Coronary artery disease risk reclassification by a new acoustic-based score.

To determine the potential of a non-invasive acoustic device (CADScor®System) to reclassify patients with intermediate pre-test probability (PTP) and clinically suspected stable coronary artery disease (CAD) into a low probability group thereby ruling out significant CAD. Audio recordings and clinical data from three studies were collected in a single database. In all studies, patients with a coronary CT angiography indicating CAD were referred to coronary angiography. Audio recordings of heart sounds were processed to construct a CAD-score. PTP was calculated using the updated Diamond-Forrester score and patients were classified according to the current ESC guidelines for stable CAD: low < 15%, intermediate 15-85% and high > 85% PTP. Intermediate PTP patients were re-classified to low probability if the CAD-score was ≤ 20. Of 2245 patients, 212 (9.4%) had significant CAD confirmed by coronary angiography ( ≥ 50% diameter stenosis). The average CAD-score was higher in patients with significant CAD (38.4 ± 13.9) compared to the remaining patients (25.1 ± 13.8; p < 0.001). The reclassification increased the proportion of low PTP patients from 13.6% to 41.8%, reducing the proportion of intermediate PTP patients from 83.4% to 55.2%. Before reclassification 7 (3.1%) low PTP patients had CAD, whereas post-reclassification this number increased to 28 (4.0%) (p = 0.52). The net reclassification index was 0.209. Utilization of a low-cost acoustic device in patients with intermediate PTP could potentially reduce the number of patients referred for further testing, without a significant increase in the false negative rate, and thus improve the cost-effectiveness for patients with suspected stable CAD.

Pure bacterial isolates that convert p-xylene to terephthalic acid.

Bacteria that grow on p-xylene, p-toluic acid, and terephthalic acid (TPA) were isolated from a wastewater bioreactor that is used to treat a waste stream that contains all three of these compounds. Although previously described aerobic bacteria degrade p-xylene by initially oxidizing a single methyl group to form p-toluic acid and then cleaving the aromatic ring, some of the bacteria isolated during this study transformed p-xylene by oxidizing both methyl groups to produce TPA.

Effect of residual acrylamide monomer from two-dimensional gels on matrix-assisted laser desorption/ionization peptide mass mapping experiments.

Residual acrylamide can cause severe suppression of signal intensity during matrix-assisted laser desorption/ionization (MALDI) peptide mass mapping experiments. This suppression phenomenon can compromise the ability to detect low picomole and subpicomolar amounts of peptides extracted from two-dimensional gels. A rapid and simple method that exploits the use of pipette tips incorporating C18 packing materials for the enhancement of MALDI signal intensity is presented. The utility of the method is demonstrated with peptide solutions incorporating residual acrylamide and/or gel monomer components.

Sample preparation for high throughput accurate mass analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

An automated sample preparation for high throughput accurate mass determinations by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been developed. Sample preparation was performed with an automated workstation and automated mass analyses were performed with a commercial MALDI-TOF mass spectrometer. The method was tested with a 41-sample library. MALDI-TOFMS was found to give the needed sensitivity, accurate mass measurement, and soft ionization necessary for structure confirmation, even of mixtures. A mass accuracy of 5 ppm or less was obtained in over 80% of known compound measurements. A mass accuracy better than 10 ppm was obtained for all measurements of known compounds. Analyses of parallel synthesis products resulted in 77% of the measurements with a mass accuracy of 5 ppm or better.

Mass accuracy and sequence requirements for protein database searching.

To elucidate the role of high mass accuracy in mass spectrometric peptide mapping and database searching, selected proteins were subjected to tryptic digestion and the resulting mixtures were analyzed by electrospray ionization on a 7 Tesla Fourier transform mass spectrometer with a mass accuracy of 1 ppm. Two extreme cases were examined in detail: equine apomyoglobin, which digested easily and gave very few spurious masses, and bovine alpha-lactalbumin, which under the conditions used, gave many spurious masses. The effectiveness of accurate mass measurements in minimizing false protein matches was examined by varying the mass error allowed in the search over a wide range (2-500 ppm). For the "clean" data obtained from apomyoglobin, very few masses were needed to return valid protein matches, and the mass error allowed in the search had little effect up to 500 ppm. However, in the case of alpha-lactalbumin more mass values were needed, and low mass errors increased the search specificity. Mass errors below 30 ppm were particularly useful in eliminating false protein matches when few mass values were used in the search. Collision-induced dissociation of an unassigned peak in the alpha-lactalbumin digest provided sufficient data to unambiguously identify the peak as a fragment from alpha-lactalbumin and eliminate a large number of spurious proteins found in the peptide mass search. The results show that even with a relatively high mass error (0.8 Da for mass differences between singly charged product ions), collision-induced dissociation can help identify proteins in cases where unfavorable digest conditions or modifications render digest peaks unidentifiable by a simple mass mapping search.

Identification of single stranded regions of DNA by enzymatic digestion with matrix-assisted laser desorption/ionization analysis.

Elucidating structure function relationships of DNA in cellular processes requires fast, reliable methods that can be applied to picomole amounts of sample. Higher order structure can be inferred by distinguishing paired and unpaired regions. It is shown here that enzymatic digestion coupled with product analysis by matrix-assisted laser desorption ionization (MALDI) is able to identify unpaired bases within structured DNA regions. The method is demonstrated with DNA duplexes having a five nucleotide mismatch as a 5' overhang, a 3' overhang, and an internal loop. Exo- and endonuclease digestions are performed under solution conditions (temperature, annealing, and enzyme buffers) which promote base pairing and specific enzyme activity. For each type of mismatch, the length and sequence of the single stranded region can be inferred from MALDI spectra taken as a function of digestion time.

Determining affinity-selected ligands and estimating binding affinities by online size exclusion chromatography/liquid chromatography-mass spectrometry.

Size exclusion chromatography (SEC) isolation of affinity-selected ligands combined with reverse phase liquid chromatography-mass spectrometry (LC-MS) is an effective means for identifying members of mixtures which form tightly bound noncovalent complexes with target proteins. A potential liability of the approach is that the SEC isolation is carried out under nonequilibrium conditions favoring protein/ligand complex dissociation. At long SEC isolation times and/or for complexes with fast off-rates the extent of dissociation can jeopardize the ability to detect the affinity-selected components. Additionally, equilibrium binding affinities cannot be exactly determined from the measured distribution of isolated ligands. We present here an online SEC/LC-MS system for determining affinity-selected members of active mixtures which reduces this liability. A kinetic model of the SEC isolation process is developed to determine the practical limits for the application of the method and to extrapolate equilibrium binding affinities from the nonequilibrium data. The utility of online SEC/LC-MS for identifying affinity-selected ligands and for estimating binding affinities is demonstrated for a small molecule mixture of compounds with known binding affinities and for a simple combinatorial mixture.

Distinguishing small molecular mass differences of proteins by mass spectrometry.

Electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry allows for high-resolution, accurate mass analysis of multiply charged ions of proteins. In the work described here, the ability of ESI-FTICR to distinguish small differences in molecular mass is evaluated. Ubiquitin was used as an internal mass calibration standard to measure the molecular mass of cytochrome c, myoglobin, and several carbonic anhydrase isoforms. Mass calibration was based on the tallest isotopic peak of each ubiquitin charge state. Ubiquitin performed well as an internal standard because its charge states covered the appropriate mass range, interference was minimal, and the tallest peak was easily identified. The peak masses of cytochrome c (12.5 kDa) and myoglobin (17 kDa) were measured to an accuracy of about 0.02 Da (<2ppm). However, errors of 1.0 Da were observed for some individual determinations because of the difficulty in identifying the tallest peak. When the technique was applied to bovine carbonic anhydrase II, even combining data from several charge states did not yield an unequivocal assignment of the tallest peak, resulting in a mass assignment of 29,023.7 or 29,024.7. Similarly, measurements of two isoforms with a mass difference of 1 Da, human carbonic anhydrase I, pI 6.0 and 6.6, yielded overlapping values for the mass of the tallest peak. However, these two isoforms were clearly distinguished by (a) identification of the tallest peak using a measurement of average mass as a guide and (b) comparison of the isotopic peak intensity patterns.

Base specificity of oligonucleotide digestion by calf spleen phosphodiesterase with matrix-assisted laser desorption ionization analysis.

Calf spleen phosphodiesterase cleaves oligonucleotide strands in a stepwise manner from the 5' end and can be used in combination with matrix-assisted laser desorption ionization (MALDI) mass spectrometry to perform ladder sequencing. The relative intensities of ladder peaks in the mass spectra of a series of 5-mers and 7-mers show that the rate of digestion is influenced by strand sequence. Sequences terminating in A or G at the 5' end are found to react two to three times faster than sequences terminating in C or T. The reactivity of the terminal base is also influenced by the sequence beyond the 5' end. When the third base from the 5' end is A or G, removal of the first and second bases is faster than when the third base is C or T. A method is described which permits reaction rates to be quantitatively determined from the time dependences of ladder peaks in the MALDI spectra. A similar approach could be used for mechanistic studies.

Oligonucleotide sequence and composition determined by matrix-assisted laser desorption/ionization.

Molecular weight measurements of several oligonucleotides ranging in size from 12 to 60 bases were performed by matrix-assisted laser desorption/ionization with a time-of-flight mass spectrometer (MALDI-TOF). In each case, the mass accuracy was better than 0.1%. Sequences for two 12-base oligonucleotides and a 24-base oligonucleotide were determined using calf spleen phosphodiesterase to sequentially cleave from the 5' end. A MALDI-TOF spectrum of the digest mixture shortly after the addition of the enzyme produced a characteristic oligonucleotide ladder. Molecular ions in the mass spectrum corresponded to the products of enzymatic cleavage, and the mass differences between these peaks identified the individual nucleotides. The resolution and mass accuracy of MALDI-TOF were sufficient to unambiguously identify the individual nucleotides in the 12- and 24-base strands.

Fundamentals of the application of matrix-assisted laser desorption-ionization mass spectrometry to low mass poly(methylmethacrylate) polymers.

Matrix-assisted laser desorption-ionization (MALDI) time of flight is shown to give a molar peak area response for isolated methylmethacrylate oligomers that have 25 and 50 repeat units when run on three different instruments in reflectron or linear mode and using three different matrix materials. In addition, fragmentation was not observed in any of the three different matrices or at higher laser power. No spectral differences were observed for syndiotactic and isotactic methylmethacrylate oligomers. These results suggest that the low most probable peak values observed for narrow distribution poly(methylmethacrylate) standards by MALDI mass spectrometry are not the result of mass discrimination or fragmentation.

The fundamentals of applying electrospray ionization mass spectrometry to low mass poly(methyl methacrylate) polymers.

Electrospray ionization (ESI) is capable of ionizing many soluble polymers. The ESI spectra are complex because of overlap of the multiply charged ions of the oligomer distribution, causing current computer transform programs to fail. However, it is possible to determine the origin of the multiply charged ions, making it feasible to write a program designed to transform ESI polymer spectra. To assess the value of such a program for polymer analysis, isolated monodisperse methyl methacrylate (MMA) oligomers (25 and 50 repeat units) were used to determine molar signal response and propensity for fragmentation.The sum of the peak areas for the multiply charged MMA 50-mer was found to be only about 66% of the summed peak areas for the 25-mer for the same molar concentration. However, conversion of the multiply charged peak areas to the singly charged representations, with peak area compression taken into account, gave equal signal responses for the 25-and 50-mers. Signal response variations due to the tacticity of the MMA oligomers were not observed. Fragmentation of the MMA oligomers also was shown not to occur under normal ESI conditions. Therefore, transformation of the polymer spectra to the singly charged molecular ion distribution should allow accurate calculation of average molecular weights, polydispersity, end group mass, and repeat unit mass.

Identification of histidine at the catalytic site of the photosynthetic oxygen-evolving complex.

The molecular oxygen in our atmosphere is a product of a water-splitting reaction that occurs in the oxygen-evolving complex of photosystem II of oxygenic photosynthesis. The catalytic core of the oxygen-evolving complex is an ensemble of four manganese atoms arranged in a cluster of undetermined structure. The pulsed electron paramagnetic resonance (EPR) technique of electron spin-echo envelope modulation (ESEEM) can be used to measure nuclear spin transitions of nuclei magnetically coupled to paramagnetic metal centers of enzymes. We report the results of ESEEM experiments on the cyanobacterium Synechocystis PCC 6803 selectively labeled with 15N at the two nitrogen sites of the imidazole side chain of histidine residues. The experiments demonstrate that histidine is bound to manganese in the oxygen-evolving complex.

Escherichia coli: derived murine interleukin-1 beta with N-terminus partially N alpha-acetylated.

Two forms of murine recombinant interleukin-1 beta (mrIL-1 beta) from Escherichia coli were purified by ion exchange column chromatography; each exhibited equivalent biological activity in the murine thymocyte proliferation assay. It was determined by mass spectrometry of tryptic peptides that both retained the initiating methionine but one form was N alpha-acetylated at the N-terminus.

Isolation and characterization of the outer membrane proteins of Serratia marcescens W225.

The study addressed the general problem of fractionating cell envelopes in order to isolate the outer membranes of gram-negative bacteria. Whereas the cells are normally transformed into spheroplasts prior to disintegration and membrane separation, Serratia marcescens was found to be resistant to spheroplast formation using the procedures available, which were originally developed for Escherichia coli. An efficient technique for spheroplasting S. marcescens was therefore developed; this comprised combining osmotic shock and lysozyme-EDTA treatment of sucrose-conditioned cells. Spheroplasting efficiency and the amount of outer membrane protein recovered were highly dependent on the spheroplasting technique used. Separation of the outer and inner membranes was performed by two methods, isopyenic centrifugation and selective detergent solubilization with Sarkosyl. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the analysis of specific inner membrane marker enzymes revealed that the protein obtained by detergent solubilization was much purer than that obtained by isopycnic centrifugation. The outer membrane isolated accounted for 60% of the envelope proteins and had a buoyant density of 1.2502 g/cm3. The protein profile of the outer membrane determined by SDS-PAGE resolved into 12 distinct protein bands, 3 of which represented major proteins.

An electrospray ion source for magnetic sector mass spectrometers.

Electro spray mass spectra of carbonic anhydrase (MW ∼ 29000) and ovalbumin (MW ∼ 45000) were obtained on a double focusing magnetic secter mass spectrometer by using a single stage of mechanical pumping in the interface between atmospheric pressure and high vacuum. Full scan spectra of lysozyme were recorded on 15 fmoles consumed. In addition, accurate mass measurement was demonstrated for peptides and proteins, and resolution in excess of 10,000 (m /△m, 10% valley) was observed. These results clearly show that high performance magnetic sector mass spectrometers can be advantageously interfaced to an atmospheric pressure electrospray ion source.

Fermentation studies of the secretion of Serratia marcescens nuclease by Escherichia coli.

The secretion of a Serratia marcescens nuclease was followed by fermentation with Escherichia coli. A plasmid, p403-SD2, carrying a 1.3-kilobase-pair insert with a 0.4-kilobase-pair region upstream of the nuclease gene caused a growth-phase-regulated expression of nuclease in E. coli in the same way as that seen in S. marcescens. Deletion of the regulatory gene generating plasmid p403-Rsa1 resulted in a constitutive expression of the nuclease. Anaerobiosis stimulated the expression from p403-SD2 in stationary growth phase by a factor of 10 compared with expression stimulated by cultivation in aerobic conditions; no such effect was found for plasmid p403-Rsa1. Different nutritional factors caused the expression level and the amount of extracellular nuclease to vary more when nuclease was expressed from plasmid p403-SD2 than when it was expressed from plasmid p403-Rsa1. A correlation between the regulatory gene and the extracellular secretion of nuclease is proposed.

A mass spectrometric study of the structure of sterol carrier protein SCP2 from rat liver.

The amino acid sequence of Sterol Carrier Protein2 (SCP2) isolated from rat has been investigated. Using a novel mass spectrometric mapping approach, the C-terminus was found to be extended beyond the previously published sequence. Carbohydrate analysis of SCP2 samples suggest the presence of tightly bound mannose oligosaccharide of 5-10 residues, although probably not in a glycoprotein linkage.