Dataset Information

High-Throughput Liquid Chromatography-Tandem Mass Spectrometry Quantification of Glycosaminoglycans as Biomarkers of Mucopolysaccharidosis II.

ABSTRACT: We recently developed a blood-brain barrier (BBB)-penetrating enzyme transport vehicle (ETV) fused to the lysosomal enzyme iduronate 2-sulfatase (ETV:IDS) and demonstrated its ability to reduce glycosaminoglycan (GAG) accumulation in the brains of a mouse model of mucopolysaccharidosis (MPS) II. To accurately quantify GAGs, we developed a plate-based high-throughput enzymatic digestion assay coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to simultaneously measure heparan sulfate and dermatan sulfate derived disaccharides in tissue, cerebrospinal fluid (CSF) and individual cell populations isolated from mouse brain. The method offers ultra-high sensitivity enabling quantitation of specific GAG species in as low as 100,000 isolated neurons and a low volume of CSF. With an LOD at 3 ng/mL and LLOQs at 5-10 ng/mL, this method is at least five times more sensitive than previously reported approaches. Our analysis demonstrated that the accumulation of CSF and brain GAGs are in good correlation, supporting the potential use of CSF GAGs as a surrogate biomarker for brain GAGs. The bioanalytical method was qualified through the generation of standard curves in matrix for preclinical studies of CSF, demonstrating the feasibility of this assay for evaluating therapeutic effects of ETV:IDS in future studies and applications in a wide variety of MPS disorders.

SUBMITTER: Wang J

PROVIDER: S-EPMC7432392 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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High-Throughput Liquid Chromatography-Tandem Mass Spectrometry Quantification of Glycosaminoglycans as Biomarkers of Mucopolysaccharidosis II.

Wang Junhua J Bhalla Akhil A Ullman Julie C JC Fang Meng M Ravi Ritesh R Arguello Annie A Thomsen Elliot E Tsogtbaatar Buyankhishig B Guo Jing L JL Skuja Lukas L LL Dugas Jason C JC Davis Sonnet S SS Poda Suresh B SB Gunasekaran Kannan K Costanzo Simona S Sweeney Zachary K ZK Henry Anastasia G AG Harris Jeffrey M JM Henne Kirk R KR Astarita Giuseppe G

International journal of molecular sciences 20200730 15

We recently developed a blood-brain barrier (BBB)-penetrating enzyme transport vehicle (ETV) fused to the lysosomal enzyme iduronate 2-sulfatase (ETV:IDS) and demonstrated its ability to reduce glycosaminoglycan (GAG) accumulation in the brains of a mouse model of mucopolysaccharidosis (MPS) II. To accurately quantify GAGs, we developed a plate-based high-throughput enzymatic digestion assay coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to simultaneously measure heparan ...[more]

PMID: 32751752

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Project description:Spontaneous preterm birth (PTB) before 37 completed weeks of gestation resulting from preterm labor (PTL) is a leading contributor of perinatal morbidity and mortality. Early identification of at-risk women by reliable screening tests could alleviate this health issue; however, conventional methods such as obstetric history and clinical risk factors, uterine activity monitoring, biochemical markers, and cervical sonography for screening women at risk for PTB have proven unsuccessful in lowering the rate of PTB. Cervicovaginal fluid (CVF) might prove to be a useful, readily available biological fluid for identifying diagnostic PTB biomarkers. Human columnar epithelial endocervical-1 (End1) and vaginal (Vk2) cell secretomes were employed to generate a stable isotope labeled proteome (SILAP) standard to facilitate characterization and relative quantification of proteins present in CVF. The SILAP standard was prepared using stable isotope labeling by amino acids in cell culture (SILAC) of End1 and Vk2 through seven passages. The labeled secreted proteins from both cell lines were combined and characterized by liquid-chromatography-tandem mass spectrometry (LC-MS/MS). In total, 1211 proteins were identified in the End1-Vk2 SILAP standard, with 236 proteins being consistently identified in each of the replicates analyzed. Individual proteins were found to contain <0.5% of the endogenous unlabeled forms. Identified proteins were screened to provide a set of 15 candidates that have either previously been identified as potential PTB biomarkers or could be linked mechanistically to PTB. Stable isotope dilution LC-multiple reaction monitoring (MRM/MS) assays were then developed for conducting relative quantification of the 15 candidate biomarkers in human CVF samples from term and PTB cases. Three proteins were significantly elevated in PTB cases (desmoplakin isoform 1, stratifin, and thrombospondin 1 precursor), providing a foundation for further validation in larger patient cohorts.

Dataset Information

High-Throughput Liquid Chromatography-Tandem Mass Spectrometry Quantification of Glycosaminoglycans as Biomarkers of Mucopolysaccharidosis II.

Publications

High-Throughput Liquid Chromatography-Tandem Mass Spectrometry Quantification of Glycosaminoglycans as Biomarkers of Mucopolysaccharidosis II.

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