Preparation And Sequencing Of N-Unsubstituted Heparin Oligosaccharides Library And Their Fragmentation Pattern In LC/MS-IT-TOF Analysis

The rare N-unsubstituted glucosamine(GlcNH3+)residues in heparan sulfate(HS)have important biological and pathophysiological roles.Because of their low natural abundance,it's difficult to obtain HS with GlcNH3+ residues from organisms.The main contents of this thesis include:(1)preparation of N-unsubstitued heparin oligosaccharides library by chemically modified,(2)developing a sensitive and effective sequencing approach for N-unsubstituted heparin/HS oligosaccharide,(3)preliminary study on the fragmentation pattern of heparin oligosaccharides with GlcNH3+ residues in LC/MS-IT-TOF analysis.This thesis consists of six chapters.The first chapter focuses on the basic knowledge about heparin/HS oligosaccharides containing GlcNH3+ residue,the common characterization methods for the structural analysis of heparin/HS oligosaccharides,and the present sequencing status for heparin/HS oligosaccharides.In chapter 2,heparin oligosaccharides(dp4-dp20)were separated and collected from low molecular weight heparin by Bio-Gel P-10 chromatography.Collected heparin tetrasaccharide(dp4)and hexasaccharide(dp6)were further analyzed using SAX-HPLC,the results show that they all have three main components,named dp4A,dp4B,dp4C,dp6A,dp6B and dp6C,respectively.Disaccharide component analysis show that dp4C and dp6C are all composed of?HexA(2S)-GlcNS(6S),i.e.,highly sulfated.Dp4A and dp6A are mainly consisted of?Hex(2S)-GlcNS and ?HexA(2S)-GlcNS(6S),however,dp4B and dp6B mainly consisted of?Hex-GlcNS(6S)and ?HexA(2S)-GlcNS(6S).In chapter 3,series of heparin dp2,dp4 and dp6 containing GlcNH3+ residues were prepared by partial de-N-sulfation of highly sulfated heparin oligosaccharides,and then the partially de-N-sulfated reaction were optimized.The stuctures of the heparin oligosaccharides were characterized by size-exclusion HPLC(SE-HPLC),heparin enzymatic and reversed-phase ion-pair liquid-chromatography hybrid ion-trap/time-of-flight mass spectrometry(RPIP-LC/MS-IT-TOF),respectively.The results show that prepared heparin oligosaccharide are all high purity,which is an important guarantee for the study of the structure and function of heparin/HS oligosaccharides with ClcNH3+ residues.In chapter 4,we developed and validated a simple and sensitive method for the sequence analysis of N-unsubstituted heparin/HS oligosaccharides.This protocol involves pH 4.0 nitrous acid(HNO2)degradation,SE-HPLC,and RPIP-LC/MS-IT-TOF.We unexpectedly found that absorbance at 232 nm(normally used for specific detection of C4-C5 unsaturated oligosaccharides)was,in most cases,still sufficiently sensitive to also simultaneously detect saturated oligosaccharides during HPLC,thus simplifying the positional analysis of GIcNH3+residues.The RPIP-LC/MS-IT-TOF system can supply further structural information leading to full sequence determination of the original oligosaccharide.This new methodology has been used to separate and sequence a variety of chemically-generated,N-unsubstituted dp6 species containing between 1 to 3 GlcNH3+ residues per oligosaccharide in different positional combinations.This strategy offers possibilities for the sequencing of natural N-unsubstituted oligosaccharides from HS,and should also be applicable,with minor modification,for sequencing at N-sulfated residues using alternative pH1.5 HNO2 scission.In chapter 5,it is well known that the saturated structure in the non-reducing end(NRE)of heparin/HS oligosaccharides is lacking ultraviolet(UV)232 nm absorbance(normally used for specific detection of C4-C5 unsaturated oligosaccharides),which poses a significant challenge for structure analysis.However,we found that saturated heparin oligosaccharides were able to be detected at UV 232 nm with sufficient sensitivity in our previous research.In order to further study the UV 232 nm absorbance of the saturated structure,four saturated heparin disaccharides were successfully prepared,and then their UV 232 nm absorptions were analyzed by RPIP-LC/MS-IT-TOF with photodiode array detector.The results shown that the saturated heparin disaccharides were able to be detected with sufficient sensitivity by absorbance at UV 232 nm,which still have about 7%-40%of UV 232 nm intensity of heparin unsaturated disaccharides.It seems that the UV 232 nm intensity might be affected by the sulfate groups in the disaccharides.In addition,comparing to the common unsaturated heparin/HS disaccharides as references,the disaccharides with GlcNH3+ residues also showed less sensitivity at UV 232 nm.Finally,we simplified the sequencing method of the N-unsubstituted dp6 oligosaccharides by a simple UV detection method,combined with pH 4.0 HNO2 scission and RPIP-LC/MS-IT-TOF analysis.This strategy offers possibilities for sequencing at N-sulfated residues using alternative pH 1.5 HNO2 scissiorn.In chapter 6,heparin dp4s and dp6s possessing different numbers of GlcNH3+ residues were further analyzed by LC/MS-IT-TOF.The results of Extracted Ion Chromatogram(EIC)-MS and MS2 showed different fragmentation patterns of dp4s and dp6s with different GlcNH3+residues using the same MS parameters.This provides a foundation for further structural identification and quantification of GlcNH3+-oligosaccharides by mass spectrum analysis,which could lead to a greater understanding of the biological roles of GlcNH3+ residues in HS/heparin.