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Biomedical subjects

J W Jorgenson

Publications and source records attributed to J W Jorgenson.

At least 19 recordsLinked to original sources
PubMed1999

Ultrahigh-pressure reversed-phase capillary liquid chromatography: isocratic and gradient elution using columns packed with 1.0-micron particles.

Fused-silica capillaries with inner diameters of 33 microns and lengths of 25-50 cm are slurry-packed with 1.0-micron nonporous octadecylsilane-modified (C18) silica spheres. These columns are used to perform ultrahigh-pressure reversed-phase liquid chromatographic analyses in both isocratic and gradient elution modes. Mobile-phase pressures as high as 5000 bar (72,000 psi) are applied to column inlets to generate more than 200,000 theoretical plates in 6 min (k' approximately 1) for small, organic analytes. Average capacity factors of analytes are found to increase linearly with applied pressure. An electrically driven constant-flow syringe pump capable of generating mobile-phase pressures as high as 9000 bar (130,000 psi) is described. This pump is used in conjunction with an exponential dilution method for the gradient separation of peptides from a tryptic digest on a 27-cm-long capillary packed with 1.0-micron particles. A peak capacity of 300 is demonstrated for a 30-min analysis.

Chromatography, High Pressure Liquid↗
PubMed1999

Automated measurement of peak widths for the determination of peak capacity in complex chromatograms.

The peak capacity was measured for an ultrahigh-pressure gradient elution chromatogram of a fluorescently tagged tryptic digest of ovalbumin. The peak widths in the chromatogram were determined by measuring the peak height and the second derivative at the peak maximum. This approach for measuring peak widths was programmed into a computer, and the software accurately determined the general progression of peak widths by measuring 47 peaks throughout the chromatogram in under 10 s. Peak capacity was determined by taking the definite integral of the plot of reciprocal base peak width versus retention time. This calculation of peak capacity is a linear transformation with respect to separation space, so the method is more rigorously accurate than previous methods. The peak capacity for the chromatogram was calculated to be 316.

Chromatography↗
PubMed1998

Pressure-induced retention variations in reversed-phase alternate-pumping recycle chromatography.

The progressions of peak width and peak separation in reversed-phase alternate-pumping (AP) recycle chromatography are found to be inconsistent with conventional chromatographic theory. These discrepancies are explained by subtle pressure-induced variations of solute retention that become amplified by AP recycling. The presence of these retention variations is demonstrated by multiply injecting a single solute into an AP system at offset times. As the serially injected peaks are recycled, the separation time between the peaks is shown to vary significantly, indicating that the retention of the solute is dependent upon the position of the peak. A new model of chromatographic retention that appropriately accounts for this variable retention is presented. When this retention model is applied to an AP system for the binary separation of phenylalanine and a pentadeuterated phenylalanine, the model accurately describes the experimentally observed progressions of peak width and peak separation. Furthermore, the retention model predicts that the improvement of resolution in AP recycling closely matches the expectations of conventional theory, so the effectiveness of AP recycling is not significantly compromised by the variations in retention.

Chromatography, High Pressure Liquid↗
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

Separation and identification of peptides in single neurons by microcolumn liquid chromatography-matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and postsource decay analysis.

Microcolumn liquid chromatography (LC) was interfaced with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for separation and identification of peptides present in single neurons from the brain of the snail Lymnaea stagnalis. The nanoliter microcolumn LC effluent, mixed off-line with nanoliter matrix solution, was deposited onto the sample target every 60 s, producing fractions of approximately 145 nL in volume, which, upon drying, produced spots of approximately 1 mm in size. At the end of the chromatographic separation, fractions from the sample target were scanned by MALDI-TOF-MS. Identification of peptide peaks was achieved on the basis of LC elution order and mass information. Further identification based on sequence information was carried out for a native peptide fractionated by microcolumn LC from a single neuron with the postsource decay technique.

Animals↗
PubMed1998

Quantal corelease of histamine and 5-hydroxytryptamine from mast cells and the effects of prior incubation.

Corelease of histamine and 5-hydroxytryptamine from individual mast cells has been measured with fast-scan cyclic voltammetry using a carbon-fiber electrode placed next to a single cell. Release events, induced by exposure of the cells to the calcium ionophore, A23187, were resolved at the level of individual exocytotic events. Changes in the relative concentrations secreted from individual granules were observed after incubation with 5-hydroxytryptamine, histamine, and tryptophan. In contrast, an alteration in individual cell content after such incubations, analyzed with capillary chromatography, was only found after incubation with 5-hydroxytryptamine. Cells incubated with 5-hydroxytryptamine or its precursor, tryptophan, released more 5-hydroxytryptamine and less histamine per secretory event relative to controls. Coincubation of the cells with pargyline and 5-hydroxytryptamine further reduced the release of histamine. Since cell content of histamine is unchanged, the reduction in its release must be due to its displacement to a nonreleasable compartment induced by 5-hydroxytryptamine granular uptake. Incubation with histamine increased histamine secretion and, surprisingly, also increased 5-hydroxytryptamine release without changing its cell content. This result is consistent with a relaxation of the storage matrix accompanying histamine granular uptake allowing more 5-hydroxytryptamine to be released. These results demonstrate that the intragranular mode of storage as well as granular uptake of biogenic amines affects the stoichiometry of their release.

Animals↗
PubMed1997

Determination of enzyme activity in single bovine adrenal medullary cells by separation of isotopically labeled catecholamines.

A microcolumn liquid chromatography method for determining norepinephrine (NE), epinephrine (E), and phenylethanolamine N-methyltransferase (PNMT) enzyme activity in single bovine adrenal medullary cells is presented. Single cells were isolated and treated with excess deuterated substrate, D3-NE (0.05 mM) for enzyme reaction. After 6 h, the reaction was quenched and the product, D3-E, was quantified along with endogenous NE and E. Separation and detection of deuterated and protiated NE and E were achieved with microcolumns (110-125 cm long, 25 microns inner diameter) packed with 3 microns octadecylsilane-modified particles and operated with amperometric detection. Of the 33 cells reported, most cells containing predominantly E have enzyme activity while cells containing predominantly NE and cells containing a mixture of both NE and E show no enzyme activity. After incubation with 10 microM hydrocortisone, of the 17 cells reported, most cells containing predominantly E and cells containing a mixture of both NE and E have enzyme activity while cells containing predominantly NE have no enzyme activity. Detection limits for NE and E were 42 and 48 amol, respectively.

Adrenal Medulla↗
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

Two-dimensional SEC/RPLC coupled to mass spectrometry for the analysis of peptides.

A two-dimensional liquid chromatography system is described here which uses size exclusion liquid chromatography (SEC) followed by reversed phase liquid chromatography (RPLC) to separate the mixture of peptides resulting from the enzymatic digestion of a protein. A novel LC/LC interface, using two RPLC columns in parallel rather than storage loops, joins the two chromatographic dimensions. This new interface design permits the use of conventional analytical diameter HPLC columns, 7.8 mm for SEC and 4.6 mm for RPLC, making construction and maintenance of this system very easy. The reversed phase chromatography utilizes 1.5 microns diameter, nonporous C-18 modified silica particles, which produce fast and efficient analyses. Following the high-resolution two-dimensional chromatographic separation, an electrospray mass spectrometer detects the peptide fragments. The mass spectrometer scans a 2000 m/z range to identify the analytes from their molecular weights. The analyses of tryptic digests of ovalbumin and serum albumin are each described.

Chromatography, Liquid↗
PubMed1997

Comprehensive on-line LC/LC/MS of proteins.

This is a description of a comprehensive two-dimensional liquid chromatography (LC) system for the separation of protein mixtures. This system uses cation-exchange chromatography followed by reversed-phase chromatography (RPLC). The two LC systems are coupled by an eight-port valve equipped with two storage loops and under computer control. The RPLC effluent is sampled by both a UV detector and an electrospray mass spectrometer. In this way, complex mixtures of large biomolecules can be rapidly separated, desalted, and analyzed for molecular weight in less than 2 h. The system's utility is demonstrated with a mixture of standards and an Escherichia coli cell lysate.

Chromatography, Ion Exchange↗
PubMed1997

Ultrahigh-pressure reversed-phase liquid chromatography in packed capillary columns.

The use of extremely high pressures in liquid chromatography can improve the efficiency and reduce analysis time for columns packed with small particles. In this work, fused-silica capillaries with inner diameters of 30 microns are slurry packed with 1.5 microns nonporous octadecylsilane-modified silica particles. These columns are prepared in lengths up to 66 cm with packing pressures as high as 4100 bar (60,000 psi). Near the optimum flow rate, columns generate as many as 300,000 theoretical plates for lightly retained compounds (k' < 0.5) and over 200,000 plates for more retained compounds (k' approximately 2). These translate to plate heights (Hmin) as low as 2.1 microns. The pressures required to run at optimum flow rates are on the order of 1400 bar (20,000 psi). Analysis times at these pressures are on the order of 30 min (k' approximately 2) and can be reduced to less than 10 min at higher than optimum flow rates. Capacity factors are observed to increase linearly with applied pressure.

Chromatography, High Pressure Liquid↗
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↗
PubMed1997

A multidimensional approach to protein characterization.

When mass spectrometry (MS) is used to study protein primary structure, it is used in a "static" mode. That is, the information is derived from a single MS or MS-MS spectrum. Information about more complex protein structure or protein interactions can also be gained via MS. If a series of mass spectra is collected as something else in the experiment is changing, we increase the "dimensionality" of the MS data. For example, measuring mass spectra as a function of time after exposure of a protein to deuterated solvents can provide information about protein structure. Likewise, by measuring mass spectra of a protein as the concentration of a binding ligand is changed, one can infer the stoichiometry of the complex. Another important, but fundamentally different way of increasing the dimensionality of mass spectral data is by coupling the mass spectrometer to a one- or two-dimensional separation technique.

Mass Spectrometry↗
PubMed1996

Preparation and evaluation of slurry-packed liquid chromatography microcolumns with inner diameters from 12 to 33 microns.

Fused silica capillary liquid chromatography columns with inner diameters between 12 and 33 microns were slurry packed with 5 microns octadecylsilane-modified silica particles. Column efficiencies and van Deemter coefficients were compared. A linear decrease of the A term as column diameter was decreased was the most significant contributor to a lower overall plate height at the optimum velocity.

Chemical Phenomena↗
PubMed1995

Electrochemical detection of histamine and 5-hydroxytryptamine at isolated mast cells.

The electrochemical oxidation of histamine has been investigated as an analytical tool. In a physiological buffer, histamine is oxidized at carbon fiber microelectrodes at potentials close to the background in a chemically irreversible process. Cylindrical carbon fiber electrodes were used as amperometric detectors for histamine separated with a reversed-phase capillary column, and detection limits of 240 amol were achieved. Electrodes with beveled tips were used as real-time sensors by monitoring with repetitive cyclic voltammograms at a scan rate of 800 V/s with a 16.7-ms repetition rate, and detection limits of 1.4 microM were achieved. Both techniques were used to probe histamine and 5-hydroxytryptamine (5-HT) stored in rat peritoneal mast cells. The content in single cells was measured by capillary HPLC, and both substances were found in single cells. Although the analysis revealed a large cell-to-cell variation in the amount of histamine and 5-HT, the average amount was 150 and 4 fmol of histamine and 5-HT, respectively. Release of histamine and 5-HT was measured with the electrode placed 1 microm from the cell surface. Release was observed as a series of sharp concentration spikes, consistent with corelease of the two substances from individual vesicles following exocytosis.

Animals↗
PubMed1995

Rapid comprehensive two-dimensional separations of peptides via RPLC-optically gated capillary zone electrophoresis.

Coupled-column two dimensional (2D) separation systems offer potentially high peak capacity and are amenable to automation. Reversed phase liquid chromatography (RPLC) and capillary zone electrophoresis (CZE) are complementary techniques well suited for use in a 2D system. Optically gated CZE is a means of performing rapid CZE analyses. In a 2D system, these rapid CZE analyses enable more frequent sampling of the RPLC separation and thus more freedom in control of the RPLC analysis conditions. Complete 2D separations can be done in the time usually required to do the RPLC analysis alone. With the present system, a complete 2D analysis can be done in under 10 min. Because the peak capacity of a 2D method is inherently high, some of the available peak capacity can be exchanged for speed of analysis. Acceleration of the RPLC elution gradient will decrease analysis time, but RPLC resolution will suffer. However, because it is a 2D system, some of the resolution lost in the RPLC can be regained in the second dimension CZE analysis. Here, the RPLC gradient was done over only 2 min, but CZE analyses done every 2.5 s restore some of the resolution lost in the rapid RPLC gradient.

Chromatography, Liquid↗
PubMed1995

Comprehensive three-dimensional separation of peptides using size exclusion chromatography/reversed phase liquid chromatography/optically gated capillary zone electrophoresis.

Multidimensional separation methods are attractive because of their potentially high peak capacities. Coupled-column systems in particular offer the advantages of on-line detection and automation. With the development of rapid two-dimensional (2D) analysis, it becomes possible to consider three-dimensional (3D) separation systems. In such a 3D system, effluent from a slow first dimension is repetitively sampled into a rapid 2D system. In the resultant data, each sample component has been subjected to three separative displacements, and the overall peak capacity is the product of that of each of the three dimensions. This paper demonstrates a comprehensive coupled-column 3D analysis of peptides. Size exclusion chromatography (SEC) is used as the first dimension to separate sample components by molecular weight, over an analysis time of several hours. The SEC effluent is repetitively sampled on-line into a rapid 2D reversed phase liquid chromatography/capillary zone electrophoresis (CZE) system with an analysis time of 7 min. Detection of sample zones is done only after the final CZE separation, by laser-induced fluorescence detection. Analysis data from this system consist of a series of 2D "slices" of the SEC effluent, which when stacked together give the 3D separation "volume".

Chromatography, Gel↗