Quantitative HILIC-Q-TOF-MS Analysis of Glycosaminoglycans and Non-reducing End Carbohydrate Biomarkers via Glycan Reductive Isotopic Labeling

Abstract

Glycosaminoglycans (GAGs) are linear, heterogeneous polysaccharides expressed on all animal cells. Sulfated GAGs, including heparan sulfate (HS) and chondroitin/dermatan sulfate (CS/DS), are involved in numerous physiological and pathological processes; therefore, precise and robust analytical methods for their characterization are essential to correlate structure with function. In this study, we developed a method utilizing hydrophilic interaction liquid chromatography coupled with time-of-flight mass spectrometry (HILIC-Q-TOF-MS) and glycan reductive isotopic reducing end labeling (GRIL) for the quantitative compositional analysis of HS and CS/DS polysaccharides. Lyase-generated disaccharides and commercial standards were chemically tagged on the reducing end with aniline stable isotopes, thus enabling the absolute quantification of HS and CS/DS disaccharides in complex biological samples. In addition, we adapted this workflow, in conjunction with new synthetic carbohydrate standards, for the quantification of disease-specific non-reducing end (NRE) carbohydrate biomarkers that accumulate in patients with mucopolysaccharidoses (MPS), a subclass of lysosomal storage disorders. As a proof of concept, we applied this method to measure NRE biomarkers in patient-derived MPS IIIA and MPS IIID fibroblasts, as well as in cortex tissue from a murine model of MPS VII. Overall, this method demonstrates improved sensitivity compared to previous GRIL-LC/MS techniques and, importantly, avoids the use of ion-pairing reagents, which are undesirable in certain mass spectrometry instrumentation and contexts. By combining the benefits of HILIC separation with isotopic labeling, our approach offers a robust and accessible tool for the analysis of GAGs, paving the way for advancements in understanding GAG structure and function.

1

HILIC-Q-TOF-MS method development for disaccharide analysis of glycosaminoglycans. (A) Overview of the workflow for isolation, purification, and reductive isotopic labeling for HILIC-Q-TOF-MS analysis. (B) The extracted ion chromatogram (XIC) for each of a mixture of eight HS disaccharide standards. The disaccharide structure code is described in Table S1 and ref . (C) XIC for the free molecular ion for [¹³C₆] aniline-labeled HS disaccharide, D0A0, [M-H]⁻¹ (m/z = 461). (D) Sensitivity and linear range for HS species (D0A0) shown as a correlation between picomole (pmol) amount and relative mass abundance. (E) The extracted ion chromatogram (XIC) for each of a mixture of seven CS/DS disaccharide standards. (F) XIC for the free molecular ion for [¹³C₆] aniline-labeled CS/DS disaccharide, D0a0, [M-H]⁻¹ (m/z = 461). (G) Sensitivity and linear range for CS/DS species (D0a0) are shown as a correlation between the picomole amount and relative mass abundance.

2

HILIC-Q-TOF-MS analysis of commercial GAG standards. (A) XIC chromatogram for [¹²C₆] aniline-tagged heparin disaccharides resolved by HILIC-Q-TOF-MS. (B) Corresponding mass spectra for D2S6 from the standard and heparin sample tagged with [¹³C₆] or [¹²C₆] aniline, respectively. (C) LC-MS quantification of disaccharides from heparin and CHO-S HS (n = 3 biological replicates). (D) XIC chromatogram for [¹²C₆] aniline-tagged CHO-S HS disaccharides resolved by HILIC-Q-TOF-MS. (E) Corresponding mass spectra for D0A0 from the standard and CHO-S HS sample tagged with [¹³C₆] or [¹²C₆] aniline, respectively. (F) Sulfation per disaccharide for pharmaceutical heparin and CHO-S HS (n = 3 biological replicates). (G) XIC chromatogram for [¹²C₆] aniline-tagged CS-A disaccharides resolved by HILIC-Q-TOF-MS. (H) Corresponding mass spectra for D0a0 from the standard and CS-A tagged with [¹³C₆] or [¹²C₆] aniline, respectively. (I) LC-MS quantification of disaccharides from CS-A (n = 3 biological replicates).

3

HILIC-Q-TOF-MS analysis of GAGs isolated from human cells. (A) LC-MS quantification of HS disaccharides from human malignant melanoma cells (A375) and human chondrocytes (TC28a2) (n = 3 biological replicates). (B) HS sulfation per disaccharide for A375 and TC28a2 cells (n = 3). (C) LC-MS quantification of total HS in A375 and TC28a2 cells (n = 3). (D) LC-MS quantification of CS/DS disaccharides from A375 and TC28a2 cells (n = 3). (E) CS/DS sulfation per disaccharide for A375 and TC28a2 cells (n = 3). (F) LC-MS quantification of total CS/DS in A375 and TC28a2 cells (n = 3).

4

HILIC-Q-TOF-MS analysis of GAGs isolated from murine tissue and human urine. (A) LC-MS quantification of HS disaccharides from NOD-SCID mouse whole brain and liver (n = 3 biological replicates). (B) HS sulfation per disaccharide for mouse whole brain and liver (n = 3). (C) LC-MS quantification of CS/DS disaccharides from mouse whole brain and liver (n = 3). (D) CS/DS sulfation per disaccharide for mouse whole brain and liver (n = 3). (E) LC-MS quantification of HS disaccharides from human urine (n = 3). (F) HS sulfation per disaccharide for human urine (n = 3). (G) LC-MS quantification of CS/DS disaccharides from human urine (n = 3). (H) CS/DS sulfation per disaccharide for human urine (n = 3).

5

HILIC-Q-TOF-MS analysis of non-reducing end (NRE) species from MPS IIIA/IIID fibroblasts and a mouse model of MPS VII. (A) XIC chromatogram for aniline-tagged N-sulfo-glucosamine (S0) monosaccharide resolved by HILIC-Q-TOF-MS. Inset: mass spectra for [¹²C₆] (335 m/z) and [¹³C₆] (341 m/z) aniline-tagged S0 shown in panel A. (B) LC-MS quantification of total HS levels from aged MPS IIIA patient-derived fibroblasts (GM06110) versus healthy control fibroblasts. (C) Analysis of N-sulfo-glucosamine (S0) NRE species in MPS IIIA and healthy fibroblasts. (D) XIC chromatogram for aniline-tagged glucosamine-6-sulfate (H6) monosaccharide resolved by HILIC-Q-TOF-MS. Inset: mass spectrum for [¹²C₆] (335 m/z) and [¹³C₆] (341 m/z) aniline-tagged H6 shown in panel D. (E) LC-MS quantification of total HS levels from aged MPS IIID patient-derived fibroblasts (GM05093) versus healthy control fibroblasts. (F) Quantification of glucosamine-6-sulfate (H6) NRE species in MPS IIID and healthy fibroblasts. (G) XIC chromatograph for aniline-tagged G0S0 NRE disaccharide resolved by HILIC-Q-TOF-MS. Inset: mass spectra for [¹²C₆] (511 m/z) and [¹³C₆] (517 m/z) aniline-tagged G0S0 shown in panel G. Respective sodium adducts (533, 539 m/z) are also included. (H) LC-MS quantification of total HS levels from wild-type and MPS VII mice (Gusb ^–/– ). (I) Quantification of G0S0 NRE species in wild-type and MPS VII (Gusb ^–/–) mice.