Abstract
Glycosaminoglycans (GAGs) are important biopolymers that differ in the sequence of saccharide units and in post polymerisation alterations at various positions, making these complex molecules challenging to analyse. Here we describe an approach that enables small quantities (<200 ng) of over 400 different GAGs to be analysed within a short time frame (3–4 h). Time of flight secondary ion mass spectrometry (ToF-SIMS) together with multivariate analysis is used to analyse the entire set of GAG samples. Resultant spectra are derived from the whole molecules and do not require pre-digestion. All 6 possible GAG types are successfully discriminated, both alone and in the presence of fibronectin. We also distinguish between pharmaceutical grade heparin, derived from different animal species and from different suppliers, to a sensitivity as low as 0.001 wt%. This approach is likely to be highly beneficial in the quality control of GAGs produced for therapeutic applications and for characterising GAGs within biomaterials or from in vitro cell culture.
Introduction
Glycosaminoglycans (GAGs) are polysaccharides found within cells, within the pericellular space and as a part of the extracellular matrix (ECM). GAGs regulate biological processes, such as self-renewal, differentiation, growth, inhibition, microbial invasion and defence, with their broad structural diversity and differential localisation accommodating specific interactions with hundreds of binding proteins1,2. The complexity of GAGs, including chain length (polymerisation machinery), modification (epimerisation and sulphation of the hydroxyl groups at various positions on the saccharide units) and core protein attachment is orchestrated by enzyme mediated synthesis and allows for GAGs to have greater information carrying capacity than the more commonly studied biological polymers, nucleic acids and proteins.
The five sulfated GAGs, heparin, heparan sulphate (HS), chondroitin sulphate (CS), dermatan sulphate (DS) and keratan sulphate (KS) are synthesised attached to protein cores as proteoglycans, unlike non-sulfated hyaluronan (HA) which is extruded into the pericellular space3. Heparin, in the form of a pure polysaccharide released from its core protein, is a globally used anticoagulant and antithrombotic and is currently being considered for anti-inflammatory indications such as chronic obstructive pulmonary disease4. Other GAG types are now also increasingly being applied clinically, for example, as treatments for cancer and osteoarthritis, as anti-viral therapies5 and to support wound healing6,7,8,9. The rapid and sensitive structural characterisation of GAGs is critical to maintain the standardisation and safety of these animal-derived biomolecules for medical use, as was highlighted by the contamination of heparin samples with over-sulfated CS (OSCS) that led to patient hypotension and death10. The ongoing biosecurity of heparin is a significant concern to healthcare systems around the world, necessitating continued efforts to improve heparin analysis and provide synthetic production routes.
Typically, chemical analysis of pharmaceutical GAGs is achieved using nuclear magnetic resonance (NMR) and high performance liquid chromatography (HPLC) methods11,12,13,14. Simple 1H-NMR has been shown to detect 0.1 wt% contaminating OSCS within heparin15, whilst HPLC achieved a limit of detection of 0.03 wt% for OSCS in heparin and remains the gold standard analysis technique for heparin characterisation14. However, some of these approaches require >10 mg of sample, as well as specialised equipment and expert analysis and, therefore, suffer from low throughput16. Mass spectrometry plays a leading role in GAG glycomics utilising soft ionisation techniques such as electrospray ionisation17, however, analysis of whole sulfated GAGs remains difficult18. This is particularly problematic for the characterisation of heparin as whole-molecule analysis is necessary to detect inter-species contamination of porcine-derived material used for medical applications19. If porcine sources become limited, for example as a consequence of recent outbreaks of African Swine Fever20, the relatively poor detection of non-porcine material (a limit of detection (LOD) of approximately 2 wt% for detecting a bovine contamination in porcine heparin21) is unlikely to be sufficient to protect supplies.
For biomaterial applications requiring surface analysis, X-ray photoelectron spectroscopy has been favoured due to quantitative readouts but is unable to resolve the subtle chemical difference between different GAGs22. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a promising approach for GAG analysis as spectral acquisition is rapid (≈20 s per sample) and can be applied to whole molecules without the need for purification or enzymatic digestion. ToF-SIMS has been applied to assess the modification of sugars at surfaces but has typically been limited to mono- or di-saccharides23,24,25,26,27,28. Studies of larger polysaccharides, typically heparin or HA, focussed on low mass fragments that have limited utility to discriminate between the different GAG types29,30,31,32,33,34,35.
In this study ToF-SIMS was used to analyse a microarray containing all six GAG types (analytical preparations of HS, CS, DS, KS, HA, porcine mucosal (PM) heparin, and clinical grade heparin from porcine mucosa, bovine mucosa and bovine lung). Together with principal component analysis (PCA) and partial least square (PLS) regression, this approach was used to chemically distinguish between the different GAG classes in a semi-quantitative manner, whilst notably being able to discern differences between heparin samples derived from different animal sources and different manufacturer batches. The combination of high throughput analysis with high chemical sensitivity indicated the feasibility of this method for quality control of pharmaceutical heparin, detecting possible process related impurities as well as contaminants, and for enabling the surface analysis of GAG-modified materials to facilitate the development of GAG-functional biomaterials…
