Development of a high-throughput dual-stream liquid chromatography-tandem mass spectrometry method to screen for inhibitors of glutamate carboxypeptidase II

被引:2
作者
Hoxie, Nate [1 ]
Qiu, Yixuan [1 ]
Kales, Stephen C. [1 ]
Schneider, Rick [1 ]
Hu, Xin [1 ]
Dalal, Anu [1 ]
Ford-Scheimer, Stephanie L. [1 ]
Wiseman, Robyn [2 ,3 ,4 ]
Tsukamoto, Takashi [3 ,4 ]
Wei, Huijun [3 ,4 ]
Slusher, Barbara S. [3 ,4 ]
Janiszewski, John S. [1 ]
Hall, Matthew D. [1 ]
机构
[1] Natl Ctr Adv Translat Sci, NIH, 9800 Med Ctr Dr, Rockville, MD 20850 USA
[2] Johns Hopkins Univ, Johns Hopkins Dept Pharmacol & Mol Sci, Sch Med, Baltimore, MD USA
[3] Johns Hopkins Univ, Sch Med, Johns Hopkins Drug Discovery, Baltimore, MD USA
[4] Johns Hopkins Univ, Dept Neurol, Sch Med, Baltimore, MD USA
基金
美国国家卫生研究院;
关键词
ACIDIC DIPEPTIDASE ACTIVITY; HIGH-PERFORMANCE; RAT-BRAIN; BIOANALYSIS; VALIDATION; SYSTEM; PLASMA; SILICA; LC/MS; LEVEL;
D O I
10.1002/rcm.9772
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
RationaleGlutamate carboxypeptidase II (GCPII) catalyzes the hydrolysis of N-acetylaspartylglutamate (NAAG) to yield glutamate (Glu) and N-acetylaspartate (NAA). Inhibition of GCPII has been shown to remediate the neurotoxicity of excess Glu in a variety of cell and animal disease models. A robust high-throughput liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was needed to quantify GCPII enzymatic activity in a biochemical high-throughput screening assay.MethodsA dual-stream LC/MS/MS method was developed. Two parallel eluent streams ran identical HILIC gradient methods on BEH-Amide (2 x 30 mm) columns. Each LC channel was run independently, and the cycle time was 2 min per channel. Overall throughput was 1 min per sample for the dual-channel integrated system. Multiply injected acquisition files were split during data review, and batch metadata were automatically paired with raw data during the review process.ResultsTwo 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 pmol for Glu. The Z-factor of this assay averaged 0.85. Over 36 000 compounds were screened using this method.ConclusionsA 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.
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页数:9
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