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.

Keywords: cerebrospinal fluid; dermatan sulfate; glycosaminoglycans; heparan sulfate; iduronate 2-sulfatase; liquid chromatography-tandem mass spectrometry; mucopolysaccharidosis.

Figure 1

LC-MS/MS GAGs analysis using a robotic workstation-assisted and plate-based sample preparation workflow.

Figure 2

Representative chromatograms of internal standard and disaccharide peaks from enzymatically digested CSF sample of an Ids KO mouse in a single run. (A) internal standard (I.S.) ∆4UA-2S-GlcOEt-6S, (B) D0a4, (C) D0A0, and (D) D0S0. Each species shows two epimers designated as E1 and E2. Shaded orange peaks are the E1s that were used to represent the species for quantification. Blue traces are the authentic standards in a separate run for D0a4, D0A0 and D0S0, respectively. LC-MS conditions refer to experiment section.

Figure 3

Calibration curves for D0A0, D0S0 and D0a4 based on the ratio of peak area to I.S. area using undigested human CSF as matrix. (A–C) Dynamic range 5–10,000 ng/mL, (D–F) inset for dynamic range 5–100 ng/mL. I.S. concentration 20 ng/sample. GAG concentration levels: 5, 10, 20, 100, 1000, 5000, 9000 to 10,000 ng/mL. QCH = 7500 ng/mL, QCM = 300 ng/mL, QCL = 15 ng/mL. Total QCs n = 6. Three (3) µL of standard solutions were used to mimic the mouse CSF sample preparation procedure.