Escherichia coli dihydrodipicolinate synthase: characterization of the imine intermediate and the product of bromopyruvate treatment by electrospray mass spectrometry.
Ontology highlight
ABSTRACT:
SUBMITTER: Borthwick EB
PROVIDER: S-EPMC1136393 | biostudies-other | 1995 Jan
REPOSITORIES: biostudies-other
Similar Datasets
Project description:The Escherichia coli panD gene, encoding l-aspartate-alpha-decarboxylase, was cloned by PCR, and shown to complement a panD mutant defective in beta-alanine biosynthesis. Aspartate decarboxylase is a pyruvoyl-dependent enzyme, and is synthesized initially as an inactive proenzyme (the pi-protein), which is proteolytically cleaved at a specific X-Ser bond to produce a beta-subunit with XOH at its C-terminus and an alpha-subunit with a pyruvoyl group at its N-terminus, derived from the serine. The recombinant enzyme, as purified, is a tetramer, and comprises principally the unprocessed pi-subunit (of 13.8 kDa), with a small proportion of the alpha- and beta-subunits (11 kDa and 2.8 kDa respectively). Incubation of the purified enzyme at elevated temperatures for several hours results in further processing. Using fluorescein thiosemicarbazide, the completely processed enzyme was shown to contain three pyruvoyl groups per tetrameric enzyme. The presence of unchanged serine at the N-terminus of some of the alpha-subunits was confirmed by electrospray mass spectrometry (ESMS) and N-terminal amino acid sequencing. A novel HPLC assay for aspartate decarboxylase was established and used to determine the Km and kcat for l-aspartate as 151+/-16 microM and 0.57 s-1 respectively. ESMS was also used to observe substrate and product adducts trapped on the pyruvoyl group by sodium cyanoborohydride treatment.
| S-EPMC1218368 | biostudies-other
Project description:We demonstrate the use of capillary zone electrophoresis with an electrokinetically pumped sheath-flow electrospray interface for the analysis of a tryptic digest of a sample of intermediate protein complexity, the secreted protein fraction of Mycobacterium marinum. For electrophoretic analysis, 11 fractions were generated from the sample using reverse-phase liquid chromatography; each fraction was analyzed by CZE-ESI-MS/MS, and 334 peptides corresponding to 140 proteins were identified in 165 min of mass spectrometer time at 95% confidence (FDR < 0.15%). In comparison, 388 peptides corresponding to 134 proteins were identified in 180 min of mass spectrometer time by triplicate UPLC-ESI-MS/MS analyses, each using 250 ng of the unfractionated peptide mixture, at 95% confidence (FDR < 0.15%). Overall, 62% of peptides identified in CZE-ESI-MS/MS and 67% in UPLC-ESI-MS/MS were unique. CZE-ESI-MS/MS favored basic and hydrophilic peptides with low molecular masses. Combining the two data sets increased the number of unique peptides by 53%. Our approach identified more than twice as many proteins as the previous record for capillary electrophoresis proteome analysis. CE-ESI-MS/MS is a useful tool for the analysis of proteome samples of intermediate complexity.
| S-EPMC3277681 | biostudies-literature
Project description:We report the use of the dynamic pH junction based capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) for bottom-up proteomics with an electrokinetically pumped sheath-flow nanospray capillary electrophoresis-mass spectrometry (CE-MS) interface and both LTQ-XL and LTQ-Orbitrap-Velos mass spectrometers. Conventional injection of 20 nL of a 1 mg/mL BSA digest identified 37 peptides and produced 66% sequence coverage. In contrast, pH junction injection of 130 nL (or larger) of a 0.05 mg/mL BSA digest identified 40 peptides and produced 70% coverage using a pH 6.5 sample buffer and the LTQ. A 20 nL conventional injection of a 1 mg/mL Escherichia coli digest identified 508 peptides and 199 proteins with the LTQ. A 400 nL pH junction injection of a 0.1 mg/mL E. coli digest identified 527 peptides and 179 proteins with the LTQ. Triplicate technical replicates of a 0.01 mg/mL sample with 400-nL injection volume using a pH junction identified 288 ± 9 peptides and 121 ± 5 proteins with the LTQ. There was outstanding concordance in migration time between the pH junction and normal injection. The pH junction produced narrower peaks and significant concentration for all but the most acidic components in the sample. Compared with the conventional stacking method, the pH junction method can generate comparable performance for small injection volume (20 nL) and significantly better concentration performance for a large injection volume (200 nL). We also applied the pH junction to three intact standard proteins and observed a >10× increase in peak intensity compared to conventional injection.
| S-EPMC4082393 | biostudies-literature
Project description:Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as an invaluable platform for top-down proteomics. However, the scale of top-down proteomics using CZE-MS/MS is still limited due to the low loading capacity and narrow separation window of CZE. In this work, for the first time we systematically evaluated the dynamic pH junction method for focusing of intact proteins during CZE-MS. The optimized dynamic pH junction-based CZE-MS/MS approached a 1 μL loading capacity, 90 min separation window, and high peak capacity (∼280) for characterization of an Escherichia coli proteome. The results represent the largest loading capacity and the highest peak capacity of CZE for top-down characterization of complex proteomes. Single-shot CZE-MS/MS identified about 2800 proteoform-spectrum matches, nearly 600 proteoforms, and 200 proteins from the Escherichia coli proteome with spectrum-level false discovery rate (FDR) less than 1%. The number of identified proteoforms in this work is over three times higher than that in previous single-shot CZE-MS/MS studies. Truncations, N-terminal methionine excision, signal peptide removal, and some post-translational modifications including oxidation and acetylation were detected.
| S-EPMC5726253 | biostudies-literature
Project description:A series of different types of wax esters (represented by RCOOR') were systematically studied by using electrospray ionization (ESI) collision-induced dissociation tandem mass spectrometry (MS/MS) along with pseudo MS(3) (in-source dissociation combined with MS/MS) on a quadrupole time-of-flight (Q-TOF) mass spectrometer. The tandem mass spectra patterns resulting from dissociation of ammonium/proton adducts of these wax esters were influenced by the wax ester type and the collision energy applied. The product ions [RCOOH2](+), [RCO](+) and [RCO-H2O](+) that have been reported previously were detected; however, different primary product ions were demonstrated for the three wax ester types including: (1) [RCOOH2](+) for saturated wax esters, (2) [RCOOH2](+), [RCO](+) and [RCO-H2O](+) for unsaturated wax esters containing only one double bond in the fatty acid moiety or with one additional double bond in the fatty alcohol moiety, and (3) [RCOOH2](+) and [RCO](+) for unsaturated wax esters containing a double bond in the fatty alcohol moiety alone. Other fragments included [R'](+) and several series of product ions for all types of wax esters. Interestingly, unusual product ions were detected, such as neutral molecule (including water, methanol and ammonia) adducts of [RCOOH2](+) ions for all types of wax esters and [R'-2H](+) ions for unsaturated fatty acyl-containing wax esters. The patterns of tandem mass spectra for different types of wax esters will inform future identification and quantification approaches of wax esters in biological samples as supported by a preliminary study of quantification of isomeric wax esters in human meibomian gland secretions.
| S-EPMC4526438 | biostudies-literature
Project description:Desorption electrospray ionization-mass spectrometry (DESI-MS) was evaluated for the detection of proteins ranging in molecular mass from 12 to 66 kDa. Proteins were uniformly deposited on a solid surface without pretreatment and analyzed with a DESI source coupled to a quadrupole ion trap mass spectrometer. DESI-MS parameters optimized for protein detection included solvent flow rate, temperature of heated capillary tube, incident and reflection angle, sheath gas pressure, and ESI voltage. Detection limits were obtained for all protein standards, and they were found to decrease with decreasing protein molecular mass: for cytochrome c (12.3 kDa) and lysozyme (14.3 kDa) a detection limit of 4 ng/mm2 was obtained; for apomyoglobin (16.9 kDa) 20 ng/mm2; for beta-lactoglobulin B (18.2 kDa) 50 ng/mm2; and for chymotrypsinogen A (25.6 kDa) 100 ng/mm2. The DESI-MS analysis of higher molecular mass proteins such as ovalbumin (44.4 kDa) and bovine serum albumin (66.4 kDa) yielded mass spectra of low signal-to-noise ratio, making their detection and molecular weight determination difficult. In this study, DESI-MS proved to be a rapid and robust method for accurate MW determination for proteins up to 17 kDa under ambient conditions. Finally, we demonstrated the DESI-MS detection of the bacteriophage MS2 capsid protein from crude samples with minimal sample preparation.
| S-EPMC3176668 | biostudies-literature
Project description:An assay to identify the common food-borne pathogens Salmonella, Escherichia coli, Shigella, and Listeria monocytogenes was developed in collaboration with Ibis Biosciences (a division of Abbott Molecular) for the Plex-ID biosensor system, a platform that uses electrospray ionization mass spectroscopy (ESI-MS) to detect the base composition of short PCR amplicons. The new food-borne pathogen (FBP) plate has been experimentally designed using four gene segments for a total of eight amplicon targets. Initial work built a DNA base count database that contains more than 140 Salmonella enterica, 139 E. coli, 11 Shigella, and 36 Listeria patterns and 18 other Enterobacteriaceae organisms. This assay was tested to determine the scope of the assay's ability to detect and differentiate the enteric pathogens and to improve the reference database associated with the assay. More than 800 bacterial isolates of S. enterica, E. coli, and Shigella species were analyzed. Overall, 100% of S. enterica, 99% of E. coli, and 73% of Shigella spp. were detected using this assay. The assay was also able to identify 30% of the S. enterica serovars to the serovar level. To further characterize the assay, spiked food matrices and food samples collected during regulatory field work were also studied. While analysis of preenrichment media was inconsistent, identification of S. enterica from selective enrichment media resulted in serovar-level identifications for 8 of 10 regulatory samples. The results of this study suggest that this high-throughput method may be useful in clinical and regulatory laboratories testing for these pathogens.
| S-EPMC3497367 | biostudies-literature
Project description:Biotin synthase (BS) catalyzes the oxidative addition of a sulfur atom to dethiobiotin (DTB) to generate the biotin thiophane ring. This enzyme is an S-adenosylmethionine (AdoMet) radical enzyme that catalyzes the reductive cleavage of AdoMet, generating methionine and a transient 5'-deoxyadenosyl radical. In our working mechanism, the 5'-deoxyadenosyl radical oxidizes DTB by abstracting a hydrogen from C6 or C9, generating a dethiobiotinyl carbon radical that is quenched by a sulfide from a [2Fe-2S] (2+) cluster. A similar reaction sequence directed at the other position generates the second C-S bond in the thiophane ring. Since the BS active site holds only one AdoMet and one DTB, it follows that dissociation of methionine and 5'-deoxyadenosine and binding of a second equivalent of AdoMet must be intermediate steps in the formation of biotin. During these dissociation-association steps, a discrete DTB-derived intermediate must remain bound to the enzyme. In this work, we confirm that the conversion of DTB to biotin is accompanied by the reductive cleavage of 2 equiv of AdoMet. A discrepancy between DTB consumption and biotin formation suggests the presence of an intermediate, and we use liquid chromatography and mass spectrometry to demonstrate that this intermediate is indeed 9-mercaptodethiobiotin, generated at approximately 10% of the total enzyme concentration. The amount of intermediate observed is increased when the reaction is run with substoichiometric levels of AdoMet or with the defective enzyme containing the Asn153Ser mutation. The retention of 9-mercaptodethiobiotin as a tightly bound intermediate is consistent with a mechanism involving the stepwise radical-mediated oxidative abstraction of sulfide from an iron-sulfur cluster.
| S-EPMC3380106 | biostudies-literature
Project description:A 193-nm wavelength deep ultraviolet laser was used for ambient laser ablation electrospray ionization mass spectrometry of biological samples. A pulsed ArF excimer laser was used to ablate solid samples, and the resulting plume of the desorbed material merged with charged electrospray droplets to form ions that were detected with a quadrupole time-of-flight mass spectrometer. Solutions containing peptide and protein standards up to 66-kDa molecular weight were deposited on a metal target, dried, and analyzed. No fragmentation was observed from peptides and proteins as well as from the more easily fragmented vitamin B12 molecule. The mass spectra contained peaks from multiply charged ions that were identical to conventional electrospray. Deep UV laser ablation of tissue allowed detection of lipids from untreated tissue. The mechanism of ionization is postulated to involve absorption of laser energy by a fraction of the analyte molecules that act as a sacrificial matrix or by residual water in the sample.
| S-EPMC6422691 | biostudies-literature
Project description:BackgroundMitochondrial DNA (mtDNA) mutations cause a large spectrum of clinically important neurodegenerative, neuromuscular, cardiovascular, and endocrine disorders. We describe the novel application of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) to the rapid and accurate identification of pathogenic mtDNA variants.MethodsIn a blinded study, we used ESI-FTICR MS to analyze 24 unrelated samples of total cellular DNA containing 12 mtDNA variants and compared the results with those obtained by conventional PCR-restriction fragment length polymorphism (PCR-RFLP) analysis and gel electrophoresis.ResultsFrom the 24-sample blinded panel, we correctly identified 12 of the samples as bearing an mtDNA variant and found the remaining 12 samples to have no pathogenic variants. The correlation coefficient between the 2 methods for mtDNA variant detection was 1.0; there were no false positives or false negatives in this sample set. In addition, the ESI-FTICR method identified 4 single-nucleotide polymorphisms (SNP) that had previously been missed by standard PCR-RFLP analysis.ConclusionsESI-FTICR MS is a rapid, sensitive, and accurate method for the identification and quantification of mtDNA mutations and SNPs.
| S-EPMC7108174 | biostudies-literature