Biomedical subjects

G A Anderson

Publications and source records attributed to G A Anderson.

At least 55 records · Page 3Linked to original sources

PubMed1999

Characterization of an improved electrodynamic ion funnel interface for electrospray ionization mass spectrometry.

An improved electrodynamic ion funnel for ion focusing at high pressure (> 1 Torr) has been developed for a triple quadrupole mass spectrometer and its performance compared with that of an earlier prototype previously reported. The ion funnel consists of a series of ring electrodes of progressively smaller internal diameters to which rf and dc electric potentials are co-applied. The new design utilizes ring electrodes possessing larger internal diameters that taper down to a relatively larger exit aperture. In the 1-10 Torr pressure range, the new design provides significant improvement in low m/z ion transmission. Additionally, the overall ion transmission range is improved by linked scanning of the ion funnel's rf voltage concomitantly with the scanning of the quadrupole mass analyzer. Transmission of a noncovalent complex through the interface demonstrated that excessive ion heating was not problematic. Computer simulations of ion transport support the ion funnel design and help explain the relative performance of both designs. Both ion simulations and experimental results are in accord and indicate close to 100% ion transmission efficiency for electrosprayed biopolymer ions through the interface and into the mass analyzer.

Animals↗

PubMed1999

High-mass-measurement accuracy and 100% sequence coverage of enzymatically digested bovine serum albumin from an ESI-FTICR mass spectrum.

The application of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to the analysis of polypeptide mixtures resulting from proteolytic digestion is described. A new 11.5-T FTICR mass spectrometer has been applied for the analysis of tryptic digestion mixtures of the protein bovine serum albumin (BSA). The improved cyclotron frequency stability and reduced frequency shifts observed over a wide range of trapped ion population sizes provide the ability to signal average spectra without degrading mass measurement accuracy, requiring internal calibration or advanced data processing schemes to compensate for variations in ion cyclotron signals brought about by different population sizes. A total of 100 spectra were signal-averaged leading to the observation of a total of 123 isotope distributions with a signal-to-noise ratio greater than 3:1. From those distributions, 86 can be ascribed to tryptic fragments of BSA on the basis of mass measurement errors of 10 ppm or less. Of these, 71 were within 2 ppm error limits corresponding to complete amino acid sequence coverage and an average error of 0.77 ppm. These results indicate that high-accuracy measurements are feasible for a large number of species detected simultaneously without the necessity for internal calibration and indicate the potential of such measurements, when combined with chromatographic separations, for facilitating more rapid identification of large numbers of proteins.

Amino Acid Sequence↗

PubMed1999

Probing proteomes using capillary isoelectric focusing-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

Unlike the genome, the proteome is exquisitely sensitive to cellular conditions and will consist of proteins having abundances dependent upon stage in the cell cycle, cell differentiation, response to environmental conditions (nutrients, temperature, stress etc.), or disease state(s). Therefore, the study of proteomes under well-defined conditions can provide a better understanding of complex biological processes and inference of protein function. Thus, much faster, more sensitive, and precise capabilities for the characterization of cellular constituents are desired. We describe progress in the development and initial application of the powerful combination of capillary isoelectric focusing (CIEF) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometry for measurements of the proteome of the model system Escherichia coli. Isotope depletion of the growth media has been used to improve mass measurement accuracy, and the comparison of CIEF-FTICR results for the analysis of cell lysates harvested from E. coli cultured in normal and isotopically depleted media are presented. The initial studies have revealed 400-1000 putative proteins in the mass range 2-100 kDa from total injections of approximately 300 ng of E. coli proteins in a single CIEF-FTICR analysis.

Bacterial Proteins↗

PubMed1999

Evidence that patients with rheumatoid arthritis have asymptomatic 'non-significant' Proteus mirabilis bacteriuria more frequently than healthy controls.

OBJECTIVES: patients with rheumatoid arthritis (RA) are reported to have in their sera raised levels of antibody specific to Proteus mirabilis. The aim of the study was to verify this and to determine an explanation for it by investigating the frequency of P. mirabilis urinary tract infection in RA patients and matched controls. METHODS: freshly voided urine was examined for the presence, number and identity of infecting bacteria. The levels of antibody in blood and in urine of the IgM, IgA and IgG classes to the common O serotypes of P. mirabilis and the antigens to which they reacted were determined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. RESULTS: analysis of urine from 76 patients with RA and 48 age- and gender-matched healthy controls showed that only two (4%) of the control urines but 25 (33%) of those from the RA patients were infected. The commonest infecting organism in the RA patients' urine was Proteus mirabilis which occurred twice as frequently as Escherichia coli. Proteus mirabilis was found in 52% of the infected urines of the RA patients and was always detected as a pure growth and usually in insignificant (< 10(4)/ml) numbers. It is highly improbable that this finding was the outcome of differences in age, physical ability or medication between the RA and control patient groups. Comparison of antibody levels to P. mirabilis by ELISA showed RA patients had raised (P < 0.0001, P = 0.001, P = 0.0063) levels of IgA, IgG and IgM respectively in their sera and raised (P < 0.0001, P < 0.0001, P = 0.0001) levels of IgG, IgM and IgA respectively in their urine compared with the control group. It was not possible to detect an antibody reacting to a P. mirabilis antigen that was specific to the RA patients. CONCLUSION: the results confirm that RA patients have raised levels of antibody to P. mirabilis not only in blood but also in urine and suggest that this arises because RA patients have an asymptomatic, non-significant P. mirabilis bacteriuria more frequently or more prolonged than control patients. This may be the trigger for their RA condition.

Adult↗

PubMed1998

An ion funnel interface for improved ion focusing and sensitivity using electrospray ionization mass spectrometry.

To improve upon the already impressive sensitivity achievable with electrospray ionization sources, a novel electrohydrodynamic ion funnel interface has been developed and implemented with a triple-quadrupole mass spectrometer. The ion funnel interface effectively consists of a series of ring electrodes of increasingly small internal diameters to which rf and dc electric potentials are coapplied. In the 1-10-Torr pressure range, the electric fields cause the collisionally damped ions to be more effectively focused and transmitted as a collimated ion beam. This paper describes the ion funnel design and presents an evaluation of its performance using a triple-quadrupole mass spectrometer. Ion transmission and m/z discriminating parameters (resulting in both effective low- and high-m/z cutoffs) are presented based upon both ion current measurements and mass spectra. Electrospray ionization mass spectra of selected protein solutions demonstrated well over 1 order of magnitude increase in signal relative to that of the instrument operated in its standard (inlet capillary-skimmer) configuration under similar conditions. The present results suggest that it will be feasible to realize close to 100% ion transmission efficiency for analytically relevant ions through the electrospray ionization interface and into the mass analyzer.

Animals↗

PubMed1998

In-trap cleanup of proteins from electrospray ionization using soft sustained off-resonance irradiation with fourier transform ion cyclotron resonance mass spectrometry.

Electrospray ionization Fourier transform ion cyclotron resonance (FTICR) mass spectrometry is capable of producing high mass resolving power and improved mass accuracy for large proteins and noncovalent complexes when coupled with collisionally induced dissociation (CID) of noncovalent adducts and consequent minimization of ion charge density in the ICR trap during measurements. This work demonstrates the application of in-trap cleanup to several biologically relevant systems, including carbonic anhydrase, 4-oxalocrotonate tautomerase (4OT) analogue, and SecB, a chaperone from Escherichia coli. In-trap cleanup yields improved mass measurements for these systems and is expected to further enable measurements for even more complex systems where adduction levels have precluded study of intact complexes.

Bacterial Proteins↗

PubMed1998

Electrospray ionization-mass spectrometry characterization of heterotetrameric sarcosine oxidase.

Electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has been used to characterize heterotetrameric corynebacterial sarcosine oxidase. By using a conventional quadrupole mass spectrometer, no spectra for the intact complex could be obtained (i.e., electrospraying protein at neutral pH), but spectra showing the four protein subunits were obtained when electrospraying from acidic solution. Initial low resolution ESI-FTICR mass spectra of the intact heterotetramer revealed a typical narrow charge state distribution in the range 6000 < m/z < 9000, consistent with retention of a compact structure in the gas phase, and gave a mass measurement about 1000 u higher than predicted. Efficient in-trap clean up, based upon low energy collisionally induced dissociation of adducts, allowed significant improvement in mass measurement accuracy. The present results represent the largest heteromultimeric protein complex successfully analyzed using FTICR mass spectrometry, and clearly illustrate the importance of sample clean up methods for large molecule characterization.

Corynebacterium↗

PubMed1998

Electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry at 11.5 tesla: instrument design and initial results.

Initial results obtained using a new electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometer operated at a magnetic field 11.5 tesla are presented. The new instrument utilized an electrostatic ion guide between the ESI source and FTICR trap that provided up to 5% overall transmission efficiency for light ions and up to 30% efficiency for heavier biomolecules. The higher magnetic field in combination with an enlarged FTICR ion trap made it possible to substantially improve resolving power and operate in a more robust fashion for large biopolymers compared to lower field instruments. Mass resolution up to 10(6) has been achieved for intermediate size biopolymers such as bovine ubiquitin (8.6 kDa) and bovine cytochrome c (12.4 kDa) without the use of frequency drift correction methods. A mass resolution of 370,000 has been demonstrated for isotopically resolved molecular ions of bovine serum albumin (66.5 kDa). Comparative measurements were made with the same spectrometer using a lower field 3.5-tesla magnet allowing the performance gains to be more readily quantified. Further improvements in pumping capacity of the vacuum system and efficiency of ion transmission from the source are expected to lead to further substantial sensitivity gains.

Algorithms↗

PubMed1998

Autonomous Biological System (ABS) experiments.

Three space flight experiments have been conducted to test and demonstrate the use of a passively controlled, materially closed, bioregenerative life support system in space. The Autonomous Biological System (ABS) provides an experimental environment for long term growth and breeding of aquatic plants and animals. The ABS is completely materially closed, isolated from human life support systems and cabin atmosphere contaminants, and requires little need for astronaut intervention. Testing of the ABS marked several firsts: the first aquatic angiosperms to be grown in space; the first higher organisms (aquatic invertebrate animals) to complete their life cycles in space; the first completely bioregenerative life support system in space; and, among the first gravitational ecology experiments. As an introduction this paper describes the ABS, its flight performance, advantages and disadvantages.

Animals↗

PubMed1997

Length and base composition of PCR-amplified nucleic acids using mass measurements from electrospray ionization mass spectrometry.

A generally applicable algorithm has been developed to allow base composition of polymerase chain reaction (PCR) products to be determined from mass spectrometrically measured molecular weights and the complementary nature of DNA. Mass measurements of arbitrary precision for single-stranded DNA species are compatible with an increasingly large number of possible base compositions as molecular weight increases. For example, the number of base compositions that are consistent with a molecular weight of 35,000 is approximately 6000, based on a mass measurement precision of 0.01%. However, given the low misincorporation rate of standard DNA polymerases, mass measurement of both of the complementary single strands produced in the PCR reduces the number of possibilities to less than 100 at 0.01% mass precision, and base composition is uniquely defined at 0.001% mass precision. Taking into account the low misincorporation rate of standard DNA polymerases and the fact that the final PCR product also contains primers of known sequence (generally 15-20-mer in size, which flank the targeted region), this reduces the number of possible base combinations to only approximately 3 at MW = 35,000. In addition, the number of base pairs (i.e., length of the DNA molecule) is uniquely defined. We show that the use of modified bases in PCR or post-PCR modification chemistry allows unique solutions for the base composition of the PCR product with only modest mass measurement precision.

Algorithms↗

PubMed1997

A dual-trap design and its applications in electrospray ionization FTICR mass spectrometry.

A new arrangement consisting of two separate Fourier transform ion cyclotron resonance (FTICR) ion traps was used to develop methods for the manipulation of the ions produced by an electrospray ionization source (ESI). A first, "accumulation" trap, is generally maintained at a higher pressure than the second, high-performance "analyzer" trap. The manipulations developed and demonstrated include the following: (1) mass-selective ion transfers between the traps; (2) mass-selective step-wise accumulation of low-abundance ions of different mass-to-charge ratios transferred from the first trap to the analyzer trap; (3) simultaneous detection of ions in the analyzer trap and ion accumulation in the source trap; (4) simultaneous ion detection in the accumulation trap and ion storage in the analyzer trap; (5) sequential multiple transfers of the ions into the analyzer trap from the same ion population stored in the accumulation trap; (6) collision-induced dissociation of ions stored in the accumulation trap followed by mass-selective transfer of the product ions into the analyzer trap; (7) sequential transfer of the ions of different mass-to-charge ratios into the analyzer trap from the same ion population stored in the accumulation trap followed by the collision-induced dissociation of transferred ions in the analyzer trap. These ion manipulations benefit multistage studies and are projected to be useful in many biochemical applications of ESI-FTICR, including structural determination of biopolymers and study of noncovalent complexes.

Cyclotrons↗

PubMed1997

The influence of progesterone-induced proteins on glucose metabolism in early equine embryos.

The influence of different maternal plasma progesterone concentrations on embryonic glucose metabolism was studied. Uterine flushes were obtained after treating ovariectomized mares (n = 3) with 0 (control), 100 or 200 mg progesterone daily for 7 d. A group of progesterone-induced proteins (PIP) of Mr approximately 20,000 were identified in flushes from progesterone treatments by SDS-PAGE but were not observed in control flushes. Progesterone-induced proteins were removed from half the pooled flush in each treatment group by Sepharose blue CL-6B. In a 3 x 2 factorial (progesterone treatments, progesterone-induced proteins) experiment, 6 groups of Day 7 equine embryos (n = 6 per group) were incubated in culture media (MEM:DPBS; 1:3) containing radioactively-labeled glucose. Contributions of the Embden-Meyerhof pathway (EMP) and the pentose-phosphate pathway (PPP) to the total metabolism of glucose in early equine embryos were assessed separately. In the 200 mg progesterone treatment group, the presence of progesterone-induced proteins in the culture medium resulted in a 4-to 5-fold increase in the activities of the Embden-Meyerhof pathway and the pentose-phosphate pathway. These results lead to the following conclusions: 1)Addition of progesterone-induced uterine proteins from mares with high levels of circulating progesterone enhance the metabolic activities of the Embden-Meyerhof pathway and the pentose-phosphate pathway in Day 7.5 equine embryos in culture. 2)Uterine secretion of progesterone-induced proteins which is quantitatively and/or qualitatively adequate to modify embryonic glucose metabolism in vitro is dependent on a minimal concentration of maternal plasma progesterone.

Journal Article↗

PubMed1997

3-dimensional computerized tomographic reconstruction of colovesical fistulas.

PURPOSE: Use of 3-dimensional tomographic reconstruction in evaluating colovesical fistulas is discussed. MATERIALS AND METHODS: We compared 3-dimensional computerized tomographic (CT) images of colovesical fistulas to conventional CT images. RESULTS: Successful surgical repair was facilitated by preoperative radiographic data. CONCLUSIONS: Three-dimensional CT reconstruction provides superior spacial detail and can clarify complex anatomical relationships preoperatively.

Colonic Diseases↗

PubMed1996

Characterization of PCR products from bacilli using electrospray ionization FTICR mass spectrometry.

A procedure for rapid purification of polymerase chain reaction (PCR) products allowing precise molecular weight determination using electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry is described. PCR amplification utilized the DNA polymerase from Pyrococcus furiosus (Pfu) which, unlike Taq, does not incorporate a nontemplated terminal deoxyadenosine phosphate. An 89-base pair nucleotide portion of the spacer region between the 16S and 23S ribosomal rRNA genes was amplified from the genome of three members of Bacillus cereus group and a 114 nucleotide region from the Bacillus subtilis. PCR involves polymerization of nucleotide precursors using two oligonucleotide primers and an amplification enzyme, as well as the presence of metal ions. Mass spectrometric analysis greatly benefits from removal of the oligonucleotide primers (15- and 17-mers in this instance) and nucleotide precursors since they adversely affect sensitivity and metal ion adduction results in an inaccurate molecular weight determination. In the presence of guanidinium hydrochloride the PCR products bind preferentially to a silica resin, allowing removal of other components (i.e., dNTP's primers, and salts). Further removal of metal ions was accomplished using a microdialysis device, allowing samples to be pumped through a hollow cellulose fiber with external countercurrent flow of 2.5 mM ammonium acetate. Prior to injection into the mass spectrometer, the sample buffer was adjusted to 50 vol % acetronitrile, 25 mM piperidine, and 25 mM imidazole, which enhanced signal intensity. The molecular weights of the PCR products determined by nucleotide sequence and MS analysis were in excellent agreement, and several PCR products were analyzed where mass differences corresponding to single base substitutions could be accurately assigned. These assignments were possible due to the high mass precision, accuracy, and resolution FTICR inherently affords. This constitutes the first report demonstrating the ionization and detection of PCR products by mass spectrometry with mass precision and accuracy for assignment of such modifications or substitutions.

Bacillus↗

PubMed1996

Molecular weight determination of plasmid DNA using electrospray ionization mass spectrometry.

Ionization and molecular weight (MW) determination of megadalton size plasmid DNA has been achieved using electrospray ionization (ESI) with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. DNA molecules were shown to remain intact through electrospray ionization by collection on a specially prepared surface, followed by agarose gel electrophoresis. Individual highly charged ions of plasmid DNA produced by ESI were trapped in an FTICR cell for up to several hours and reacted with acetic acid to induce charge state shifts. Measurements of mass-to-charge ratios for these multiple peaks arising from charge state shifting give MW measurements of individual ions with an average accuracy of 0.2%. The MW distribution was obtained by measurements for a number of individual ions from the same sample [plasmid DNA: pGEM-5S MW(cal) = 1.946 MDa], yielding a MW(obs) of 1.95 +/- 0.07 MDa for ions clustered in the vicinity of the expected MW.

Cyclotrons↗

PubMed1996

Screening derivatized peptide libraries for tight binding inhibitors to carbonic anhydrase II by electrospray ionization-mass spectrometry.

This paper describes the use of electrospray ionization-mass spectrometry (ESI-MS) to screen two libraries of soluble compounds to search for tight binding inhibitors for carbonic anhydrase II (EC 4.2.1.1). The two libraries, H2NO2SC6H4C(O)NH-AA1-AA2-C(O)NHCH2CH2CO2H where AA1 and AA2 are L-amino acids (library size: 289 compounds) or D-amino acids (256 compounds), were constructed by attaching tripeptides to the carboxyl group of 4-carboxybenzenesulfonamide. Screening of both libraries yielded, as the tightest binding inhibitor, compound 1 (AA1 = AA2 = L-Leu; binding constant Kb = 1.4 x 10(8) M-1). The ability of ESI-MS to estimate simultaneously the relative binding affinities of a protein to soluble ligands in a library, if general, should be useful in drug development.

Amino Acid Sequence↗

PubMed1996

Coronavirus isolation from nasal swab samples in cattle with signs of respiratory tract disease after shipping.

OBJECTIVE: To monitor the prevailing viral respiratory tract infections in cattle after transportation to feedlots. ANIMALS: 100 cattle with signs of respiratory tract disease on arrival at 2 feedlots. PROCEDURES: Nasal swab samples were obtained from each animal and were used for inoculation of defined cell culture systems that detected bovine viruses known to cause respiratory tract infections, as well as viruses previously not recognized as respiratory pathogens for cattle. RESULTS: Bovine respiratory coronaviruses were isolated from 38 of the 100 cattle, including 6 of 50 cattle from California, 22 of 31 cattle from Oklahoma, 6 of 11 cattle from Texas, and 4 of 8 cattle of unknown origin. Parainfluenza 3 viruses also were isolated from 5 California cattle, but other bovine viruses were not detected. CLINICAL IMPLICATIONS: The high rate of coronavirus isolations from feedlot cattle with signs of respiratory tract disease implied wide distribution and high susceptibility among cattle to this infection, which had not been detected by use of viral isolation systems in previous etiologic evaluations of feedlot cattle affected with bovine respiratory disease complex.

Animals↗

PubMed1996

"Colored" noise waveforms and quadrupole excitation for the dynamic range expansion of Fourier transform ion cyclotron resonance mass spectrometry.

Fourier transform ion cyclotron resonance (FTICR) mass spectrometry offers unparalleled analytical performance in most regards but has a dynamic range of typically no better than 10(2)-10(3). This limitation reportedly arises from two opposing constraints, involving the maximum number of ions that can be effectively trapped (10(6)-10(7)) and the minimum number of ions required to produce a detectable signal (10(2)-10(3)). A potential solution to this dynamic range limitation is presented, based on the application of selected-ion accumulation using quadrupole excitation. We show that lower concentration species can be effectively accumulated in the FTICR trapped ion cell, while the more abundant species are continually removed by the application of quadrupolar excitation in the form of band-limited or "colored" noise waveforms. The result is that "room" is made in the cell for lower abundance species, even during extended accumulation periods. This approach was demonstrated with mixtures of the bovine proteins, insulin, ubiquitin, and cytochrome c. For normal accumulation, the dynamic range was approximately 100. The application of selected-ion accumulation in the form of colored noise allowed the extension by 2 orders of magnitude and the detection of species of 1 x 10(-8) M concentration from a solution also containing another component at 9 x 10(-5) M. With this method, a putative new low abundance variant of bovine insulin was observed, and selected-ion accumulation and subsequent collisionally activated dissociation were used for its identification. Dipolar magnetron excitation was also explored to enhance selected-ion accumulation and was found to reduce the amount of buffer gas required for complete removal of the undesired species by a factor of 5. Further possible improvements are discussed, as are the complications due to the required balance between magnetron and cyclotron damping rates.

Amino Acid Sequence↗