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

Abstract

Rationale: 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.

Methods: 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.

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 pmol 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.

FIGURE 1

GCPII reactants at physiological pH.

FIGURE 2

BEH‐Amide and Penta‐HILIC sorbent chemistries assessed in method development. [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 3

Dual‐stream plumbing diagram as shown within LeadScape™ software. The diverter valve switches the sampled stream at the mid‐point of the gradient cycle. [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 4

(A) Co‐elution of NAAG (305/148 m/z) and its collisionally induced fragment ion Glu (148/84 m/z) at 0.25 min retention time using the HILIC method described in Table 2 on BEH‐Amide. The NAAG sample was prepared as a standard in bioassay matrix (Table 1) absent of GCPII with the quench mix used as diluent. A discrete Glu peak was not present at 0.7 min demonstrating that Glu is formed post‐column and is not present at significant concentrations as a NAAG contaminant. (B) Full‐scan spectra of 1 μM NAAG standard infused at a flow rate of 0.7 mL/min in 84% acetonitrile illustrating the up‐front collision‐induced dissociation of NAAG to Glu. [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 5

IC₅₀ curves of the known GCPII inhibitor quisqualic acid taken to assess intra‐assay variation. Row D was injected through stream one of the dual‐stream method while row E was injected through stream two. The experimental IC₅₀ is in good agreement with previously reported values, and the two streams provide comparable and consistent results.

FIGURE 6

Elution profile of NAA and Glu on BEH‐Amide and Penta‐HILIC sorbents. Three step gradient profiles were tested: 12% (green trace), 16% (red trace), and 20% (blue trace) aqueous. The gradient used (Table 1) features a 0.55 min hold at low percent aqueous prior to stepping up the 55% aqueous for elution. Glu elutes much earlier and its peak shape is significantly broadened at 20% aqueous on the BEH‐Amide phase (A). The more lipophilic NAA's peak shape is broadened at low percent aqueous, higher organic content (B). These observations suggest that solute physicochemical and structural properties predict Glu and NAA retention on the BEH‐Amide phase. The stronger retention of relatively polar, but less hydrophilic, NAA compared to Glu suggests that two discrete retention mechanisms are active on Penta‐HILIC stationary phase (C, D) under these conditions. Glu's hydrophilicity allows it to interact directly with the stationary phase being retained by predominantly charge‐based polar/dipole interaction. In contrast, we hypothesize that NAA is predominantly retained by hydrogen bonding interactions with the water‐enriched sorbitol layer on Penta‐HILIC. [Color figure can be viewed at wileyonlinelibrary.com]