{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,5]],"date-time":"2026-03-05T01:55:05Z","timestamp":1772675705058,"version":"3.50.1"},"reference-count":39,"publisher":"Wiley","issue":"S1","license":[{"start":{"date-parts":[[2024,6,12]],"date-time":"2024-06-12T00:00:00Z","timestamp":1718150400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["analyticalsciencejournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Rapid Comm Mass Spectrometry"],"published-print":{"date-parts":[[2025,5]]},"abstract":"<jats:sec>\n                    <jats:title>Rationale<\/jats:title>\n                    <jats:p>\n                      Glutamate carboxypeptidase II (GCPII) catalyzes the hydrolysis of\n                      <jats:italic>N<\/jats:italic>\n                      \u2010acetylaspartylglutamate (NAAG) to yield glutamate (Glu) and\n                      <jats:italic>N<\/jats:italic>\n                      \u2010acetylaspartate (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\u2010throughput liquid chromatography\u2013tandem mass spectrometry (LC\/MS\/MS) method was needed to quantify GCPII enzymatic activity in a biochemical high\u2010throughput screening assay.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>A dual\u2010stream LC\/MS\/MS method was developed. Two parallel eluent streams ran identical HILIC gradient methods on BEH\u2010Amide (2\u00a0\u00d7\u00a030\u2009mm) columns. Each LC channel was run independently, and the cycle time was 2\u00a0min per channel. Overall throughput was 1\u00a0min per sample for the dual\u2010channel integrated system. Multiply injected acquisition files were split during data review, and batch metadata were automatically paired with raw data during the review process.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      Two LC sorbents, BEH\u2010Amide and Penta\u2010HILIC, 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\u2010Amide allowed interfering species to be diverted to waste. The limit of quantification was 0.1\u00a0pmol for Glu. The\n                      <jats:italic>Z<\/jats:italic>\n                      \u2010factor of this assay averaged 0.85. Over 36\u2009000 compounds were screened using this method.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>A fast gradient dual\u2010stream 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.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1002\/rcm.9772","type":"journal-article","created":{"date-parts":[[2024,6,12]],"date-time":"2024-06-12T23:09:46Z","timestamp":1718233786000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Development of a high\u2010throughput dual\u2010stream liquid chromatography\u2013tandem mass spectrometry method to screen for inhibitors of glutamate carboxypeptidase II"],"prefix":"10.1002","volume":"39","author":[{"given":"Nate","family":"Hoxie","sequence":"first","affiliation":[{"name":"National Center for Advancing Translational Sciences National Institutes of Health  Rockville Maryland USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yixuan","family":"Qiu","sequence":"additional","affiliation":[{"name":"National 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Translational Sciences National Institutes of Health  Rockville Maryland USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Stephanie L.","family":"Ford\u2010Scheimer","sequence":"additional","affiliation":[{"name":"National Center for Advancing Translational Sciences National Institutes of Health  Rockville Maryland USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Robyn","family":"Wiseman","sequence":"additional","affiliation":[{"name":"Johns Hopkins Department of Pharmacology and Molecular Sciences Johns Hopkins University School of Medicine  Baltimore Maryland USA"},{"name":"Johns Hopkins Drug Discovery and Department of Neurology Johns Hopkins University School of Medicine  Baltimore Maryland USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Takashi","family":"Tsukamoto","sequence":"additional","affiliation":[{"name":"Johns Hopkins Drug Discovery and Department of Neurology Johns Hopkins University School of Medicine  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