A New Method For The Analysis Of Heparan Sulfate By Electrophoresis-mass Spectrometry

Heparan sulfate(HS)is a type of linear and complex polysaccharide.It belongs to the family of glycosaminoglycans(GAGs).HS is mainly at the cell surface and in the extracellular matrix as the form of proteoglycans.HS is involved in a variety of physiological processes,such as cell growth,signal transduction,cell adhesion and differentiation,angiogenesis and viral invasion.HS consists of repeating disaccharide units of uronic acid(HexA)-(1-4)-D-glucosamine(GlcN).HexA is either gluronic acid(GlcA)or the iduronic acid(IdoA),and the HexA can be sulfated at C2 position.The GlcN residue is N-sulfated and 6-O-sulfated.The structural complexity of HS makes it more challenging to characterize.HS is polydisperse and heterogeneous,but the molecular weight distribution and the sulfation degree in different degree of polymerization of HS are not clear.There is still no effective and direct method to study these aspects.Because HS is heterogeneous macro biomolecules,it is usually depolymerized into oligosaccharides or disaccharides by enzymatical or chemical degradation,following by liquid chromatography(LC),liquid chromatography-mass spectrometry(MS),nuclear magnetic(NMR)and LC-MS analysis.Although these analytical techniques can provide rich structural informations in oligosaccharides and disaccharides level,they are not able to give the detailed structural characterization in different degree of polymerization of HS.In this work,we have established a in-gel digestion of polyacrylamide gel electrophoresis(PAGE)coupled with LC-MS to determine the molecular weight distribution of HS and provide the fine structural characterization in different molecular weight of HS.Compared with the traditional analytical techniques,this method is able to provide the detailed structural information of HS in a specific molecular weight range.And this method can also be applied to analyze the GAGs isolated from biological samples due to its high sensitivity.The in-gel digestion of PAGE has overcome the problem that GAGs PAGE can not be compatible with MS.We applied this method to characterize heparin drug,our results demonstrated that the molecular weight distribution of heparin is continuous,and the sulfation degree does not show significant difference in different molecular weight ranges.We also applied the method to characterize the heparan sulfate from mouse lungs.Compared to the heparin,the HS extracted from mouse lung presents a discontinuous molecular weight distribution,and it is divided into three sections based on the molecular weight.And the sulfation degree also show significance difference between the sections,the degree of sulfation is lower with the chain growing.This paper provides intuitive and in-depth characterization of heparin drug and HS structure,which providing a means for more comprehensive control of drug quality and ensuring drug safety.In addition,it provides a new strategy for the fine structure characterization of HS in biological samples,especially the low-aboundance samples due to its sensitivity and accuracy,and it will promote the process of study on the structure-function relationship of HS.