. Chromatographic separation was achieved using a gradient elution, with a mobile phase of acetonitrile, formic acid, and ammonium acetate, at pH 3.6. A Thermo Hypersil C8 HyPurity Advance column (100x2.1 mm i.d., 3 microm) was used with a flow rate of 0.3 ml/min). Anandamide (AEA), 2-arachidonyl glycerol (2-AG), 2-arachidonylglyceryl ether (noladin ether), O-arachidonyl ethanolamide (virodhamine), 2-linoleoyl glycerol (2-LG), arachidonyl glycine, oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA) were quantified by positive ion tandem electrospray ionization mass spectrometry. Internal standards were deuterated AEA, deuterated 2-AG, and heptadecanoyl ethanolamide (HEA). Linearity was proven over the range of 25 fmol to 250 pmol, with a limit of detection of 25 fmol on column for all analytes except 2-AG, noladin ether, and 2-LG (250 fmol). This corresponded to a limit of quantification in biological tissue of 10 pmol/g for all analytes except 2-AG (100 pmol/g). Intra- and interday precision in biological tissue was routinely approximately 20% or lower, and accuracy was between 65% and 155%. This method was used to quantitatively profile regional differences in nine discrete rat brain regions for AEA, 2-AG, 2-LG, OEA, PEA, noladin ether, virodhamine, and arachidonyl glycine.     

An assay combining high-performance liquid chromatography and mass spectrometry to measure DNA interstrand cross-linking efficiency in oligonucleotides of varying sequences

The main method of evaluating the DNA interstrand cross-linking ability of cancer chemotherapeutic agents in naked DNA currently involves the electrophoresis of relatively long radiolabeled duplex DNA fragments (typically approximately 2000 bp) on neutral gels after incubation with the agent of interest. Denaturation by heating is carried out prior to loading, and a neutral gel allows reannealing of cross-linked DNA. To avoid the use of radioactivity we have developed a new method based on ion pair reversed phase liquid chromatography (RPLC) and mass spectrometry (MS) that allows characterization and quantitation of drug-DNA interstrand cross-links formed within short oligonucleotide duplexes (i.e., 12 bp). Advantages of this assay include rapid throughput, as compared to electrophoretic methods, and the use of readily available short nonradiolabeled oligonucleotides of any sequence, thereby facilitating investigation of sequence selectivity. A further advantage is that all species separated by the chromatographic process can be positively identified by MS. Using this new method, we have investigated the rate of DNA cross-linking and sequence selectivity of the interstrand cross-linking agent SJG-136, a pyrrolobenzodiazepine (PBD) dimer currently in phase I clinical trials. The assay was found to be sufficiently sensitive and selective to allow separation of the unbound and drug-bound oligonucleotide species by high-performance liquid chromatography (HPLC) and to allow positive identification of these individual species by MS. A further benefit, as compared with electrophoretic methods, is that kinetic information can be obtained and compared for different binding sequences.     

Application of electrospray ionization mass spectrometry to study the hydrophobic interaction between the epsilon and theta subunits of DNA polymerase III

The interactions between the N-terminal domain of the epsilon (epsilon186) and theta subunits of DNA polymerase III of Escherichia coli were investigated using electrospray ionization mass spectrometry. The epsilon186-theta complex was stable in 9 M ammonium actetate (pH 8), suggesting that hydrophobic interactions have a predominant contribution to the stability of the complex. Addition of primary alkanols to epsilon186-theta in 0.1 M ammonium acetate (pH 8), led to dissociation of the complex, as observed in the mass spectrometer. The concentrations of methanol, ethanol, and 1-propanol required to dissociate 50% of the complex were 8.9 M, 4.8 M, and 1.7 M, respectively. Closer scrutiny of the effect of alkanols on epsilon186, theta, and epsilon186-theta showed that epsilon186 formed soluble aggregates prior to precipitation, and that the association of epsilon186 with theta stabilized epsilon186. In-source collision-induced dissociation experiments and other results suggested that the epsilon186-theta complex dissociated in the mass spectrometer, and that the stability (with respect to dissociation) of the complex in vacuo was dependent on the solution from which it was sampled.     

Oligosaccharide structure of a functional unit RvH1-b of Rapana venosa hemocyanin using HPLC/electrospray ionization mass spectrometry

In the present study the structures of two glycopeptides (G1 and G1'), isolated from FU RvH(1)-b and two glycopeptides (G2 and G3), isolated from the structural subunit RvH(1) of Rapana venosa hemocyanin, were determined. To structurally characterize the site-specific carbohydrate heterogeneity and binding site of the N-linked glycopeptide(s), a combination of capillary reversed-phase chromatography and ion trap mass spectrometry was used. The amino acid sequences of glycopeptides G1 and G1' determined by Edman degradation and MS/MS sequencing demonstrated that the oligosaccharides are linked to N-glycosylation sites. Two peptides (a glycosylated (G1) and non-glycosylated one) were identified in this fraction and no linkage sites were observed in the latter one. Based on the sequencing of the glycosylated fractions G1, G1', G2 and G3, the carbohydrate structure Man(alpha1-6)Man(alpha1-3)Man(beta1-4)GlcNAc(beta1-4)[Fuc(alpha1-6)]GlcNAc-R could be identified for glycopeptides G1 and G3, and only the typical core structure Man(alpha1-6)Man(alpha1-3)Man(beta1-4)GlcNAc(beta1-4)GlcNAc-R was found for G1' and G2. The Fuc residue found in glycopeptides G1 and G3 is attached to N-acetyl-glucosamine of the carbohydrate core, as often found in other glycoproteins.     

The simultaneous measurement of nicotinamide adenine dinucleotide and related compounds by liquid chromatography/electrospray ionization tandem mass spectrometry

We have developed a liquid chromatographic-tandem mass spectrometric method that is sensitive and specific and that simultaneously measures cellular NAD(+) and related compounds. Using this method, NAD(+), NAAD, NMN, NAMN, NAM, NA, ADPR, and 5'AMP were first separated over a reverse-phase high-performance liquid chromatography resin in a mobile ammonium formate-methanol linear gradient. Then each compound was ionized at an electrospray source and detected in the positive multiple reaction monitoring mode of a triple-quadrupole tandem mass spectrometer. We found a good linear response for each NAD(+)-related compound. The limits of quantification for NAD(+) and related compounds range from 0.1 to 1 pmol. The extraction efficiency of NAD(+) and related compounds from mouse erythrocytes is between 84 and 114%. The coefficients of variation for the analyses are all less than 6%. Using our method, we measured, in a single analysis, the amounts of NMN, NAMN, NAD(+), and 5'AMP present in mouse erythrocytes. Additionally, this is the first report of a direct determination of the amounts of NMN and NAMN present in any type of cell. These results indicate that our method sensitively, specifically, and simultaneously measures cellular NAD(+) and related compounds.     

Analysis of hyaluronan content in chondroitin sulfate preparations by using selective enzymatic digestion and electrospray ionization mass spectrometry

Two important glycosaminoglycans (GAGs) with structural roles in the body's cartilage are hyaluronan (HA) and chondroitin sulfate (CS). A simple mass spectrometric method for determining the amount of HA that may be present in isolated CS samples is presented in this article. Samples are subjected to selective enzymatic digestion using a bacterial hyaluronidase (HA lyase, EC 4.2.2, from Streptococcus dysgalactiae) specific for HA. Undigested CS GAG is then removed by centrifugal filtration, and digested HA left in the filtrate is quantified by electrospray ionization mass spectrometry using an internal standard and selected ion monitoring. The described method was applied to the analysis of several CS samples prepared for use in nutritional supplements.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry protocol for monitoring the progress of enzymatic (13)C/(15)N-labeled DNA syntheses

We demonstrate that a simple matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry protocol provides a rapid and accurate method for monitoring the stage and completeness of enzymatic DNA syntheses. A crucial step of these syntheses is to quench the reaction at the desired nucleotide length. This is especially important when expensive, e.g., (13)C/(15)N-labeled DNA segments, are synthesized for multinuclear magnetic resonance purposes to reveal detailed structural information. The analyses of three templates for a human telomeric 22-mer, a wild type, and a mutant human c-MYC promoter (18- and 22-mer) DNA and their reactions with the 3'-5' exo(-) Klenow fragment of DNA polymerase I demonstrate the usefulness of our protocol. Small amounts of samples (ca. 1-2 microl each) were taken from the reaction mixtures at different times and analyzed promptly by MALDI-TOF, applying our successive on-plate desalting method that eliminates the insensitivity of the MALDI technique at high salt content. The progress of the reaction was detected by monitoring the relative intensity ratios of ions corresponding to the desired products and the primer-template complexes. The effectiveness of NH(3) cleavage leading to final products was also followed by MALDI-TOF in successful enzymatic reactions.     

Chiral recognition of verapamil by cyclodextrins studied with capillary electrophoresis,NMR spectroscopy,and electrospray ionization mass spectrometry

Capillary electrophoresis (CE) allows the observation of the opposite affinities of the enantiomers of (±)‐verapamil [2‐isopropyl‐2,8‐bis(3,4‐dimethoxyphenyl)‐6‐methyl‐6‐azaoctannitrile, VP] toward β‐cyclodextrin (β‐CD) and heptakis(2,3,6‐tri‐O‐methyl)‐β‐CD (TM‐β‐CD). In addition, in the presence of β‐CD in the background electrolyte, longer migration times and lower separation factors were observed compared to TM‐β‐CD. The binding constants of (+)‐ and (−)‐VP with β‐CD and TM‐β‐CD determined using ¹³C NMR spectroscopy explain the results observed in CE. Electrospray ionization mass spectrometry (ESI‐MS) was used as an alternative technique for the characterization of VP‐CD complexes. Chirality 11:635–644, 1999. © 1999 Wiley‐Liss, Inc.