The Syntheses Of Novel Functional Two-dimensional Molybdenum Disulfide Nanomaterials And Their Applications In Exosome Glycoproteomics

Exosomes,as a type of living cells,secrete small vesicles with a particle size of 30-150 nm and contain a lot of biological information carried by their mother cells.More and more studies have shown that the generation and transfer of exosomes in vivo and organisms are closely related to the physiological processes of many diseases,so they have become an important source of disease mechanism research and biomarker discovery.At present,the research on exosomes mainly has the following aspects: first,the method of exosome separation analysis,second,the study of exosomes genomics,third,the study of exosomes proteomics.With the continuous innovation and development of biotechnology,the research on exosomes has made great progress in recent years.As a potential source of biomarkers,monitoring the state of proteins contained in diseased cells’ secreted exosomes and the dynamic process of posttranslational modification and mining of biological information are even more important.On the one hand,the discovery of biological information can enable people to further understand the occurrence and development of diseases;on the other hand,in-depth exploration of disease mechanisms can help to discover new drug targets and provide professional and reliable information for the development of new drugs.However,due to the small size of exosomes,low extraction of biological samples,low functional protein content,and low ionization efficiency of glycosylated modified peptides during mass spectrometry analysis,the identification of glycoproteins in exosomes by mass spectrometry(MS)is still It is a difficult task.Therefore,the development of convenient and efficient exosome protein / peptide separation and enrichment methods and high-throughput large-scale proteomics methods are of great significance for the study of exosome proteomics.Aiming at the problems of low abundance of glycosylated proteins in exosomes and poor ionization efficiency in MS analysis,a hydrophilic magnetic two-dimensional molybdenum disulfide nanocomposite(Mo S2-Fe3O4-Au / NWs-GSH)was prepared.In this work,a new type of magnetic hydrophilic material was prepared by connecting ultrathin two-dimensional molybdenum disulfide with Fe3O4 nanoparticles,nano-gold wires and glutathione and functionalizing them,which can be used to effectively enrich Nglycopeptides.First,magnetic Fe3O4 nanoparticles make the materials magnetic,which makes them easier to enrich,separate,and elute,this greatly simplifies the enrichment process.Secondly,the modification of ultra-fine nano-gold wires further expands the specific surface area of the nanocomposite materials,and their characteristic of easily reacting with thiol groups provides a large number of reactive sites for hydrophilic groups.Third,the highly hydrophilic glutathione can specifically retain the N-glycopeptides,which improve the hydrophilicity and enrichment capacity of the materials.The use of Mo S2-Fe3O4-Au/NWs-GSH to enrich standard protein digests with(Ig G,BSA),and femoral detection sensitivity and 1: 1000 enrichment selectivity could be achieved by MS.The materials were used in the study of human urine exosome glycoproteomics,and a total of 1,250 N-glycosylated peptides in 613 glycoproteins were identified.The materials were further used in human serum glycoproteome research,and a total of 489 Nglycopeptides of 258 glycoproteins were identified.The data sets of N-glycoproteomes show that Mo S2-Fe3O4-Au/NWs-GSH functional nanocomposites have great advantages in enriching and separating glycosylated peptides,which can effectively solve the problem of low glycoprotein abundance in exosomes or other biological samples.In addition,another nanocomposite(Mo S2/Au/4-MPB)with the surface modification of 4-mercaptophenylboronic acid was prepared based on the principle of boric acid chemistry.In this study,a simple and convenient two-step reactions between nano gold wires(Au),4-mercaptophenylboronic acid(4-MPB)and ultra-thin twodimensional molybdenum disulfide(2D-Mo S2)were successfully performed.A novel functional nanomaterial(Mo S2/Au/4-MPB)for enriching N-glycosylated peptides.Layered and nanostructured molybdenum disulfide has smaller intermolecular resistance,which can effectively reduce non-specific adsorption of functional materials and mass transfer resistance during enrichment and elution.Moreover,boronic acid groups in 4-mercaptophenylboronic acid make indiscriminate selection of N-glycopeptides.This improves the enrichment ability of the material in biological samples.The digests of human immunoglobulin G and bovine serum albumin digested with trypsin were used to evaluate the enrichment performance of N-glycosylated peptides of the new functional nanomaterials.The detection sensitivity reached 5 fmol and the selectivity reached 1: 1000.The material was further used for the enrichment of N-glycosylated peptides in biological samples.A total of 768 N-glycopeptides belonged to 377 proteins were identified from 50 μg of urine exosome proteins digested with trypsin.These results indicate that the new functional nanomaterials have a great application potential for the selective enrichment of N-glycopeptides in complex biological samples.These studies provide new methods for the functional studies of exosome glycoproteomics.