Hoxie Nate, Qiu Yixuan, Kales Stephen C, Schneider Rick, Hu Xin, Dalal Anu, Ford-Scheimer Stephanie L, Wiseman Robyn, Tsukamoto Takashi, Wei Huijun, Slusher Barbara S, Janiszewski John S, Hall Matthew D
National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland, USA.
Johns Hopkins Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Rapid Commun Mass Spectrom. 2025 May;39 Suppl 1(Suppl 1):e9772. doi: 10.1002/rcm.9772. Epub 2024 Jun 12.
Glutamate 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.
A dual-stream LC/MS/MS method was developed. Two parallel eluent streams ran identical HILIC gradient methods on BEH-Amide (2 × 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.
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 pmol for Glu. The Z-factor of this assay averaged 0.85. Over 36 000 compounds were screened using this method.
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.
谷氨酸羧肽酶II（GCPII）催化N - 乙酰天门冬氨酰谷氨酸（NAAG）水解生成谷氨酸（Glu）和N - 乙酰天门冬氨酸（NAA）。在多种细胞和动物疾病模型中，抑制GCPII已被证明可缓解过量谷氨酸的神经毒性。在生化高通量筛选试验中，需要一种强大的高通量液相色谱 - 串联质谱（LC/MS/MS）方法来定量GCPII酶活性。
开发了一种双流LC/MS/MS方法。两条平行的洗脱液流在BEH - 酰胺（2×30 mm）柱上运行相同的亲水作用色谱（HILIC）梯度方法。每个LC通道独立运行，每个通道的循环时间为2分钟。双通道集成系统的总通量为每个样品1分钟。多次进样采集文件在数据审查期间进行拆分，并且在审查过程中批处理元数据会自动与原始数据配对。
测试了两种LC吸附剂，BEH - 酰胺和五氟苯基 - 亲水作用色谱（Penta - HILIC），以从生物测定基质中的等压干扰物和离子抑制剂中分离NAAG裂解产物Glu。NAAG和NAA在BEH - 酰胺上的早期洗脱使得干扰物质被转移到废液中。Glu的定量限为0.1皮摩尔。该试验的Z因子平均为0.85。使用该方法筛选了超过36000种化合物。
开发并验证了一种用于GCPII生化筛选试验样品中Glu定量的快速梯度双流LC/MS/MS方法。亲水作用色谱分离化学为Glu、NAA和NAAG的靶向正模式定量提供了强大的性能和独特的选择性。
