| Glycosaminoglycans (GAGs) have critical roles in a number of biological processes, including anticoagulation, development, angiogenesis, axonal growth and cancer progression. The characterization and identification of GAGs in the biological samples will reveal the relationship of their structure and the biological functions. In addition to their physiological and pathophysiological roles, these natural complex polysaccharides are active biological and pharmaceutical agents. Therefore several GAG-based drugs are already clinically used. In recent years, the GAGs-like sulfated polysaccharides from the marine organisms have been intensively studied for their special biological activities and the potential pharmaceutical values. The direct analysis of intact GAGs is difficult because of their relatively high molecular mass and polydispersity. As a consequence, controlled enzymatic or chemical depolymerization is often required to lower their molecular weight and simplify these polydisperse mixtures before analysis. The fundamental biological, pathological, pharmacological, and therapeutic roles of GAGs have challenged researchers to devise new processes to prepare these critical polysaccharides and novel methods to decode their fine complex structure, which is needed to establish the structure-activity relationships. A successful combination of controlled enzymatic or chemical depolymerization, separation, detection, and spectral analysis provides the critical advantage to establish the GAG structure.In the study, the hydrophilic interaction chromatography (HILIC) combined the high resolution fourier transform mass spectrometry (FT-MS) is applied for the identification and quantification of GAGs-derived oligosaccharides; On the other hand, the multiple reaction monitoring is used to analyze the trace amount of the GAGs in the microscale biological samples.Online HILIC-FTMS was applied to analyze the oligosaccharide fragments of LMWHs generated by heparin lyase Ⅱ digestion, offering a new method for the bottom-up analysis of LMWHs. Bioinformatics software Decon2LS and GlycResoft were applied in the process of the raw data generated by LC-MS. This method is so sensitive that it can detect and identify as little as 0.01%components within about 7-min chromatographic separation window. The current method is relatively high throughput, but compromised some LC resolution. Based on this method, more than 40 digested components were observed. The primary unsaturated oligosaccharides,3-O-sulfo containing tetrasaccharides,1,6-anhydro rings, saturated uronic acid at the non-reducing end and oxidized linkage region oligosaccharides coming from some chain reducing ends were used to compare three different sources of LMWHs. This bottom-up approach provides rich detailed structural analysis and quantitative information with high accuracy and reproducibility and is suitable for quality control and/or validation of a LMWH production.Based on the above method and the micro-free radical reaction for the depolymerization of GAGs developed, four different types of GAGs, N-sulfated heparosan, chondroitin sulfate A (CSA), heparin and oversulfated chondroitin sulfate (OSCS), were depolymerized and profiled by HILIC-FTMS. The results indicated the features of the free radical depolymerization and the retention mechanism of different GAG-derived oligosaccharides on the HILIC column. These strategies were more appropriately applied in the study of the GAGs, which there was no-enzyme available for, chemically modified GAGs and fucosylated chondroitin sulfate from sea cucumber. Based on the specific di-or tri-saccharides of these GAGs generated by the radical depolymerization, the potential oversulfated GAGs contaminants in the heparin were identified and quantified, including the OSCS, oversulfated heparan sulfate (OSHS) and N-sulfated oversulfated chondroitin sulfate (NSOSCS). This method confirms that OSCS was the sole contaminant in Chinese heparin contaminated in 2008 and that OSHS was not present. This method is also capable of detecting a new potential contaminant, NSOSCS, which cannot be detected by current USP assays, and has been recently reported present in heparin API produced in Asia.The composition and structure of GAGs have a direct relationship to the biological functions. Some critical illnesses are marked by degradation of the endothelial glycocalyx, a layer of glycosaminoglycans lining the vascular lumen. It was hypothesized that different pathophysiologic insults would produce characteristic patterns of released glycocalyx fragments. We collected plasma from healthy donors as well as from subjects with respiratory failure due to altered mental status (intoxication, ischemic brain injury), indirect lung injury (non-pulmonary sepsis, pancreatitis), or direct lung injury (aspiration, pneumonia). Mass spectrometry was employed to determine the quantity and sulfation patterns of circulating glycosaminoglycans. It was found that circulating heparan sulfate fragments were significantly (23-fold) elevated in patients with indirect lung injury, while circulating hyaluronic acid concentrations were elevated (32-fold) in patients with direct lung injury. Degree of sulfation of heparan disaccharides were significantly increased in patients with indirect lung injury. Chondroitin disaccharide sulfation was suppressed in all groups with respiratory failure. Plasma heparan sulfate concentrations directly correlated with intensive care unit length of stay. These findings demonstrate that circulating glycosaminoglycans are elevated in patterns characteristic of the etiology of respiratory failure and may serve as diagnostic and/or prognostic biomarkers of critical illness.Rapid and sensitive LC-MSMS method was developed for analyzing trace amount of GAGs in the micro-scale biological samples. The classical disaccharide analysis is time-consuming (1-2 weeks) and labor intensive, requiring recovery and multi-step purification, prior to the enzymatic/chemical digestion of GAGs, and their analysis. New disaccharide recovery method was established using the Chinese hamster ovary cells (CHO). CHO cells were lysed using a commercial surfactant reagent, sonicated and digested with polysaccharide lyases. The resulting disaccharides were recovered by centrifugal filtration, labeled by reductive amination with 2-aminoacridone (AMAC), and analyzed by liquid chromatography (LC)-mass spectrometry (MS). A highly sensitive MS method developed using multiple reaction monitoring (MRM) detector utilized an Agilent 1200 LC separation system coupled with a TSQ Quantum UltraTM triple quadrupole mass spectrometer. The limit of detection for each disaccharide was reduced to 0.16-1.3 pg in MRM mode compared with 0.1-1.0 ng obtained in LC-MS mode. Sample preparation time was reduced from 1-2 weeks to 1-2 days and the number of cells required was reduced to 5000-10000 cells for complete GAG characterization to as few as 500 CHO cells for the characterization of the major GAG disaccharide components. Our survey of the glycosaminoglycanomes of 20 cell lines reveal major differences in GAG types and compositions, suggesting the utility of this method in cellular glycobiology. |
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