Biomedical subjects

J E Bruce

Publications and source records attributed to J E Bruce.

At least 19 recordsLinked to original sources

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↗

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

The observation of chaperone-ligand noncovalent complexes with electrospray ionization mass spectrometry.

Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) was applied for the study of noncovalent chaperone SecB-ligand complexes produced in solution and examined in the gas phase with the aid of electrospray ionization (ESI). Since chaperone proteins are believed to recognize and bind only with ligands with nonnative tertiary structure, this work required careful unfolding of the ligand and subsequent reaction with the intact chaperone (the noncovalent tetrameric protein, SecB). A high denaturant concentration was employed to produce nonnative structures of the OppA, and microdialysis of the resulting solutions containing the chaperone-ligand complexes was carried out to rapidly remove the denaturant prior to analysis. Multistage mass spectrometry was essential to the successful study of these complexes since the initial mass spectra indicated extensive adduction that precluded mass measurements, even after microdialysis. However, low energy collisional activation of the ions in the FTICR trap proved useful for adduct removal, and careful control of excitation level preserved the intact complexes of interest, revealing a 1:1 SecB:OppA stoichiometry. To our knowledge, these results present the first direct observation of chaperone-ligand noncovalent complexes and the highest molecular weight heterogeneous noncovalent complex observed to date by mass spectrometry. Furthermore, these results highlight the capabilities of FTICR for the study of such complex systems, and the development of a greater understanding of chaperone interactions in protein export.

Bacterial Proteins↗

PubMed1998

The interaction between the chaperone SecB and its ligands: evidence for multiple subsites for binding.

The chaperone protein SecB is dedicated to the facilitation of export of proteins from the cytoplasm to the periplasm and outer membrane of Escherichia coli. It functions to bind and deliver precursors of exported proteins to the membrane-associated translocation apparatus before the precursors fold into their native stable structures. The binding to SecB is characterized by a high selectivity for ligands having nonnative structure but a low specificity for consensus in sequence among the ligands. A model previously presented (Randall LL, Hardy SJS, 1995, Trends Biochem Sci 20:65-69) to rationalize the ability of SecB to distinguish between the native and nonnative states of a polypeptide proposes that the SecB tetramer contains two types of subsites for ligand binding: one kind that would interact with extended flexible stretches of polypeptides and the other with hydrophobic regions. Here we have used titration calorimetry, analytical ultracentrifugation, and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry to obtain evidence that such distinguishable subsites exist.

Aprotinin↗

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↗

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

Geriatric urolithiasis.

PURPOSE: We define the differences between geriatric patients with urinary stone disease compared to a younger cohort. MATERIALS AND METHODS: A data base, including serum biochemical profiles, 24-hour urinalyses and standardized questionnaires, was retrospectively evaluated from more than 6,000 consecutive patients with urinary stone disease. RESULTS: Geriatric stone formers comprised 12% (721) of all stone patients. Two-thirds of these elderly patients had aberrant urinary values and 29% had isolated hypocitraturia compared to 17% in the younger group. Of geriatric stone forming patients 76% had recurrent urinary stones (mean 3.5 stone episodes), which was similar to the younger comparable group (77%, mean 3.3 stone episodes). The severity of urinary stone disease was similar between the 2 groups based on the need for urological intervention. Geriatric stone patients, in general, experienced the first stone episode later in life (after age 50 years) compared with younger patients. Elderly patients had an increased incidence of uric acid stones, but had a similar incidence of struvite calculi. Geriatric stone patients underwent parathyroid surgery more frequently (2.7 versus 0.7%). Geriatric stone forming patients rarely had renal failure. CONCLUSIONS: The incidence, recurrence and severity of recurrent urinary stone disease were similar between geriatric and younger stone forming patients. Geriatric stone patients had an increased incidence of isolated hypocitraturia, uric acid calculi and previous parathyroidectomy. The geriatric stone population is not merely an extension of younger stone forming patients presenting at an older age. Rather, geriatric patients commonly experience the first symptomatic stone episode later in life.

Age Distribution↗

PubMed1996

Mass spectrometric characterization of sequence-specific complexes of DNA and transcription factor PU.1 DNA binding domain.

Electrospray ionization mass spectrometry (ESI-MS) has been used to study the noncovalent interaction of the 13.5-kDa DNA binding domain of PU.1 (PU.1-DBD) with specific double-stranded DNA (dsDNA) target molecules. Mixtures of PU.1-DBD protein and wild-type target DNA sequence yielded ESI-MS spectra showing only protein-dsDNA complex ions of 1:1 stoichiometry and free dsDNA. When PU.1-DBD protein, wild type target DNA, and a mutant target DNA lacking the consensus sequence were mixed, only the 1:1 complex with the wild-type DNA was observed, consistent with gel electrophoresis mobility shift assay results, demonstrating the observation of sequence-specific protein-dsDNA complexes using ESI-MS.

Binding Sites↗

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

"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↗

PubMed1996

Analysis of double-stranded polymerase chain reaction products from the Bacillus cereus group by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

The analysis of polymerase chain reaction (PCR) products by electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR) has been achieved. Specifically, a 105 base-pair nucleotide portion of the ribosomal spacer region was amplified in two members of the B. cereus group (i.e. B. thuringiensis and B. cereus) using PCR. These amplified regions were then analyzed by gel electrophoresis and ESI-FTICR. Based on the predicted sequence of the PCR products for each organism, the mass measurement using ESI-FTICR matched the theoretical mass within experimental error and was consistent with gel electrophoresis results. In contrast, for the typical several hour time-scale of the gel electrophoresis experiment, the mass spectrometric analysis was completed in a matter of minutes. To our knowledge, this constitutes the first report demonstrating the ionization and detection of a double-stranded PCR product by ESI-MS. This preliminary result indicates the potential use of ESI-MS to analyze PCR products on a rapid time-scale, with potential for medical and taxonomic applications.

Bacillus cereus↗

PubMed1996

Transrectal ultrasound guided prostatic nerve blockade eases systematic needle biopsy of the prostate.

PURPOSE: We assessed the effect of transrectal ultrasound guided prostatic nerve blockade on the discomfort associated with systematic needle biopsy of the prostate. MATERIALS AND METHODS: A prospective randomized double-blind study was performed of 64 patients requiring systematic biopsy of the prostate. Patients were randomly assigned to receive an injection of 5 ml. 1% lidocaine or 5 ml. saline (0.9% sodium chloride) at the vascular pedicle on 1 side of the prostate only. They were then asked to score the severity of discomfort of the injection and subsequent biopsies on each side. RESULTS: Mean pain scores were significantly lower on the side with than the side without lidocaine injection (1.6 +/- 0.9 versus 2.4 +/- 1.2, p < 0.0001) and not significantly different when saline was injected (2.9 +/- 1.2 versus 3.0 +/- 1.1, p = 0.52). Pain scores were significantly different when the lidocaine injected side was compared to the saline solution injected side (p < 0.0001) but the difference was not significant between the noninjected sides of the 2 groups (p = 0.076). Of the patients in the lidocaine group 68% reported that they would prefer to undergo biopsy with the injection compared to only 41% in the placebo group (p = 0.037). During the study no patient in either group had any adverse effect from the injection. CONCLUSIONS: Transrectal ultrasound guided nerve blockade before prostatic biopsy results in a more comfortable procedure for the patient.

Biopsy, Needle↗

PubMed1996

Efficacy of radiographic imaging in pediatric blunt renal trauma.

PURPOSE: We sought to determine whether radiographic imaging can effectively detect significant renal injuries in children with blunt trauma who do not have significant hematuria. MATERIALS AND METHODS: We reviewed the records of 180 children who presented to our hospital for suspected renal trauma between 1977 and 1995. Results of excretory urography or abdominal computerized tomography were correlated with urinalysis findings and clinical outcome. RESULTS: Of 147 patients with microscopic hematuria after blunt trauma 77 underwent imaging. Only 1 patient had a significant renal injury (grade 2 or greater) and 76 had normal findings or renal contusions only, including 11 with microscopic hematuria and shock. Of the 74 patients who did not undergo imaging a clinical diagnosis of renal contusion was made and followup was available for 57 (77%). All patients healed without adverse sequelae. Of 33 patients with gross hematuria significant renal injuries were found in 9, including 3 who required immediate surgical repair of a major renal laceration or vascular injury. Combining our results with those of other reported series revealed significant renal injuries in only 11 of 548 children (2%) with less than 50 red blood cells per high power field on presenting urinalysis after blunt abdominal trauma. These patients were likely to have multiple associated injuries. CONCLUSIONS: Significant renal injuries are unlikely in pediatric patients with blunt renal trauma but no gross or substantial microscopic hematuria. Shock does not appear to be a clinically useful indicator.

Algorithms↗

PubMed1995

Bio-affinity characterization mass spectrometry.

A new approach, bio-affinity characterization mass spectrometry (BACMS), aimed at providing a more rapid, sensitive and potentially more flexible alternative to techniques presently employed for the characterization of noncovalent interactions in mixtures, such as would be encountered in combinatorial chemistry, in presented. BACMS avoids some of the difficulties and potential artifacts associated with affinity chromatography since the noncovalent associations occur in solution; thus, BACMS avoids the requirement of solid support media and the development of non-interfering linker species. This paper describes the conceptual basis for the methodology and its potential use in applications which include the screening of high affinity ligands in support of new drug development. BACMS exploits new Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry technologies which, when coupled to electrospray ionization (ESI), allow the investigation of specific noncovalent complexes formed in solution. BACMS utilizes the well-known attributes of FTICR, such as the high resolution mass analysis and (MS)n (n > or = 2) capabilities; however, it is even more directly a result of recently developed techniques involving quadrupolar excitation, such as selected-ion accumulation. These tools are demonstrated and the results illustrate the extraordinary sensitivity achievable (solution concentration of 1 x 10-9 M without the use of separations prior to ESI). Thus, the new capabilities demonstrated here, in conjunction with ESI, will be useful for the investigation of very low relative concentration noncovalent association directly from solution, and promote a faster alternative for combinatorial mixture screening and analysis.

Animals↗

PubMed1995

Nephrectomy for traumatic renal injuries.

During a 16-year period 2,521 patients presented to our institution with renal trauma. Renal exploration was performed in 195 patients (202 renal units), with 31 units requiring exploration alone, 145 repair and 26 nephrectomy, yielding an overall nephrectomy rate of 13%. A detailed review of these 26 patients identified them as a unique population: all had major renal injuries, and as a group they demonstrated significantly higher rates of shock, injury severity scores, transfusion requirements and death rates than those in whom exploration with renal salvage was possible. In no case was nephrectomy required as a complication of exploration or attempted repair. In the properly staged patient undergoing surgery for appropriate indications with early vascular control exploration of renal injuries is safe. It is not the exploration that results in the nephrectomy but the injury itself.

Adult↗

PubMed1995

Sono-urethrography in the evaluation of anterior urethral strictures.

During a 7-year period 123 paired urethrographic and sono-urethrographic studies were performed on 101 patients with 110 urethral strictures. In all but 3 cases the urethra was subsequently evaluated either cystoscopically or at open operation. Sono-urethrography readily identified urethral calculi, diverticula and false passages. It correctly identified the stricture and its site in every case. There was a significant difference between stricture length as measured by urethrography compared to that measured by sono-urethrography (p < 0.003). However, if the strictures were grouped based on anatomical location, there was good correlation and no significant difference in the penile urethra (correlation coefficient = 0.94, p = 0.74) but poor correlation and the significant difference remained in the urethral bulb (correlation coefficient = 0.64, p < 0.007). Similarly, when urethrographic and sono-urethrographic stricture lengths were compared with operative lengths, in the penile urethra the correlation coefficients were close (correlation coefficient = 0.91 versus 0.98) but in the urethral bulb the poor correlation persisted (correlation coefficient = 0.69 versus 0.89). Although sono-urethrography certainly identifies periurethral tissue, it was unreliable in predicting the depth of spongiofibrosis when compared with full depth biopsies in 36 patients with histopathological correlation. Finally, in 16% of the patients sono-urethrography correctly indicated a reconstructive procedure different from that originally suggested by conventional urethrography. Sono-urethrography is a dynamic 3-dimensional study that accurately identifies stricture site, number and caliber. Compared with conventional urethrography, it more accurately measures stricture length and diameter, and identifies periurethral tissue, making it a valuable adjunct in the evaluation of patients with suspected anterior urethral strictures.

Biopsy↗