Biomedical subjects

M A Moseley

Publications and source records attributed to M A Moseley.

13 recordsLinked to original sources

PubMed1998

Comprehensive two-dimensional high-performance liquid chromatography for the isolation of overexpressed proteins and proteome mapping.

A two-dimensional liquid chromatographic system is described here which uses size-exclusion liquid chromatography (SEC) followed by reversed-phase liquid chromatography (RPLC) to separate the mixture of proteins resulting from the lysis of Escherichia coli cells and to isolate the proteins that they produce. The size-exclusion chromatography can be conducted under either denaturing or nondenaturing conditions. Peaks eluting from the first dimension are automatically subjected to reversed-phase chromatography to separate similarly sized proteins on the basis of their various hydrophobicities. The RPLC also serves to desalt the analytes so that they can be detected in the deep ultraviolet region at 215 nm regardless of the SEC mobile phase used. The two-dimensional (2D) chromatograms produced in this manner then strongly resemble the format of stained 2D gels, in that spots are displayed on a X-Y axis and intensity represents quantity of analyte. Following chromatographic separation, the analytes are deposited into six 96-well (576 total) polypropylene microtiter plates via a fraction collector. Interesting fractions are analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) or electrospray mass spectrometry (ESI/MS) depending on sample concentration, which both yield accurate (2 to 0.02%) molecular weight information on intact proteins without any additional sample preparation, electroblotting, destaining, etc. The remaining 97% of a fraction can then be used for other analyses, such Edman sequencing, amino acid analysis, or proteolytic digestion and sequencing by tandem mass spectrometry. This 2D HPLC protein purification and identification system was used to isolate the src homology (SH2) domain of the nonreceptor tyrosine kinase pp60c-src and beta-lactamase, both inserted into E. coli, as well as a number of native proteins comprising a small portion of the E. coli proteome.

Bacterial Proteins↗

PubMed1998

The EMBL nucleotide sequence database.

The EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl. html ) constitutes Europe's primary nucleotide sequence resource. DNA and RNA sequences are directly submitted from researchers and genome sequencing groups and collected from the scientific literature and patent applications (Fig. 1). In collaboration with DDBJ and GenBank the database is produced, maintained and distributed at the European Bioinformatics Institute. Database releases are produced quarterly and are distributed on CD-ROM. EBI's network services allow access to the most up-to-date data collection via Internet and World Wide Web interface, providing database searching and sequence similarity facilities plus access to a large number of additional databases.

Base Sequence↗

PubMed1997

Sensitive analysis of [D-Pen2,5]enkephalin in rat serum by capillary electrophoresis and laser-induced fluorescence detection.

A highly sensitive analytical method based on capillary zone electrophoresis (CZE) coupled with a laser-induced fluorescence (LIF) detector was explored for the analysis of [D-Pen2,5]enkephalin (DPDPE) in rat serum. DPDPE and the internal standard Phe-Leu-Glu-Glu-Ile (P9396) were extracted from serum samples with C18 solid-phase extraction disk cartridges, followed by derivatization with tetramethylrhodamine-5-isothiocyanate (TRITC) isomer G before introduction onto the capillary column. Complete resolution of DPDPE and the internal standard from other serum components was achieved within 20 min on a 140 cm x 50 microns I.D. capillary column with borate buffer (25 mM. pH 8.3). With the current method, it is possible to detect 1.3E-18 mol of DPDPE on column. The results suggest that CZE-LIF is a promising method for the sensitive and specific quantitation of therapeutic peptides in biological matrices.

Animals↗

PubMed1997

Rapid separation and characterization of protein and peptide mixtures using 1.5 microns diameter non-porous silica in packed capillary liquid chromatography/mass spectrometry.

Octadecyl-modified 1.5 microns diameter non-porous silica particles were packed in 150 microns i.d. (360 microns o.d.) capillaries with lengths of 20 cm which were used to separate proteins and peptides generated from enzymatic digests of proteins. Gradients were produced using an exponential dilution method at pressures of 520 Bar (7500 psi) and electrospray ionization mass spectrometry was used for detection. This system was similar to packed capillary perfusion chromatography with respect to chromatographic resolution and analysis time and had a limit of detection comparable to traditional packed capillaries which use 5 microns diameter porous particles. The analyses required as little as 250 femtomol of protein or 500 femtomol of peptide on-column in approximately 30 min. This technique was then applied to verify the existence of an overexpressed protein in an E. coli cell lysate and to confirm the presence of four glycoforms of a peptide generated in the proteolytic digest of an antibody.

Chromatography, Ion Exchange↗

PubMed1996

CYP3A-like cytochrome P450-mediated metabolism and polarized efflux of cyclosporin A in Caco-2 cells.

Transport of cyclosporin A (CsA) across Caco-2 cells is modulated by its directional efflux, mediated by a p-glycoprotein-like pump (Augustijns et al., Biochem. Biophys. Res. Comm. 197:360-365, 1994). In addition to this unidirectional flux, oxidative metabolism of CsA by cytochrome P450 is likely to influence the absorption of this cyclic peptide across intestinal mucosa. Thus, metabolism of CsA in the in vitro Caco-2 cell culture system was investigated. Formation of several metabolites was observed during the course of CsA transport across Caco-2 cell monolayers. Results from LC/MS/MS experiments revealed that the major metabolite was 1eta-hydroxy CsA (M-17), one of the three major metabolites produced by CYP3A4 present in both the liver and small intestine in humans. Preincubation of Caco-2 cell monolayers with troleandomycin, a specific inhibitor for the microsomal CYP3A protein, reduced the formation of the metabolite M-17, suggesting that an enzyme that functionally resembles CYP3A is responsible for the formation of this metabolite. However, formation of only the M-17 metabolite suggests that the isozyme present in the Caco-2 cells is distinct from CYP3A4, which also catalyzes the formation of significant quantities of the metabolites 9gamma-hydroxy cyclosporin A (M-1) and 4N-desmethyl cyclosporin A (M-21) from CsA. Interestingly, the amount of M-17 accumulating on the apical (AP) side was much greater than that on the basolateral (BL) side during the AP --> BL transport of CsA across Caco-2 cell monolayers. This is consistent with p-glycoprotein pump-mediated efflux of the metabolite to the apical side. Furthermore, formation of the M-17 metabolite on the AP side of cell monolayers during the AP --> BL transport of CsA was much greater than that during the BL --> AP transport. This result suggests that the p-glycoprotein efflux pump causes an increase in the metabolism of CsA during the course of its AP --> BL transport by effectively slowing down the transport of CsA molecules across Caco-2 cells. Thus, Caco-2 cells serve as an excellent model to dissect the relative roles played by p-glycoprotein-mediated efflux and CYP3A-catalyzed oxidation in modulating the overall absorption of CsA and other such compounds.

Anti-Bacterial Agents↗

PubMed1991

Nanoscale separations combined with tandem mass spectrometry.

High-efficiency separations of peptide mixtures, tryptic digest and other biological compounds have been achieved using nanoscale packed capillaries and capillary zone electrophoresis (CZE). The coaxial continuous-flow fast atom bombardment design is an excellent interface for coupling these separation techniques with mass spectrometry (MS). In addition, this interface is very useful for the acquisition of MS-MS data from compounds separated by nanoscale packed capillary liquid chromatography and CZE. Structurally informative daughter-ion spectra can be obtained at the low picomole to femtomole level.

Amino Acid Sequence↗

PubMed1991

Nanoscale packed-capillary liquid chromatography coupled with mass spectrometry using a coaxial continuous-flow fast atom bombardment interface.

Nanoscale packed-capillary liquid chromatography (LC) columns have been coupled with mass spectrometry (MS) using a coaxial continuous-flow fast atom bombardment interface. The combined system has been applied to the analysis of mixtures of peptides, including synthetic mixtures of bioactive peptides and tryptic digests of proteins. Nanoscale packed-capillary columns offer two principal advantages for LC/MS analysis--high chromatographic separation efficiencies and low mobile-phase flow rates. The high separation efficiencies facilitate the separation of complex mixtures, and the low mobile-phase flow rates reduce problems with coupling the LC effluent with the high-vacuum, high-voltage environment of sector MS ion sources. The columns used in this work were 50- or 75-micron i.d., 1-2 m long, packed with 10-micron C18 particles, using mobile-phase flow rates of 50-350 nL/min.

Animals↗

PubMed1991

Determination of bioactive peptides using capillary zone electrophoresis/mass spectrometry.

Mixtures of bioactive peptides have been analyzed by capillary zone electrophoresis/mass spectrometry (CZE/MS) using an on-line coaxial continuous-flow fast atom bombardment interface. High separation efficiencies (up to 410,000 theoretical plates) were obtained from low femtomole levels of peptides. The analysis of basic peptides was accomplished by using aminopropyl-silylated CZE columns to minimize zone broadening due to adsorption effects. CZE/MS/MS data were acquired from femtomole levels of peptides in electrophoretic real time.

Absorption↗

PubMed1990

Capillary zone electrophoresis-mass spectrometry using a coaxial continuous-flow fast atom bombardment interface.

Mixtures of peptides have been analyzed by capillary zone electrophoresis in conjunction with mass spectrometry (MS) using an on-line coaxial continuous-flow fast atom bombardment interface. MS and MS-MS spectra have been acquired in electrophoretic real time from femtomole levels of the peptides, while maintaining separation efficiencies in excess of 100,000 theoretical plates.

Amino Acids↗

PubMed1989

Coaxial continuous flow fast atom bombardment in conjunction with tandem mass spectrometry for the analysis of biomolecules.

The capability of interfacing coaxial continuous flow fast atom bombardment (CF-FAB) with tandem mass spectrometry (MS/MS) is demonstrated. The goal of this research is to demonstrate the ability of obtaining on-the-fly (i.e. chromatographic real time) MS/MS spectra of biomolecules and to demonstrate the feasibility of using open tubular CF-FAB as a means of introducing and maintaining a constant flux of analyte into the mass spectrometer over long periods of time. On-the-fly MS/MS spectra of a tripeptide, Met-Leu-Phe, were obtained on a 220-pg injection and a 22-pg injection. With a total acquisition time of 2 s, fragment ions resulting from common backbone cleavages were observed. With a 50 microns i.d. packed microcapillary column, the separation of a mixture was obtained and the MS/MS spectra were acquired as the analytes were eluting from the column. Through the use of the coaxial CF-FAB interface to deliver a constant flow of analyte, MS/MS spectra of a variety of compounds, including peptides, sugars, fatty acids, phospholipids, and steroids, were obtained as well as an MS/MS/MS spectrum of a tetrapeptide.

Carbohydrates↗

PubMed1989

Coupling of capillary zone electrophoresis and capillary liquid chromatography with coaxial continuous-flow fast atom bombardment tandem sector mass spectrometry.

The coaxial continuous-flow fast atom bombardment (FAB) system has proven to be very useful for interfacing capillary liquid chromatography and capillary zone electrophoresis (CZE) with sector mass spectrometry (MS). The interface can be used for the acquisition of both MS and MS-MS spectra from femtomole levels of non-volatile and/or thermally labile analytes while maintaining separation efficiencies of hundreds of thousands of plates. The use of coaxial fused-silica capillary columns to independently deliver the microcolumn effluent and the FAB matrix to the tip of the FAB probe offers the following advantages: the composition and flow-rates of the two liquid streams can be independently optimized; the FAB matrix does not effect the microcolumn separation process; peak broadening is minimized since the two liquid streams do not mix until they reach the tip of the FAB probe where ion desorption occurs; and, with CZE, active electrophoretic transport delivers the analytes directly to the FAB probe tip. These features combine to make this coaxial continuous flow fast atom bombardment interface particularly well suited for use with microcolumn separation methods.

Chromatography, Liquid↗

PubMed1989

Capillary-zone electrophoresis/fast-atom bombardment mass spectrometry: design of an on-line coaxial continuous-flow interface.

An on-line coaxial continuous-flow capillary-zone electrophoresis/fast-atom bombardment mass spectrometry (CZE/FAB-MS) interface is described. This interface is shown to be capable of acquiring mass spectra in an on-line fashion from low femtomole amounts of peptides while maintaining high (hundreds of thousands of plates) electrophoretic separation efficiencies. Active electrophoretic transport of the analytes directly to the FAB probe tip obviates the need for a transfer line from the end of the CZE capillary to this point, and thereby precludes the zone broadening that would otherwise occur both within such a transfer line and in the connections between the CZE column and the transfer line. The capability of acquiring an on-line tandem mass spectrometry (MS/MS) spectrum of an electrophoretically separated analyte using this interface is also demonstrated.

Electrophoresis↗

PubMed1985

Recent advances in the analysis of polychlorinated biphenyls in environmental and biological media.

Analytical advances in the detection, identification and quantification of polychlorinated biphenyl isomers (PCBs) are reviewed. High-resolution gas chromatography, with specific reference to capillary column development and support "phases", methodologies, detector systems and the comparative advantages and limitations of each combination, is covered. Problems associated with instrument calibration, general non-availability of primary PCB standards and the use of secondary standards are discussed. Typical applications of these newer methods to environmental, biological and process stream samples are presented.

Aroclors↗