Characterization Of Active Oligosaccharide Chains In Low Molecular Weight Heparin Using Mass Spectrometry And In Silico Sequencing

Although heparin was discovered by a medical student as early as 100 years ago,its structure as a clinical anticoagulant has not been fully elucidated so far.Heparin(and related heparan sulfate)is a linear polysaccharide composed of 1→4 glycosidically linked repeating disaccharide units consisted of hexuronic acid(L-iduronic acid or D-glucuronic acid)and D-glucosamine.L-iduronic acid may undergo 2-O-sulfation,and D-glucosamine may undergo 6-O-sulfation,3-O-sulfation,N-sulfation,or N-acetylation.Heparin is mainly used clinically in thromboembolic diseases,cardiovascular surgery,cardiac catheterization,myocardial infarction,hemodialysis and cardiopulmonary bypass.Low molecular weight heparin is a new type of anticoagulant obtained by enzymatic degradation or chemical cleavage of heparin.Compared with heparin,it has stronger antithrombotic effect,higher bioavailability and longer plasma half-life.The low molecular weight heparin obtained through β-elimination degradation is called enoxaparin sodium.Enoxaparin sodium is combined with antithrombin III(AT III)through a special pentasaccharide sequence to enhance the inhibition of coagulation factors(mainly FIIa and FXa)and achieve anticoagulation.Therefore,only the components with binding ability to AT Ⅲ,which account for 30%after a severe degradation process,have anticoagulant activity.Currently,the analysis of intact chains,basic units as well as oligosaccharides,and the qualitative as well as quantitative analysis of monosaccharide and substituent positions are limited to the overall characterization of enoxaparin sodium.Components with anticoagulant activity have not been separated,which are not conducive to revealing the structure-activity relationship of enoxaparin sodium.With the development of soft ionization technology,tandem mass spectrometry is playing an increasingly important role in the sequencing of low-molecular-weight heparin.However,the lack of tolerance of non-volatile salt ions by mass spectrometry has limited the use of strong anion exchange chromatography(SAX-HPLC)with it.The combination of offline SAX-HPLC and electrospray ionization mass spectrometry(ESI-MS-MS)requires multiple collection and desalting of a single component of the sample,which is time-consuming and laborious.Therefore,it is necessary to establish a database for simulation sequencingIn order to characterize the active components of enoxaparin sodium and solve the disadvantages of offline LC-MS sequencing,in this study we used affinity chromatography to separate the affinity components of enoxaparin sodium at different salt ion concentrations and HILIC-ESI-MS was used for the analyzing fingerprint of intact suger chains and basic building blocks.SEC was used to separate the low-polymerization affinity components.The affinity oligomerization components were subjected to enzymolysis and nitrous acid degradation to obtain the contents of disaccharides,tetrasaccharides and uronic acid.Finally database simulation was used to determine the possible sequences of low-polymerization affinity components.This study provides a new method for deep understanding of the structure-activity relationship of low-molecular-weight heparin,and has application value for improving the level of drug evaluation and ensuring drug safety.