Development Of Novel Boronate Affinity Materials And Their Coupling With Bio-Mass Spectrometry For Glycoproteomics Analysis

Protein glycosylation is one of the most important post-translational modifications of proteins.In mammalian systems,more than 50%of the total proteins are glycoproteins.Protein glycosylation plays essential roles in many biological processes,such as molecular recognition,inter-and intra-cell signaling,immune response,sperm-egg interaction,and regulation of development.Thus,glycoproteomics analysis is of great importance and has attracted more and more attentions.In recent years,as the rapid development of bio-mass spectrometry(bio-MS)techniques,glycoproteomics analysis has also achieved significant breakthroughs.However,glycoproteomics analysis is still challenging,such as complex structure of proteins caused by glycan heterogeneity,low abundance of glycoproteins in bio-samples and poor ionization efficiency of glycoproteins and glycopeptides.In recent years,the hyphenations of boronate affinity materials and bio-MS have proved to be powerful tools in glycoproteomics analysis.However,the reported hyphenations still suffered several drawbacks,including low affinity and poor selectivity.Most importantly,it is quite difficult to analyze the specific glycoproteins/glycopeptides through existing approaches.Besides,once a glycoprotein was digested in vivo,it's almost impossible to recognize them by conventional specific recognition techniques.In this paper,aiming at the present issues,we developed several novel boronate affinity materials.By combining them with bio-MS,a series hyphenation platforms for glycoproteomics analysis were successfully consturcted.First,aiming at the exiting drawbacks of conventional boronate affinity materials,an improved Wulff-type boronic acids(benzoboroxoles)functionalized monolithic column were designed and synthesised.By combining this novel boronate affinity monolithic column with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS),an off-line hyphenation was developed to analyze glycoproteins and glycopeptides in complex samples.The approach was first applied to the analysis of glycopeptides in the tryptic digest of horseradish peroxidase(HRP).Totally 22 glycopeptides were identified.To the best of our knowledge,this is the best performance among all the boronic acid-functionalized materials.We further employed this approach to the analysis of intact proteins in human saliva.Totally 6 intact glycoproteins were successfully identified.This approach exhibited several significant advantages,including excellent extraction selectivity,no need of pH adjustment,minimal sample volume required and easy manipulation.Second,aiming at the problems for unstable glycoprotein detection.By combining boronate affinity and molecular imprinting techniques,we present a new strategy,named boronate-affinity glycan-oriented surface imprinting,for producing glycans-imprinting MIPs that can specifically recognize an intact glycoprotein and its characteristic fragments.In this work,RNase B was used as the model protein to validate the method.Finally,transferrin(TRF)glycan-imprinted materials were coupled with MALDI-TOF MS to directly analyze TRF from human serum.Compared to antibodies,aptamers and lectins,glycan-imprinted MIPs are much easier to prepare,more cost-efficient,and more stable.Furthermore,target release can be carried out under gentle conditions.Because of the difficulty in purification of glycans and its poor immunogenicity,it's quite hard to get the antibody for glycoproteins.To overcome this,we developed a novel high-efficient and general method,in situ dual-enzymatic digestion assisted boronate-affinity glycopeptides-oriented surface imprinting.This new method was not only preserved the unique recognition mechanism of the original glycan-imprinted materials,but also applicable to all glycoproteins.More importantly,because of the employment of the glycans with peptide motifs as the imprinting templates,the obtained MIPs exhibit better selectivity and stronger binding affinity towards target glycoproteins and glycopeptides.Based on this novel method,HRP glycopeptides-imprinted materials were successfully prepared.And the prepared materials had specifically recognized intact HRP and its characteristic fragments glycopeptides.Finally,the human fetuin A(AHSG)glycopeptide-imprinted materials were coupled with MALDI-TOF MS to directly analyze AHSG from human serum.This general and high efficient method could prove to be promising assets for important applications such as glycoproteomics and medical diagnostics.