Jennifer Cunliffe - Academia.edu (original) (raw)

Papers by Jennifer Cunliffe

Research paper thumbnail of A CE assay for the detection of agonist-stimulated adenylyl cyclase activity

Electrophoresis, Jun 1, 2007

A CE assay was developed for the detection of adenylyl cyclase (AC) activity stimulated at the AC... more A CE assay was developed for the detection of adenylyl cyclase (AC) activity stimulated at the AC and G protein-coupled receptor (GPCR) level. In the assay, cell membranes overexpressing GPCR and/or AC were incubated with modulators and substrate ATP to produce cAMP in a dose-dependent manner. In both the CE-UV and a radiochemical assay, the addition of forskolin (FSK) resulted in a two-to three-fold maximum increase in AC activity with EC 50 s of 4.2 6 0.7 and 2.4 6 0.7 mM, respectively, demonstrating that similar results were obtained by both assays. GPCR activation was also detected using cell membranes overexpressing AC and the b 2 -adrenergic receptor (b 2 AR) fused to the stimulatory G protein. Terbutaline (b 2 AR agonist) increased the basal rate of cAMP formation 1.7 6 0.1-fold resulting in an EC 50 of 62 6 10 nM. The assay's ability to detect antagonists is demonstrated by the expected right-shifted EC 50 of terbutaline by the b 2 AR antagonist propranolol. The CE-UV assay offers advantages over the traditional radioactivity assay in terms of safety and labor.

Research paper thumbnail of Evaluation and comparison of very high pressure liquid chromatography systems for the separation and validation of pharmaceutical compounds

Journal of Separation Science, 2007

Evaluation and comparison of very high pressure liquid chromatography systems for the separation ... more Evaluation and comparison of very high pressure liquid chromatography systems for the separation and validation of pharmaceutical compounds Chromatography using sub-2 lm particles is becoming increasingly popular due to the potential for increased speed, resolution, sensitivity, and peak capacity. To meet the demand, various vendors have re-engineered traditional LC systems to operate at pressures of up to 15 000 psi to accommodate the elevated backpressures associated with using sub-2 lm particles. This report investigates and compares the performance of three very high pressure LC (VHPLC) systems: Waters Acquity, Agilent 1200 SL, and Thermo Accela. Specifications for the pump, autosampler, column compartment, detector, and software for each instrument are presented. To assess the chromatographic performance of the three instruments, method development and validation were performed for three pharmaceutical compounds and the results are compared and discussed. The material presented herein serves to highlight the different features of the VHPLC instruments, and assess their suitability for the analysis of pharmaceutical compounds.

Research paper thumbnail of Fused-core particle technology as an alternative to sub-2-μm particles to achieve high separation efficiency with low backpressure

Journal of Separation Science, 2007

Fused-core particle technology as an alternative to sub-2-lm particles to achieve high separation... more Fused-core particle technology as an alternative to sub-2-lm particles to achieve high separation efficiency with low backpressure Fused-Core particles have recently been introduced as an alternative to using sub-2lm particles in chromatographic separations. Fused-Core particles are composed of a 1.7 lm solid core surrounded by a 0.5 lm porous silica layer (d p = 2.7 lm) to reduce mass transfer and increase peak efficiency. The performance of two commercially available Fused-Core particles (Advanced Materials Technology Halo C18 and Supelco Ascentis Express C18) was compared with sub-2-lm particles from Waters, Agilent, and Thermo Scientific. Although the peak efficiencies were only L80% of those obtained by the Waters Acquity particles, the 50% lower backpressure allowed columns to be coupled in series to increase peak efficiency to 92 750 plates. The low backpressure and high efficiencies of the Fused-Core particles offer a viable alternative to using sub-2-lm particles and very-high-pressure LC instrumentation.

Research paper thumbnail of Using temperature to optimize resolution and reduce analysis times for bioanalytical diastereomer LC–MS/MS separations

Journal of Pharmaceutical and Biomedical Analysis, 2011

A rugged and reproducible liquid chromatographic tandem mass spectrometric bioanalytical method w... more A rugged and reproducible liquid chromatographic tandem mass spectrometric bioanalytical method was developed for the quantitation of drug stereoisomers in human plasma. Column temperature was shown to be an important variable toward optimizing diastereomer selectivity, resolution and analysis cycle time. Non-linear Van't Hoff plots and changes in peak shape with temperature suggested that selectivity was governed by multiple retention mechanisms. The high temperature chromatography method was validated and used to analyze samples from human clinical trials. Utilization of high temperature chromatography offered alternative selectivity and is a viable approach for difficult separations in regulated bioanalysis.

Research paper thumbnail of A high-throughput LC–MS/MS method for the quantitation of posaconazole in human plasma: Implementing fused core silica liquid chromatography

Journal of Pharmaceutical and Biomedical Analysis, 2009

A rapid and robust liquid chromatographic tandem mass spectrometric (LC-MS/MS) method for the det... more A rapid and robust liquid chromatographic tandem mass spectrometric (LC-MS/MS) method for the determination of posaconazole concentrations in human plasma was validated. Posaconazole was extracted from human plasma using mixed-mode cation exchange solid phase extraction in a 96-well plate format followed by gradient separation on a fused-core Halo C18 column. The analyte and its corresponding internal standard were detected using a Sciex API 4000 triple quadrupole LC-MS/MS system equipped with a TurboIonSpray ionization source operated in the positive ion mode. The calibration range of the method was 5.00-5000ng/mL using a 50microL aliquot of plasma. The assay inter-run accuracy and precision were-4.6-2.8% and 2.3-8.7%, respectively (n=18). The results from method validation indicate the method to be sensitive, selective, accurate, and reproducible. The method was successfully applied to the routine analysis of clinical samples with the fused-core silica columns providing excellent reproducibility for greater than 1000 injections per column.

Research paper thumbnail of Use of a native affinity ligand for the detection of G proteins by capillary isoelectric focusing with laser-induced fluorescence detection

ELECTROPHORESIS, 2004

Affinity probe capillary isoelectric focusing (CIEF) with laser-induced fluorescence was explored... more Affinity probe capillary isoelectric focusing (CIEF) with laser-induced fluorescence was explored for detection of Ras-like G proteins. In the assay, a fluorescent BOD-IPY  FL GTP analogue (BGTPgS) and G protein were incubated resulting in formation of BGTPgS-G protein complex. Excess BGTPgS was separated from BGTPgS-G protein complex by CIEF using a 3-10 pH gradient and detected in whole-column imaging mode. In other cases, a single point detector was used to detect zones during the focusing step of CIEF using a 2.5-5 pH gradient. In this case, analyte peaks passed the detector in , 5 min at an electric field of 350 V/cm. Detection during focusing allowed for more reproducible assays at shorter times but with a sacrifice in sensitivity compared to detection during mobilization. Resolution was adequate to separate BGTPgS-Ras and BGTPgS-Rab3A complexes. Formation of specific complexes was confirmed by adding GTPgS to samples containing BGTPgS-G protein. GTPgS competed with BGTPgS for G protein binding sites resulting in decreased BGTPgS-G protein peak heights. The concentrating effect of CIEF enabled detection limits of 30 pM.

Research paper thumbnail of A CE assay for the detection of agonist-stimulated adenylyl cyclase activity

ELECTROPHORESIS, 2007

A CE assay was developed for the detection of adenylyl cyclase (AC) activity stimulated at the AC... more A CE assay was developed for the detection of adenylyl cyclase (AC) activity stimulated at the AC and G protein-coupled receptor (GPCR) level. In the assay, cell membranes overexpressing GPCR and/or AC were incubated with modulators and substrate ATP to produce cAMP in a dose-dependent manner. In both the CE-UV and a radiochemical assay, the addition of forskolin (FSK) resulted in a two-to three-fold maximum increase in AC activity with EC 50 s of 4.2 6 0.7 and 2.4 6 0.7 mM, respectively, demonstrating that similar results were obtained by both assays. GPCR activation was also detected using cell membranes overexpressing AC and the b 2 -adrenergic receptor (b 2 AR) fused to the stimulatory G protein. Terbutaline (b 2 AR agonist) increased the basal rate of cAMP formation 1.7 6 0.1-fold resulting in an EC 50 of 62 6 10 nM. The assay's ability to detect antagonists is demonstrated by the expected right-shifted EC 50 of terbutaline by the b 2 AR antagonist propranolol. The CE-UV assay offers advantages over the traditional radioactivity assay in terms of safety and labor.

Research paper thumbnail of Fluorescence-Based Adenylyl Cyclase Assay Adaptable to High Throughput Screening

Combinatorial Chemistry & High Throughput Screening, 2007

The second messenger cAMP has been implicated in numerous cellular processes such as glycogen met... more The second messenger cAMP has been implicated in numerous cellular processes such as glycogen metabolism, muscle contraction, learning and memory, and differentiation and development. Genetic evidence suggests that the enzyme that produces cAMP, adenylyl cyclase (AC), may be involved in pathogenesis in many of these cellular processes. In addition, these data suggest that membrane-bound ACs may be valuable targets for therapeutics to treat pathogenesis of these processes. The development of a robust real-time adenylyl cyclase assay that can be scalable to high-throughput screening could help in the development of novel therapeutics. Here we report a novel fluorescence-based cyclase assay using Bodipy FL GTPgammaS (BGTPgammaS). The fluorescence of the Bodipy moiety of BGTPgammaS was dramatically enhanced by incubation with the minimal catalytic core of wild-type-AC (wt-AC) and a mutant with decreased purine selectivity (mut-AC), in an AC activation-dependent manner. No increase in fluorescence was observed using Bodipy FL ATPgammaS (BATPgammaS) as substrate for either wt-AC or mut-AC. Using BGTPgammaS, forskolin, Gsalpha.GTPgammaS and the divalent cation Mn(2+) potently enhanced the rate of fluorescence increase in a concentration-dependent manner. The fluorescence enhancement of the Bodipy moiety was inhibited by known inhibitors of AC such as 2'deoxy,3'AMP and 2',5'-dideoxy-3'ATP. Furthermore, the fluorescence assay is adaptable to 96-well and 384-well multiplate format and is thus applicable to high throughput screening methodologies.

Research paper thumbnail of Detection of Adenylyl Cyclase Activity Using a Fluorescent ATP Substrate and Capillary Electrophoresis

Analytical Chemistry, 2006

A capillary electrophoresis laser-induced fluorescence (CE-LIF) assay was developed for detection... more A capillary electrophoresis laser-induced fluorescence (CE-LIF) assay was developed for detection of adenylyl cyclase (AC) activity using BODIPY FL ATP (BATP) as substrate. In the assay, BATP was incubated with AC and the resulting mixture of BATP and enzyme product (BODIPY cyclic AMP, BcAMP) separated in 5 min by CE-LIF. Substrate depletion and product accumulation were simultaneously monitored during the course of the reaction. The rate of product formation depended upon the presence of AC activators forskolin or Galpha(s)-GTPgammaS as evidenced by a more rapid BATP turnover to BcAMP compared to basal levels. The CE-LIF assay detected EC50 values for forskolin and Galpha(s)-GTPgammaS of 27 +/- 6 microM and 317 +/- 56 nM, respectively. These EC50 values compared well to those previously reported using [alpha-32P]ATP as substrate. When AC was concurrently activated with 2.5 microM forskolin and 25 nM Galpha(s)-GTPgammaS, the amount of BcAMP formed was 3.4 times higher than the additive amounts of each activator alone indicating a positively cooperative activation by these compounds in agreement with previous assays using radiolabeled substrate. Inhibition of AC activity was also demonstrated using the AC inhibitor 2'-(or-3')-O-(N-methylanthraniloyl) guanosine 5'-triphosphate with an IC50 of 9 +/- 6 nM. The use of a fluorescent substrate combined with CE separation has enabled development of a rapid and robust method for detection of AC activity that is an attractive alternative to the AC assay using radioactive nucleotide and column chromatography. In addition, the assay has potential for high-throughput screening of drugs that act at AC.

Research paper thumbnail of Phospholipid Bilayer Coatings for the Separation of Proteins in Capillary Electrophoresis

Analytical Chemistry, 2002

The double-chained, zwitterionic phospholipid 1,2-dilauroyl-sn-phosphatidylcholine (DLPC, C12) wa... more The double-chained, zwitterionic phospholipid 1,2-dilauroyl-sn-phosphatidylcholine (DLPC, C12) was investigated for its use as a wall coating for the prevention of protein adsorption in capillary electrophoresis. DLPC forms a semipermanent coating at the capillary wall, which allows excess phospholipid to be removed from the capillary prior to electrophoretic separation. A DLPC-coated capillary allowed for the separation of both cationic and anionic proteins with efficiencies as high as 1.4 million plates/m. Migration time reproducibility was less than 1.3% RSD from run to run and less than 4.0% RSD from day to day. Protein recovery was as high as 93%. Cationic and anionic proteins could be separated over a pH range of 3-10, all yielding good efficiencies (N up to 1 million plates/m). The chain length of the phospholipid affected the performance of the wall coating. The C10 analogue of DLPC (DDPC) did not form a coating on the capillary wall while the C14 analogue of DLPC (DMPC) formed a stable coating that prevented protein adsorption to the same extent as its C12 counterpart.

Research paper thumbnail of Detection of G Proteins by Affinity Probe Capillary Electrophoresis Using a Fluorescently Labeled GTP Analogue

Analytical Chemistry, 2003

An affinity probe capillary electrophoresis (APCE) assay for guanine-nucleotide-binding proteins ... more An affinity probe capillary electrophoresis (APCE) assay for guanine-nucleotide-binding proteins (G proteins) was developed using BODIPY FL GTPgammaS (BGTPgammaS), a fluorescently labeled GTP analogue, as the affinity probe. In the assay, BGTPgammaS was incubated with samples containing G proteins and the resulting mixtures of BGTPgammaS-G protein complexes and free BGTPgammaS were separated by capillary electrophoresis and detected with laser-induced fluorescence detection. Separations were completed in less than 30 s using 25 mM Tris, 192 mM glycine at pH 8.5 as the electrophoresis buffer and applying 555 V/cm over a 4-cm separation distance. BGTPgammaS-Galpha(o) peak heights increased linearly with Galpha(o) up to approximately 200 nM using a 50 nM BGTPgammaS probe. The detection limit for Galpha(o) was 2 nM, corresponding to a mass detection limit of 3 amol. The high speed of the APCE assays allowed reaction kinetics and the dissociation constant (Kd) to be determined. The on-rate and off-rate of BGTPgammaS to Galpha(o) were 0.0068 +/- 0.0004 and 0.000 23 +/- 0.000 01 s(-1), respectively. The half-life of the BGTPgammaS-Galpha(o) complex was 3060 +/- 240 s and Kd was 8.6 +/- 0.7 nM. The estimates of these parameters are in good agreement with those obtained using established techniques, indicating the suitability of this method for such measurements. Lowering the temperature of the separation improved the detection of the complex, allowing the assay to be performed on a commercial instrument with longer separation times. Additionally, the capability of the technique to detect several G proteins based on their binding to BGTPgammaS was demonstrated with assays for Galpha and Galpha(i1) and for Ras and Rab3A.

Research paper thumbnail of Detection of G Protein Coupled Receptor Mediated Adenylyl Cyclase Activity by Capillary Electrophoresis Using Fluorescently Labeled ATP

Analytical Chemistry, 2007

A capillary electrophoresis (CE) laser-induced fluorescence (LIF) assay was developed for the det... more A capillary electrophoresis (CE) laser-induced fluorescence (LIF) assay was developed for the detection of G protein coupled receptor mediated adenylyl cyclase (AC) activity using BODIPY FL ATP (BATP) as substrate. In the assay, cell membranes coexpressing the stimulatory G protein fused to the beta2 adrenergic receptor (beta2AR) and AC were incubated with BATP, the resultant mixture injected, and BATP separated from product BODIPY FL cAMP (BcAMP) by CE. AC activity was quantified by measuring the rate of BcAMP formation. beta2AR agonists isoproterenol and terbutaline increased basal AC activity with EC50s of 2.4 +/- 0.2 and 60 +/- 9 nM, respectively. The antagonist propranolol competed with terbutaline for beta2AR binding sites and expectedly right-shifted the terbutaline dose-response curve to 8 +/- 3 microM. The high sensitivity of the assay was demonstrated by detection of small changes in AC activity, with the partial agonist alprenolol increasing (22 +/- 1%) and the inverse agonist ICI 118,551 decreasing (19 +/- 2%) basal activity. The simplicity and automation of the CE-LIF assay offers advantages over the more traditional assay using radiochemical ATP and column chromatography.

Research paper thumbnail of Implementation of high-temperature superficially porous technologies for rapid LC–MS/MS diastereomer bioanalysis

Bioanalysis, 2011

The fast-paced nature of the pharmaceutical industry requires robust bioanalytical methods to end... more The fast-paced nature of the pharmaceutical industry requires robust bioanalytical methods to endure the high-throughput sample demands of the production environment. A rapid, accurate and precise LC-MS/MS method was developed for the quantitation of a diastereomer quartet in human plasma. Virtually all of the phosphatidylcholine and most of the lysophosphatidylcholine from human plasma were removed using a phospholipid-removing protein precipitation 96-well plate. An Agilent Poroshell SB-C18 2.1 × 50 mm superficially porous column was used at 100°C and 1.2 ml/min to separate a diastereomer quartet in <2.5 min. Peak shape, retention and resolution were maintained over nearly 200 extracted bioanalytical samples under these separation conditions. The method was tested for accuracy and precision; the assay inter-run accuracy and precision were minus 7.2-0.7% and 2.1-11.9%, respectively (n = 18). The application of the superficially porous column resulted in twofold response increase and a 2.6-fold reduction in cycle time compared with a 3.5-µm column performing under comparable resolution conditions.

Research paper thumbnail of A CE assay for the detection of agonist-stimulated adenylyl cyclase activity

Electrophoresis, Jun 1, 2007

A CE assay was developed for the detection of adenylyl cyclase (AC) activity stimulated at the AC... more A CE assay was developed for the detection of adenylyl cyclase (AC) activity stimulated at the AC and G protein-coupled receptor (GPCR) level. In the assay, cell membranes overexpressing GPCR and/or AC were incubated with modulators and substrate ATP to produce cAMP in a dose-dependent manner. In both the CE-UV and a radiochemical assay, the addition of forskolin (FSK) resulted in a two-to three-fold maximum increase in AC activity with EC 50 s of 4.2 6 0.7 and 2.4 6 0.7 mM, respectively, demonstrating that similar results were obtained by both assays. GPCR activation was also detected using cell membranes overexpressing AC and the b 2 -adrenergic receptor (b 2 AR) fused to the stimulatory G protein. Terbutaline (b 2 AR agonist) increased the basal rate of cAMP formation 1.7 6 0.1-fold resulting in an EC 50 of 62 6 10 nM. The assay's ability to detect antagonists is demonstrated by the expected right-shifted EC 50 of terbutaline by the b 2 AR antagonist propranolol. The CE-UV assay offers advantages over the traditional radioactivity assay in terms of safety and labor.

Research paper thumbnail of Evaluation and comparison of very high pressure liquid chromatography systems for the separation and validation of pharmaceutical compounds

Journal of Separation Science, 2007

Evaluation and comparison of very high pressure liquid chromatography systems for the separation ... more Evaluation and comparison of very high pressure liquid chromatography systems for the separation and validation of pharmaceutical compounds Chromatography using sub-2 lm particles is becoming increasingly popular due to the potential for increased speed, resolution, sensitivity, and peak capacity. To meet the demand, various vendors have re-engineered traditional LC systems to operate at pressures of up to 15 000 psi to accommodate the elevated backpressures associated with using sub-2 lm particles. This report investigates and compares the performance of three very high pressure LC (VHPLC) systems: Waters Acquity, Agilent 1200 SL, and Thermo Accela. Specifications for the pump, autosampler, column compartment, detector, and software for each instrument are presented. To assess the chromatographic performance of the three instruments, method development and validation were performed for three pharmaceutical compounds and the results are compared and discussed. The material presented herein serves to highlight the different features of the VHPLC instruments, and assess their suitability for the analysis of pharmaceutical compounds.

Research paper thumbnail of Fused-core particle technology as an alternative to sub-2-μm particles to achieve high separation efficiency with low backpressure

Journal of Separation Science, 2007

Fused-core particle technology as an alternative to sub-2-lm particles to achieve high separation... more Fused-core particle technology as an alternative to sub-2-lm particles to achieve high separation efficiency with low backpressure Fused-Core particles have recently been introduced as an alternative to using sub-2lm particles in chromatographic separations. Fused-Core particles are composed of a 1.7 lm solid core surrounded by a 0.5 lm porous silica layer (d p = 2.7 lm) to reduce mass transfer and increase peak efficiency. The performance of two commercially available Fused-Core particles (Advanced Materials Technology Halo C18 and Supelco Ascentis Express C18) was compared with sub-2-lm particles from Waters, Agilent, and Thermo Scientific. Although the peak efficiencies were only L80% of those obtained by the Waters Acquity particles, the 50% lower backpressure allowed columns to be coupled in series to increase peak efficiency to 92 750 plates. The low backpressure and high efficiencies of the Fused-Core particles offer a viable alternative to using sub-2-lm particles and very-high-pressure LC instrumentation.

Research paper thumbnail of Using temperature to optimize resolution and reduce analysis times for bioanalytical diastereomer LC–MS/MS separations

Journal of Pharmaceutical and Biomedical Analysis, 2011

A rugged and reproducible liquid chromatographic tandem mass spectrometric bioanalytical method w... more A rugged and reproducible liquid chromatographic tandem mass spectrometric bioanalytical method was developed for the quantitation of drug stereoisomers in human plasma. Column temperature was shown to be an important variable toward optimizing diastereomer selectivity, resolution and analysis cycle time. Non-linear Van't Hoff plots and changes in peak shape with temperature suggested that selectivity was governed by multiple retention mechanisms. The high temperature chromatography method was validated and used to analyze samples from human clinical trials. Utilization of high temperature chromatography offered alternative selectivity and is a viable approach for difficult separations in regulated bioanalysis.

Research paper thumbnail of A high-throughput LC–MS/MS method for the quantitation of posaconazole in human plasma: Implementing fused core silica liquid chromatography

Journal of Pharmaceutical and Biomedical Analysis, 2009

A rapid and robust liquid chromatographic tandem mass spectrometric (LC-MS/MS) method for the det... more A rapid and robust liquid chromatographic tandem mass spectrometric (LC-MS/MS) method for the determination of posaconazole concentrations in human plasma was validated. Posaconazole was extracted from human plasma using mixed-mode cation exchange solid phase extraction in a 96-well plate format followed by gradient separation on a fused-core Halo C18 column. The analyte and its corresponding internal standard were detected using a Sciex API 4000 triple quadrupole LC-MS/MS system equipped with a TurboIonSpray ionization source operated in the positive ion mode. The calibration range of the method was 5.00-5000ng/mL using a 50microL aliquot of plasma. The assay inter-run accuracy and precision were-4.6-2.8% and 2.3-8.7%, respectively (n=18). The results from method validation indicate the method to be sensitive, selective, accurate, and reproducible. The method was successfully applied to the routine analysis of clinical samples with the fused-core silica columns providing excellent reproducibility for greater than 1000 injections per column.

Research paper thumbnail of Use of a native affinity ligand for the detection of G proteins by capillary isoelectric focusing with laser-induced fluorescence detection

ELECTROPHORESIS, 2004

Affinity probe capillary isoelectric focusing (CIEF) with laser-induced fluorescence was explored... more Affinity probe capillary isoelectric focusing (CIEF) with laser-induced fluorescence was explored for detection of Ras-like G proteins. In the assay, a fluorescent BOD-IPY  FL GTP analogue (BGTPgS) and G protein were incubated resulting in formation of BGTPgS-G protein complex. Excess BGTPgS was separated from BGTPgS-G protein complex by CIEF using a 3-10 pH gradient and detected in whole-column imaging mode. In other cases, a single point detector was used to detect zones during the focusing step of CIEF using a 2.5-5 pH gradient. In this case, analyte peaks passed the detector in , 5 min at an electric field of 350 V/cm. Detection during focusing allowed for more reproducible assays at shorter times but with a sacrifice in sensitivity compared to detection during mobilization. Resolution was adequate to separate BGTPgS-Ras and BGTPgS-Rab3A complexes. Formation of specific complexes was confirmed by adding GTPgS to samples containing BGTPgS-G protein. GTPgS competed with BGTPgS for G protein binding sites resulting in decreased BGTPgS-G protein peak heights. The concentrating effect of CIEF enabled detection limits of 30 pM.

Research paper thumbnail of A CE assay for the detection of agonist-stimulated adenylyl cyclase activity

ELECTROPHORESIS, 2007

A CE assay was developed for the detection of adenylyl cyclase (AC) activity stimulated at the AC... more A CE assay was developed for the detection of adenylyl cyclase (AC) activity stimulated at the AC and G protein-coupled receptor (GPCR) level. In the assay, cell membranes overexpressing GPCR and/or AC were incubated with modulators and substrate ATP to produce cAMP in a dose-dependent manner. In both the CE-UV and a radiochemical assay, the addition of forskolin (FSK) resulted in a two-to three-fold maximum increase in AC activity with EC 50 s of 4.2 6 0.7 and 2.4 6 0.7 mM, respectively, demonstrating that similar results were obtained by both assays. GPCR activation was also detected using cell membranes overexpressing AC and the b 2 -adrenergic receptor (b 2 AR) fused to the stimulatory G protein. Terbutaline (b 2 AR agonist) increased the basal rate of cAMP formation 1.7 6 0.1-fold resulting in an EC 50 of 62 6 10 nM. The assay's ability to detect antagonists is demonstrated by the expected right-shifted EC 50 of terbutaline by the b 2 AR antagonist propranolol. The CE-UV assay offers advantages over the traditional radioactivity assay in terms of safety and labor.

Research paper thumbnail of Fluorescence-Based Adenylyl Cyclase Assay Adaptable to High Throughput Screening

Combinatorial Chemistry & High Throughput Screening, 2007

The second messenger cAMP has been implicated in numerous cellular processes such as glycogen met... more The second messenger cAMP has been implicated in numerous cellular processes such as glycogen metabolism, muscle contraction, learning and memory, and differentiation and development. Genetic evidence suggests that the enzyme that produces cAMP, adenylyl cyclase (AC), may be involved in pathogenesis in many of these cellular processes. In addition, these data suggest that membrane-bound ACs may be valuable targets for therapeutics to treat pathogenesis of these processes. The development of a robust real-time adenylyl cyclase assay that can be scalable to high-throughput screening could help in the development of novel therapeutics. Here we report a novel fluorescence-based cyclase assay using Bodipy FL GTPgammaS (BGTPgammaS). The fluorescence of the Bodipy moiety of BGTPgammaS was dramatically enhanced by incubation with the minimal catalytic core of wild-type-AC (wt-AC) and a mutant with decreased purine selectivity (mut-AC), in an AC activation-dependent manner. No increase in fluorescence was observed using Bodipy FL ATPgammaS (BATPgammaS) as substrate for either wt-AC or mut-AC. Using BGTPgammaS, forskolin, Gsalpha.GTPgammaS and the divalent cation Mn(2+) potently enhanced the rate of fluorescence increase in a concentration-dependent manner. The fluorescence enhancement of the Bodipy moiety was inhibited by known inhibitors of AC such as 2'deoxy,3'AMP and 2',5'-dideoxy-3'ATP. Furthermore, the fluorescence assay is adaptable to 96-well and 384-well multiplate format and is thus applicable to high throughput screening methodologies.

Research paper thumbnail of Detection of Adenylyl Cyclase Activity Using a Fluorescent ATP Substrate and Capillary Electrophoresis

Analytical Chemistry, 2006

A capillary electrophoresis laser-induced fluorescence (CE-LIF) assay was developed for detection... more A capillary electrophoresis laser-induced fluorescence (CE-LIF) assay was developed for detection of adenylyl cyclase (AC) activity using BODIPY FL ATP (BATP) as substrate. In the assay, BATP was incubated with AC and the resulting mixture of BATP and enzyme product (BODIPY cyclic AMP, BcAMP) separated in 5 min by CE-LIF. Substrate depletion and product accumulation were simultaneously monitored during the course of the reaction. The rate of product formation depended upon the presence of AC activators forskolin or Galpha(s)-GTPgammaS as evidenced by a more rapid BATP turnover to BcAMP compared to basal levels. The CE-LIF assay detected EC50 values for forskolin and Galpha(s)-GTPgammaS of 27 +/- 6 microM and 317 +/- 56 nM, respectively. These EC50 values compared well to those previously reported using [alpha-32P]ATP as substrate. When AC was concurrently activated with 2.5 microM forskolin and 25 nM Galpha(s)-GTPgammaS, the amount of BcAMP formed was 3.4 times higher than the additive amounts of each activator alone indicating a positively cooperative activation by these compounds in agreement with previous assays using radiolabeled substrate. Inhibition of AC activity was also demonstrated using the AC inhibitor 2'-(or-3')-O-(N-methylanthraniloyl) guanosine 5'-triphosphate with an IC50 of 9 +/- 6 nM. The use of a fluorescent substrate combined with CE separation has enabled development of a rapid and robust method for detection of AC activity that is an attractive alternative to the AC assay using radioactive nucleotide and column chromatography. In addition, the assay has potential for high-throughput screening of drugs that act at AC.

Research paper thumbnail of Phospholipid Bilayer Coatings for the Separation of Proteins in Capillary Electrophoresis

Analytical Chemistry, 2002

The double-chained, zwitterionic phospholipid 1,2-dilauroyl-sn-phosphatidylcholine (DLPC, C12) wa... more The double-chained, zwitterionic phospholipid 1,2-dilauroyl-sn-phosphatidylcholine (DLPC, C12) was investigated for its use as a wall coating for the prevention of protein adsorption in capillary electrophoresis. DLPC forms a semipermanent coating at the capillary wall, which allows excess phospholipid to be removed from the capillary prior to electrophoretic separation. A DLPC-coated capillary allowed for the separation of both cationic and anionic proteins with efficiencies as high as 1.4 million plates/m. Migration time reproducibility was less than 1.3% RSD from run to run and less than 4.0% RSD from day to day. Protein recovery was as high as 93%. Cationic and anionic proteins could be separated over a pH range of 3-10, all yielding good efficiencies (N up to 1 million plates/m). The chain length of the phospholipid affected the performance of the wall coating. The C10 analogue of DLPC (DDPC) did not form a coating on the capillary wall while the C14 analogue of DLPC (DMPC) formed a stable coating that prevented protein adsorption to the same extent as its C12 counterpart.

Research paper thumbnail of Detection of G Proteins by Affinity Probe Capillary Electrophoresis Using a Fluorescently Labeled GTP Analogue

Analytical Chemistry, 2003

An affinity probe capillary electrophoresis (APCE) assay for guanine-nucleotide-binding proteins ... more An affinity probe capillary electrophoresis (APCE) assay for guanine-nucleotide-binding proteins (G proteins) was developed using BODIPY FL GTPgammaS (BGTPgammaS), a fluorescently labeled GTP analogue, as the affinity probe. In the assay, BGTPgammaS was incubated with samples containing G proteins and the resulting mixtures of BGTPgammaS-G protein complexes and free BGTPgammaS were separated by capillary electrophoresis and detected with laser-induced fluorescence detection. Separations were completed in less than 30 s using 25 mM Tris, 192 mM glycine at pH 8.5 as the electrophoresis buffer and applying 555 V/cm over a 4-cm separation distance. BGTPgammaS-Galpha(o) peak heights increased linearly with Galpha(o) up to approximately 200 nM using a 50 nM BGTPgammaS probe. The detection limit for Galpha(o) was 2 nM, corresponding to a mass detection limit of 3 amol. The high speed of the APCE assays allowed reaction kinetics and the dissociation constant (Kd) to be determined. The on-rate and off-rate of BGTPgammaS to Galpha(o) were 0.0068 +/- 0.0004 and 0.000 23 +/- 0.000 01 s(-1), respectively. The half-life of the BGTPgammaS-Galpha(o) complex was 3060 +/- 240 s and Kd was 8.6 +/- 0.7 nM. The estimates of these parameters are in good agreement with those obtained using established techniques, indicating the suitability of this method for such measurements. Lowering the temperature of the separation improved the detection of the complex, allowing the assay to be performed on a commercial instrument with longer separation times. Additionally, the capability of the technique to detect several G proteins based on their binding to BGTPgammaS was demonstrated with assays for Galpha and Galpha(i1) and for Ras and Rab3A.

Research paper thumbnail of Detection of G Protein Coupled Receptor Mediated Adenylyl Cyclase Activity by Capillary Electrophoresis Using Fluorescently Labeled ATP

Analytical Chemistry, 2007

A capillary electrophoresis (CE) laser-induced fluorescence (LIF) assay was developed for the det... more A capillary electrophoresis (CE) laser-induced fluorescence (LIF) assay was developed for the detection of G protein coupled receptor mediated adenylyl cyclase (AC) activity using BODIPY FL ATP (BATP) as substrate. In the assay, cell membranes coexpressing the stimulatory G protein fused to the beta2 adrenergic receptor (beta2AR) and AC were incubated with BATP, the resultant mixture injected, and BATP separated from product BODIPY FL cAMP (BcAMP) by CE. AC activity was quantified by measuring the rate of BcAMP formation. beta2AR agonists isoproterenol and terbutaline increased basal AC activity with EC50s of 2.4 +/- 0.2 and 60 +/- 9 nM, respectively. The antagonist propranolol competed with terbutaline for beta2AR binding sites and expectedly right-shifted the terbutaline dose-response curve to 8 +/- 3 microM. The high sensitivity of the assay was demonstrated by detection of small changes in AC activity, with the partial agonist alprenolol increasing (22 +/- 1%) and the inverse agonist ICI 118,551 decreasing (19 +/- 2%) basal activity. The simplicity and automation of the CE-LIF assay offers advantages over the more traditional assay using radiochemical ATP and column chromatography.

Research paper thumbnail of Implementation of high-temperature superficially porous technologies for rapid LC–MS/MS diastereomer bioanalysis

Bioanalysis, 2011

The fast-paced nature of the pharmaceutical industry requires robust bioanalytical methods to end... more The fast-paced nature of the pharmaceutical industry requires robust bioanalytical methods to endure the high-throughput sample demands of the production environment. A rapid, accurate and precise LC-MS/MS method was developed for the quantitation of a diastereomer quartet in human plasma. Virtually all of the phosphatidylcholine and most of the lysophosphatidylcholine from human plasma were removed using a phospholipid-removing protein precipitation 96-well plate. An Agilent Poroshell SB-C18 2.1 × 50 mm superficially porous column was used at 100°C and 1.2 ml/min to separate a diastereomer quartet in <2.5 min. Peak shape, retention and resolution were maintained over nearly 200 extracted bioanalytical samples under these separation conditions. The method was tested for accuracy and precision; the assay inter-run accuracy and precision were minus 7.2-0.7% and 2.1-11.9%, respectively (n = 18). The application of the superficially porous column resulted in twofold response increase and a 2.6-fold reduction in cycle time compared with a 3.5-µm column performing under comparable resolution conditions.