Development of a High-Throughput Dual Stream LC/MS/MS method to screen
for inhibitors of Glutamate Carboxypeptidase II (GCPII)
Abstract
RATIONALE: Glutamate carboxypeptidase II (GCPII) catalyzes the
hydrolysis of N-acetyl-aspartyl-glutamate (NAAG) to yield glutamate
(Glu) and N-acetyl-aspartate (NAA). Inhibition of GCPII has been shown
to remediate the neurotoxicity of excess glutamate in a variety of cell
and animal disease models. A robust high-throughput LC/MS/MS method was
needed to quantify GCPII enzymatic activity in a biochemical
high-throughput screening assay. METHODS: A dual-stream
LC/MS/MS method was developed. Two parallel eluent streams ran identical
HILIC gradient methods on BEH-Amide (2x30mm) columns. Each LC Channel
was run independently, the cycle time was 2 min per channel. Overall
throughput was 1-minute per sample for the dual-channel integrated
system. Multiply injected acquisition files were split during data
review, batch metadata was automatically paired with raw data during the
review process. RESULTS: Two LC sorbents, BEH-Amide and Penta-
HILIC, were tested to separate the NAAG cleavage product Glu from
isobaric interference and ion suppressants in the bioassay matrix. Early
elution of NAAG and NAA on BEH-Amide allowed interfering species to be
diverted to waste. The limit of quantification was 0.1 picomoles for
Glu. The Z-factor of this assay averaged 0.85. Over 36,000 compounds
were screened using this method. CONCLUSIONS: A fast gradient
dual-stream LC/MS/MS method for Glu quantification in GCPII biochemical
screening assay samples was developed and validated. HILIC separation
chemistry offers robust performance and unique selectivity for targeted
positive mode quantification of Glu, NAA and NAAG.