Biomedical subjects

Julie A Leary

Publications and source records attributed to Julie A Leary.

At least 37 records · Page 2Linked to original sources

PubMed2005

Structural characterization of Lipoarabinomannans from Mycobacterium tuberculosis and Mycobacterium smegmatis by ESI mass spectrometry.

Structural aspects of lipoarabinomannans (LAM) from Mycobacterium tuberculosis and Mycobacterium smegmatis were investigated by using mild acid hydrolysis in combination with Fourier-transform ion cyclotron resonance (FT-ICR), and quadrupole ion trap mass spectrometry. Exact mass measurements with less than 2.5 ppm mass error confirmed the presence of a series of arabinose oligomers (Ara(n); n = 2-7) as the major components observed following mild acid hydrolysis of both M. tuberculosis and M. smegmatis LAM. However, the mass spectrum of the resulting LAM extract also revealed a highly-abundant distribution of ions that exact mass measurements identified as mannose-linked arabinose species, Ara(n)Man(m) + Na+ (n = 1-6; m = 1-3). The observed mannose caps were linked to arabinose species as mono-, di-, and trimannose units, and the ratio of the mono-, di-, and trimannose caps was determined to be 1.00:9.00:1.15, respectively, different from previous reports. Analysis of the linkage of lithiated arabinose trimer standards was accomplished with MS3 experiments and the information generated was used to identify linkages of arabinose trimers generated by mild acid hydrolysis of M. tuberculosis and M. smegmatis LAM. The MS3 spectra confirmed the linkage of arabinose trimers from M. smegmatis and M. tuberculosis LAM as predominantly alpha(1 --> 5), alpha(1 --> 5).

Antigens, Bacterial↗

PubMed2004

Identification, function and structure of the mycobacterial sulfotransferase that initiates sulfolipid-1 biosynthesis.

Sulfolipid-1 (SL-1) is an abundant sulfated glycolipid and potential virulence factor found in Mycobacterium tuberculosis. SL-1 consists of a trehalose-2-sulfate (T2S) disaccharide elaborated with four lipids. We identified and characterized a conserved mycobacterial sulfotransferase, Stf0, which generates the T2S moiety of SL-1. Biochemical studies demonstrated that the enzyme requires unmodified trehalose as substrate and is sensitive to small structural perturbations of the disaccharide. Disruption of stf0 in Mycobacterium smegmatis and M. tuberculosis resulted in the loss of T2S and SL-1 formation, respectively. The structure of Stf0 at a resolution of 2.6 A reveals the molecular basis of trehalose recognition and a unique dimer configuration that encloses the substrate into a bipartite active site. These data provide strong evidence that Stf0 carries out the first committed step in the biosynthesis of SL-1 and establish a system for probing the role of SL-1 in M. tuberculosis infection.

Binding Sites↗

PubMed2004

Mass spectrometric characterization of a three-enzyme tandem reaction for assembly and modification of the novobiocin skeleton.

The tripartite scaffold of the natural product antibiotic novobiocin is assembled by the tandem action of novobiocin ligase (NovL) and novobiocic acid noviosyl transferase (NovM). The noviosyl ring of the tripartite scaffold is further decorated by a methyltransferase (NovP) and a carbamoyltransferase (NovN), resulting in the formation of novobiocin. To facilitate kinetic evaluation of alternate substrate usage by NovL and NovM toward the creation of variant antibiotic scaffolds, an electrospray ionization/MS assay for obtaining kinetic measurements is presented for NovL and NovM separately, in each case with natural substrate and the 3-methyl-4-hydroxybenzoic acid analog. Additionally, assays of tandem two-enzyme (NovL/NovM) and three-enzyme (NovL/NovM/NovP) incubations were developed. The development of these assays allows for the direct detection of each intermediate followed by its utilization as substrate for the next enzyme, as well as the subsequent formation of final product as a function of time. This MS tandem assay is useful for optimization of conditions for chemoenzymatic generation of novobiocin and is also suitable for evaluation of competitive usage of variant substrate analogs by multiple enzymes. The studies presented here serve as a platform for the subsequent expansion of the repertoire of coumarin-based antibiotics.

Kinetics↗

PubMed2004

Kinetic measurements of phosphoglucomutase by direct analysis of glucose-1-phosphate and glucose-6-phosphate using ion/molecule reactions and Fourier transform ion cyclotron resonance mass spectrometry.

A method for the direct determination of kinetic constants for phosphoglucomutase and its phosphorylated products is described. Fourier transform ion cyclotron resonance gas-phase ion/molecule reactions between trimethyl borate and glucose phosphate, phosphorylated at either the 1 or the 6 position, generate mass spectra distinguishable with regard to product ion distribution. A multicomponent quantification method is utilized to determine the composition of a binary mixture of the two positional isomers. Using this method, the conversion between glucose-1-phosphate and glucose-6-phosphate can be directly monitored without the use of coupling enzymes. The values of K(m) for glucose-1-phosphate and glucose-6-phosphate were determined using the substrate-velocity plot and the Haldane relationship, respectively. Values of V(max) for both the forward and the reverse directions were measured, and the equilibrium constant for the reversible reaction was determined using this methodology. Kinetic parameters measured correlate well with those obtained using traditional methods. The assay was demonstrated to be accurate and particularly convenient to determine kinetic constants for enzymatic systems that involve the interconversion of phosphorylated positional isomers.

Chemistry Techniques, Analytical↗

PubMed2004

Screening and identification of acidic carbohydrates in bovine colostrum by using ion/molecule reactions and Fourier transform ion cyclotron resonance mass spectrometry: specificity toward phosphorylated complexes.

A screening method was developed for the identification of acidic saccharides from biological mixtures utilizing gas-phase derivatization and mass spectrometry. Phosphorylated compounds were differentiated from other acidic species by exploiting the selective reactivity of chlorotrimethylsilane with the phosphate ions (phosphorylated compounds shift by 72 Da, allowing rapid compound detection). A 13-component mock mixture was used to demonstrate the viability of the method, and a detection limit of 600 nM (30 fmol) was determined. This method was applied to the identification of acidic compounds from bovine colostrum. To further verify the selectivity of the ion/molecule reaction, exact mass measurements were used to determine the elemental composition of 14 compounds. Eight novel acidic carbohydrate species were observed in bovine colostrum, six of which have never been reported previously in milks. Tandem mass spectrometric experiments allowed compound characterization for two of these components.

Animals↗

PubMed2004

Determination of enzyme/substrate specificity constants using a multiple substrate ESI-MS assay.

The traditional method used to investigate the reaction specificity of an enzyme with different substrates is to perform individual kinetic measurements. In this case, a series of varied concentrations are required to study each substrate and a non-regression analysis program is used several times to obtain all the specificity constants for comparison. To avoid the large amount of experimental materials, long analysis time, and redundant data processing procedures involved in the traditional method, we have developed a novel strategy for rapid determination of enzyme substrate specificity using one reaction system containing multiple competing substrates. In this multiplex assay method, the electrospray ionization mass spectrometry (ESI-MS) technique was used for simultaneous quantification of multiple products and a steady-state kinetics model was established for efficient specificity constant calculation. The system investigated was the bacterial sulfotransferase NodH (NodST), which is a host specific nod gene product that catalyzes the sulfate group transfer from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to natural Nod factors or synthetic chitooligosaccharides. Herein, the reaction specificity of NodST for four chitooligosaccharide acceptor substrates of different chain length (chitobiose, chitotriose, chitotetraose, and chitopentaose) was determined by both individual kinetic measurements and the new multiplex ESI-MS assay. The results obtained from the two methods were compared and found to be consistent. The multiplex ESI-MS assay is an accurate and valid method for substrate specificity evaluation, in which multiple substrates can be evaluated in one assay.

Carbohydrate Sequence↗

PubMed2004

Delineating mechanisms of dissociation for isomeric heparin disaccharides using isotope labeling and ion trap tandem mass spectrometry.

Heparin and heparan sulfate (HS) glycosaminoglycans have been identified as important players in many physiological as well as pathophysiological settings. A better understanding of the biosynthesis and structure of these molecules is critical for further elucidation of their biological function. We have demonstrated the successful use of negative electrospray ionization tandem mass spectrometry in the differentiation of all twelve standard heparin-building blocks, including the potentially important N-unsubstituted disaccharides. Collision induced dissociation of each of the isomeric disaccharides provided unique product ion spectra, useful for identification and quantification of the relative amounts of each isomer present. In the research presented herein, isotopic labeling studies using (18)O and (2)H were used to determine the origins of each of the neutral losses observed in the product ion spectra, and mechanisms of dissociation consistent with the observed data were postulated. The general mechanisms postulated were for the generation of B, Y, and Z ions formed from glycosidic cleavages, as well as A and X ions formed from cross-ring cleavages. The eight isomeric heparin disaccharides all underwent cross-ring cleavage to form (0,2)X(1) and (0,2)A(2) ions, and further experiments suggest that the mechanisms of formation of these ions are through a charge-remote process. The tandem mass spectrometry data presented herein also provide a foundation for further developments towards a practical analysis tool for the structural elucidation of larger, biologically important heparin/HS oligosaccharides by using mass spectrometry.

Deuterium↗

PubMed2004

Characterization of noncovalent protein-ligand complexes and associated enzyme intermediates of GlcNAc-6-O-sulfotransferase by electrospray ionization FT-ICR mass spectrometry.

In this study, a GlcNAc-6-O-Sulfotransferase, NodST and its complexation with the substrate 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and the inhibitor 3'-phosphoadenosine 5'-phosphate (PAP) were studied using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. In addition, using isotopically labeled substrate, we have successfully confirmed a sulfated enzyme intermediate, which was predicted by the MS kinetic measurement. It is also shown that information regarding solution binding affinities can be obtained using electrospray ionization (ESI)-FTICR mass spectrometry. The relative binding constants, Kd(PAPS)/Kd(PAP), derived from the solution and gas phase were very similar, which suggests that the binding domain of this particular enzyme system, given known structures of other sulfotransferases, may be preserved during the transmission of the complex from solution to the gas phase.

Disaccharides↗

PubMed2004

Observation of a hybrid random ping-pong mechanism of catalysis for NodST: a mass spectrometry approach.

An efficient enzyme kinetics assay using electrospray ionization mass spectrometry (ESI-MS) was initially applied to the catalytic mechanism investigation of a carbohydrate sulfotransferase, NodST. Herein, the recombinant NodST was overexpressed with a His(6)-tag and purified via Ni-NTA metal-affinity chromatography. In this bisubstrate enzymatic system, an internal standard similar in structure and ionization efficiency to the product was chosen in the ESI-MS assay, and a single point normalization factor was determined and used to quantify the product concentration. The catalytic mechanism of NodST was rapidly determined by fitting the MS kinetic data into a nonlinear regression analysis program. The initial rate kinetics analysis and product inhibition study described support a hybrid double-displacement, two-site ping-pong mechanism of NodST with formation of a sulfated NodST intermediate. This covalent intermediate was further isolated and detected via trypsin digestion and Fourier transform ion cyclotron resonance mass spectrometry. To our knowledge, these are the first mechanistic data reported for the bacterial sulfotransferase, NodST, which demonstrated the power of mass spectrometry in elucidating the reaction pathway and catalytic mechanism of promising enzymatic systems.

Amino Acid Sequence↗

PubMed2003

Compositional analysis and quantification of heparin and heparan sulfate by electrospray ionization ion trap mass spectrometry.

A new method using a combination of electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MSn) was developed for the identification and quantitative analysis of eight heparan sulfate (HS)- and heparin-derived delta-disaccharides obtained by enzymatic depolymerization. The compositional analysis of nonisomeric disaccharide constituents of heparin/HS was achieved from full-scan MS1 spectra using an internal standard and a calculated response factor for each disaccharide. Diagnostic product ions from MSn spectra of isomeric disaccharides were used for the quantitative analysis of the relative amounts of each of the isomers in mixtures. The protocol was validated using several quality control samples and showed satisfactory accuracy and precision. The analysis is rapid, accurate, and uses no purification or separation steps prior to analysis by MS, thus reducing sample consumption and analysis time of traditional methods. Using this quantitative analysis procedure, percentages of disaccharide compositions for heparins from porcine and bovine intestinal mucosa and heparan sulfate from bovine kidney were determined.

Animals↗

PubMed2003

MmpL8 is required for sulfolipid-1 biosynthesis and Mycobacterium tuberculosis virulence.

Mycobacterium tuberculosis, the causative agent of human tuberculosis, is unique among bacterial pathogens in that it displays a wide array of complex lipids and lipoglycans on its cell surface. One of the more remarkable lipids is a sulfated glycolipid, termed sulfolipid-1 (SL-1), which is thought to mediate specific host-pathogen interactions during infection. However, a direct role for SL-1 in M. tuberculosis virulence has not been established. Here we show that MmpL8, a member of a large family of predicted lipid transporters in M. tuberculosis, is required for SL-1 production. The accumulation of an SL-1 precursor, termed SL(1278), in mmpL8 mutant cells indicates that MmpL8 is necessary for an intermediate step in the SL-1 biosynthesis pathway. We use a novel fractionation procedure to demonstrate that SL-1 is present on the cell surface, whereas SL(1278) is found exclusively in more internal layers. Importantly, we show that mmpL8 mutants are attenuated for growth in a mouse model of tuberculosis. However, SL-1 per se is not required for establishing infection as pks2 mutants, which are defective in an earlier step in SL-1 biosynthesis, have no obvious growth defect. Thus, we hypothesize that either MmpL8 transports molecules in addition to SL-1 that mediate host-pathogen interactions or the accumulation of SL(1278) in mmpL8 mutant cells interferes with other pathways required for growth during the early stages of infection.

Bacterial Proteins↗

PubMed2003

Multiplex inhibitor screening and kinetic constant determinations for yeast hexokinase using mass spectrometry based assays.

An electrospray ionization mass spectrometry based assay was developed for kinetic measurements and inhibitor screening of yeast hexokinase. There is considerable discrepancy in the literature as to the accuracy of kinetic data obtained for hexokinase. In the assay described herein, the product, glucose 6-phosphate was directly monitored by ion trap mass spectrometry and quantified using an internal standard, 2 deoxy-glucose 6-phosphate. The kinetic parameters, K(M) and V(max) for the two substrates were determined without using a coupling enzyme as is normally employed in the traditional spectrophotometric assay for systems lacking a chromophore. In addition, hexokinase was successfully immobilized onto an amino-link gel, and a mock library was screened against the immobilized enzyme for the identification of possible inhibitors. After comparing the mass spectra of the library before and after incubation, trehalose 6-phosphate, ADP, and oxidized glutathione were differentiated from other weak or non-inhibitors. Inhibition behavior of ADP with respect to ATP was further evaluated with the ESI-MS assay and the value of K(i) was determined. This ESI-MS assay was demonstrated to be both accurate and precise for determining kinetic constants and for identifying enzyme inhibitors.

Drug Evaluation, Preclinical↗

PubMed2003

Probing isomeric differences of phosphorylated carbohydrates through the use of ion/molecule reactions and FT-ICR MS.

Through the use of ion/molecule reactions and tandem mass spectrometry, phosphate position is assigned in both phosphorylated monosaccharides and oligosaccharides. In previous work phosphate moieties of monosaccharides were stabilized under collisional activation, by first derivatizing the deprotonated monosaccharide with trimethyl borate through an ion/molecule reaction, and the phosphate position determined through marker ions generated in tandem mass spectra. In this work, the methodology is extended to larger phosphorylated oligomers employing chlorotrimethylsilane (TMSCl) as the ion/molecule reagent. Phosphorylated monosaccharides were first investigated to determine diagnostic ions for phosphate linkage in monomeric standards. It was observed that the diagnostic ions showed both linkage and some monosaccharide stereochemical information. Furthermore, it was observed that TMS addition stabilized the phosphate moiety under collisionally activated conditions. Upon identification of the diagnostic ions, the methodology was applied to lactose-1-phosphate. It was found that TMSCl, stabilized the phosphate moiety upon collisional activation, and furthermore, the phosphate linkage could be determined through tandem mass spectrometric analysis. As a further extrapolation to biologically relevant problems, the methodology was applied to a lipophosphoglycan analog from the protozoan parasite Leishmania. This sample contains bridging phosphates which were converted to terminal phosphates through collision induced dissociation. The sample was then analyzed in the same manner as lactose-1-phosphate, yielding phosphate linkage information and stereochemical information. This study showed that, using the developed methodology, phosphate linkage can be determined from both monosaccharides and larger oligosaccharides; furthermore it is applicable to samples in which the phosphates are either terminating or bridging.

Animals↗

PubMed2003

Investigation of ion/molecule reactions as a quantification method for phosphorylated positional isomers. an FT-ICR approach.

A rapid and accurate method of quantifying positional isomeric mixtures of phosphorylated hexose and N-acetylhexosamine monosacchrides by using gas-phase ion/molecule reactions coupled with FT-ICR mass spectrometry is described. Trimethyl borate, the reagent gas, reacts readily with the singly charged negative ions of phosphorylated monosaccharides to form two stable product ions corresponding to the loss of one or two neutral molecules of methanol from the original adduct. Product distribution in the ion/molecule reaction spectra differs significantly for isomers phosphorylated in either the 1- or the 6-position. As a result, the percents of total ion current of these product ions for a mixture of the two isomers vary with its composition. In order to determine the percentage of each isomer in an unknown mixture, a multicomponent quantification method is utilized in which the percents of total ion current of the two product ions for each pure monosaccharide phosphate and the mixture are used in a two-equation, two-unknown system. The applicability of this method is demonstrated by successfully quantifying mock mixtures of four different isomeric pairs: Glucose-1-phosphate and glucose-6-phosphate; mannose-1-phosphate and mannose-6-phosphate; galactose-1-phosphate and galactose-6-phosphate; N-acetylglucosamine-1-phosphate and N-acetylglucosamine-6-phosphate. The effects of mixture concentrations and ion/molecule reaction conditions on the quantification are also discussed. Our results demonstrate that this assay is a fast, sensitive, and robust method to quantify isomeric mixtures of phosphorylated monosaccharides.

Fourier Analysis↗

PubMed2002

Discovery of sulfated metabolites in mycobacteria with a genetic and mass spectrometric approach.

The study of the metabolome presents numerous challenges, first among them being the cataloging of its constituents. A step in this direction will be the development of tools to identify metabolites that share common structural features. The importance of sulfated molecules in cell-cell communication motivated us to develop a rapid two-step method for identifying these metabolites in microorganisms, particularly in pathogenic mycobacteria. Sulfurcontaining molecules were initially identified by mass spectral analysis of cell extracts from bacteria labeled metabolically with a stable sulfur isotope (34SO 4 2-). To differentiate sulfated from reduced-sulfur-containing molecules, we employed a mutant lacking the reductive branch of the sulfate assimilation pathway. In these sulfur auxotrophs, heavy sulfate is channeled exclusively into sulfated metabolites. The method was applied to the discovery of several new sulfated molecules in Mycobacterium tuberculosis and Mycobacterium smegmatis. Because a sulfur auxotrophic strain is the only requirement of the approach, many microorganisms can be studied in this manner. Such genetic engineering in combination with stable isotopic labeling can be applied to various metabolic pathways and their products.

Cysteine↗

PubMed2002

Kinetic analysis of NodST sulfotransferase using an electrospray ionization mass spectrometry assay.

A novel and efficient enzyme kinetics assay using electrospray ionization mass spectrometry was developed and applied to the bacterial carbohydrate sulfotransferase (NodST). NodST catalyzes the sulfuryl group transfer from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to chitobiose, generating 3'-phosphoadenosine 5'-phosphate (PAP) and chitobiose-6-OSO(3)(-) as products. Traditional spectrophotometric assays are not applicable to the NodST system since no shift in absorption accompanies sulfuryl group transfer. Alternative assays have employed thin-layer chromatography, but this procedure is time-consuming and requires radioactive materials. The ESI-MS assay presented herein requires no chromophoric substrate or product, and the analysis time is very short. The ESI-MS assay is used to determine NodST kinetic parameters, including K(M), V(max), and K(i) (for PAP). In addition, the mode of inhibition for PAP was rapidly determined. The results were in excellent agreement with those obtained from previous assays, verifying the accuracy and reliability of the ESI-MS assay. This unique technique is currently being used to investigate the enzymatic mechanism of NodST and to identify sulfotransferase inhibitors.

Bacterial Proteins↗

PubMed2002

Analysis of phosphate position in hexose monosaccharides using ion-molecule reactions and SORI-CID on an FT-ICR mass spectrometer.

Through the use of ion-molecule reactions and SORI-CID, the phosphate position in hexose phosphate monosaccharides has been determined in the negative ion mode. Trimethyl borate was used as a reagent gas and was found to react readily with the phosphorylated hexose monosaccharides. After reaction of the reagent gas with the hexose phosphate, ion activation of the precursor by SORI-CID yielded different MS/MS spectra. Different diagnostic ions were generated for the two isomers, thus enabling differentiation and linkage position determination of the phosphate moiety.

Fourier Analysis↗

PubMed2002

Solvent effects in tandem mass spectrometry: mechanistic studies indicating how a change in solvent conditions and pH can dramatically alter CID spectra.

Dramatically different CID (collision-induced dissociation) spectra are obtained when the complex [Zn(dien-glucose)](+) is electrosprayed from acidic and basic solutions. To understand this peculiar phenomenon, an in-depth mechanistic study was performed on one of the product ions that is present when the initial complex is diluted in basic solution but absent when the complex is diluted with acidic solution. On the basis of the results of this study, the differences in the CID spectra can be rationalized by the fact that the complex electrosprayed from basic solution was kinetically trapped, with the deprotonation site distal from the metal center. Under acidic conditions, the deprotonation site is at a hydroxyl group coordinated to the metal ion. A variety of experiments support this hypothesis. The studies herein underscore the importance of using identical solvent conditions when comparing sets of CID spectra. The data also highlight a very interesting phenomenon involving deprotonation of a hydroxyl group, which was several atoms away from the Zn(II) metal center.

Glucose↗