GAG-NRE Profiling by Nano-LC-MS/MS for MPS Subtypes

Title: A glycomic workflow for LC–MS/MS analysis of urine glycosaminoglycan biomarkers in mucopolysaccharidoses

Journal: Glycoconjugate Journal

Published: 2023

Background

Mucopolysaccharidoses (MPS) are inherited lysosomal storage disorders caused by enzyme deficiencies that impair glycosaminoglycan (GAG) degradation, leading to accumulation of GAGs and progressive tissue and organ damage. Accurate biomarkers are essential for diagnosing and monitoring these diseases, particularly with the emergence of advanced therapies like enzyme replacement, stem cell, and gene therapies. Traditional urinary GAG assays lack specificity, and genetic testing requires functional confirmation. This study introduces a nano-LC–MS/MS glycomic workflow based on GAG domain mapping (GAGDoMa) to identify specific urinary GAG non-reducing ends (GAG-NREs) in MPS patients. The method enhances sensitivity through 2-aminobenzamide labeling and enables subtype-specific biomarker detection in a single analysis, aiming to improve MPS diagnosis and treatment monitoring in clinical labs.

Materials & Methods

Patient Samples

Urine samples were collected from 10 patients diagnosed with various MPS types (I, II, IIIc, IVa, VI) and 8 age-matched controls at the Clinical Chemistry Laboratory, Sahlgrenska University Hospital, Sweden. MPS diagnoses were confirmed through enzymatic and genetic testing. Controls were ruled out for MPS via urinary GAG analysis. Samples were processed and stored at –20 °C following standardized protocols. Ethical approval was obtained (EPN 2021–01858); informed consent was waived.

Reagents and Enzymes

Key materials included SAX spin columns, 10 kDa centrifugal filters, and 2-aminobenzamide (2-AB). Enzymes used were chondroitinase ABC, chondroitinase B, and heparinases II and III, prepared in ammonium acetate buffers and stored at –80 °C.

Sample Preparation

Urine samples were centrifuged, pH-adjusted, and applied to SAX columns. Eluted GAGs were desalted via ultrafiltration, dried, and stored at –20 °C. GAGs were quantified using the DMB assay.

GAG Depolymerization and Labeling

GAGs were enzymatically digested and labeled with 2-AB in NaBH₃CN/DMSO/acetic acid at 60 °C. Labeled samples were diluted for LC–MS analysis.

nLC–MS/MS Analysis

Samples were analyzed using an Easy-nLC 1200 coupled to an LTQ Orbitrap Elite. Separation was performed on C18 columns with ion-pair reversed-phase chromatography. MS1 spectra were acquired at 120,000 resolution; MS2 used HCD fragmentation with stepped collision energies (60–80%).

Data Processing

Features were identified using Proteome Discoverer 2.4 and matched to a custom GAG database (Suppl. Table 2) via in-house Python scripts. Quantification was based on normalized peak intensities of internal disaccharides and NREs.

Results

Release and Analysis of HS- and CS in Urine

A nLC-MS/MS method for GAG-NRE profiling of CS/DS and HS in urine was developed, utilizing 2-AB labeling for enhanced sensitivity and resolution. GAGs from 1 mL urine samples were enriched using SAX columns and depolymerized with chondroitinase ABC and heparinases II and III. Depolymerization occurred within 3 hours, generating internal disaccharides (dp2) and GAG-NRE saccharides, which were labeled with 2-AB. The yield of internal disulfated disaccharides (dp2S2) was 91±2%. An automated algorithm for the identification and quantification of 2-AB-labeled saccharides was developed.

Fragmentation Analysis of GAG-NREs and Internal dp2 Saccharides

LC-MS/MS fragmentation revealed the structure of internal dp2 glycoforms like ΔUAGalNAc and ΔUAGlcNAc. MPS-specific GAG-NREs were identified through de novo fragmentation. For MPS I, IdoAGalNAc4S was dominant, while MPS II patients showed IdoA2SGlcNS and IdoA2SGalNAc4S. MPS IIIc patients exhibited unique GlcNH2-containing NREs, and MPS IVa had elevated GalNAc6S/GalNAc4S ratios. MPS VI showed a slight elevation of GalNAc4S, normalizing after HSCT.

Relative Quantification of GAG-NREs in Urine

GAG-NREs were detected in both MPS and control samples, with distinct biomarkers for each MPS subgroup. MPS I (n=4) exhibited elevated IdoAGlcNS, IdoAGalNAc4S, and IdoAGlcNS(6S). MPS II (n=2) showed IdoA2SGlcNS and IdoA2SGalNAc4S. MPS IIIc (n=1) had GlcNH2-containing NREs, while MPS IVa (n=2) showed altered GalNAc6S/GalNAc4S ratios. MPS VI exhibited elevated GalNAc4S, normalizing after HSCT. This method offers potential for treatment monitoring.

GAG-NREs in Urine – A Semi-Quantitative Approach

Internal disaccharides were the predominant products in urine samples, with GAG-NREs comprising 10.3±1.1% of the total saccharides in controls. Relative quantification using internal disaccharides as references confirmed that GAG-NRE/dp2S1 ratios effectively distinguished MPS subtypes. MPS IH and MPS VI biomarkers normalized post-HSCT, suggesting the method's potential for monitoring disease progression and treatment response.

LC–MS/MS of GAG-NRE disaccharide (dp2) glycoforms (Nilsson, Jonas, et al., 2023).

LC–MS/MS of GAG-NRE trisaccharide glycoforms (Nilsson, Jonas, et al., 2023).

Reference

Nilsson, Jonas, et al. "A glycomic workflow for LC–MS/MS analysis of urine glycosaminoglycan biomarkers in mucopolysaccharidoses." Glycoconjugate Journal 40.5 (2023): 523-540. https://doi.org/10.1007/s10719-023-10128-5

* For Research Use Only. Not for use in diagnostic procedures.

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