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Pharmaceutical Discovery, May 1, 2005 
Identification of Glycosylated Peptides Using a Linear Ion Trap Mass Spectrometer
By Gargi Choudhary , Jae Schwartz , Diane Cho
High-throughput LC-based Assays for Screening of Caspase Inhibitors
May 1, 2005
By: Jeff Koehler, Courtney Coyne
Pharmaceutical Discovery

Introduction Separation-based assays performed using liquid chromatography (LC) offer several advantages when compared to assays performed using plate readers. LC-based assays reduce interference by separating quenchers and fluorescent compounds from the substrate. Also, simultaneous detection by ultraviolet (UV) absorbance and fluorescence provides additional assay information that is not available from a plate reader. While the throughput of conventional LC instrumentation limits its value for assay detection, micro parallel liquid chromatography (µPLC) increases analytical throughput by permitting analysis of 24 samples at the same time.

 


The Nanostream® (Pasadena, CA, USA) Veloce™ µPLC system — used in conjunction with 24-column Brio™ cartridges — facilitates assay development, offers quantitative information about hits and provides additional information on compound purity and solubility. The Veloce system enables scientists to incorporate separations prior to detection when performing screening studies. This application note demonstrates the utility of the Veloce system for high-throughput LC-based screening of caspase inhibitors and compares results to those obtained using a plate reader.

Experimental Conditions Equipment. Veloce µPLC system with binary gradient module, 24 UV absorbance detectors, 24 fluorescence detectors and eight-head autosampler for use with 384-well plates; Brio cartridge with 24 parallel LC columns [80 × 0.5 mm] packed with C18 (Nanostream); Analyst AD 96-384 plate reader (Molecular Devices, Sunnyvale, CA, USA) and the EnzChek Caspase-3 Assay Kit #2 (cat # E-13184, Molecular Probes, Eugene, OR, USA).

Assay conditions. Enzyme concentration: 5.7 nM; substrate concentration: 3 µM; 10 mM PIPES (pH 7.4), 2 mM EDTA, 0.1% CHAPS, 2.5 mM TCEP; 25 °C; 45 min reaction time.

Results and Discussion A screening study was performed for five inhibitors and one inhibitor with an introduced contaminant using both the Veloce µPLC system and an Analyst AD plate reader. Compared to the plate reader, the Veloce system delivered more accurate results and more information due to its ability to perform separation-based assays and the availability of simultaneous UV detection.

 

Figure 1. Percent inhibition for five inhibitors and one contaminated inhibitor, as determined using the Veloce system and a plate reader. Inset: a fluorescence chromatogram obtained using the Veloce µPLC system reveals the presence of a contaminant, thereby enabling accurate determination of the percent inhibition, compared to results from a plate reader. Analytical conditions: injection volume: 5 µL; Mobile phase A: 5:95 acetonitrile:water plus 0.1% formic acid; B: acetonitrile plus 0.085% formic acid. Separation conditions: flow rate: 12.5 µL/min (for each column); gradient: 0 to 2 min., 10% B; 2 to 10 min, 10 to 80% B; 10 to 11 min., 80% B; 11 to 14 min., 10% B. Fluorescence detection: excitation λ = 485 nm; emission λ = 530 nm.
Improved accuracy. Both systems found similar inhibitory effects for the pure inhibitors. However, when a contaminant was introduced to one sample, the plate reader results would have led the researcher to conclude an inaccurate percent inhibition (Figure 1). Using µPLC, the product peaks were isolated from the contaminant peaks, and an accurate inhibition level was determined for the pure inhibitor.

 

Figure 2. The percent inhibition and inhibitor peak area found using the Veloce system for two concentrations of inhibitor I1. The example illustrates how additional information from µPLC reduces the chance of overlooking potential drugs that may have low IC50 values.
Additional information from the assay. The Veloce system additionally offered simultaneous UV absorbance detection, which allowed for analysis of the inhibitor's UV absorbance peak area, thereby enabling determination of the actual concentration of the inhibitor present. Figure 2 depicts how false negatives could arise when the actual inhibitor concentration was lower than the expected concentration.

Conclusions The Veloce system allows for identification of hits that would be missed in cases where:

1) impurities are present with the inhibitor and/or

2) the inhibitor concentration is lower than expected due to degradation or precipitation.

By offering the ability to identify and analyze contaminated samples and to verify inhibitor concentration, the Veloce µPLC system offers improved accuracy and versatility compared to conventional screening techniques.

Nanostream Inc.
580 Sierra Madre Villa Ave.
Pasadena, CA 91107 USA
Tel. 626-351-8200; Fax 626-351-8201
www.nanostream.com