The Synthesis Of Novel Functional Two-dimensional Molybdenum Disulfide Nanomaterials And Their Application In Proteome Research

In bottom-up proteomics strategy,the enzymatic digestion of proteins is undoubtedly one of the most crucial steps in the high-performance liquid chromatography-mass-spectrometry-based proteomics.However,the conventional protein digestion in solution is often time-consuming,incomplete digestion,enzyme autolysis and many other shortcomings,and usually hinders highly efficient,high-throughput and in-depth analysis of proteomes.In addition,phosphorylation and glycosylation of proteins are the two types of most common and complex post-translational modifications(PTMs).Unfortunately,in-depth profiling of phosphoproteomes and glycoproteome faces challenges,including the inherent low abundance and high dynamic range of phosphorylated and glycosylated proteins in complex biological samples,the low ionization efficiency of phosphopeptides or glycopeptides and serious signal suppression caused by the co-existence of abundant unmodified peptides and salts.To solve the above-mentioned problems,the specific enrichment methods developed for low-abundance post-translational modified protein/peptides with functionalized materials are the key to comprehensive and in-depth analysis of the post-translation modified proteomes.Based on all mentioned above,this paper focuses on the preparation and functionalizition of ultra-thin two-dimensional molybdenum disulfide nanomaterials and their application in proteomics research as described in following.Firstly,a novel Mo S2-based immobilized trypsin reactor was designed and prepared in this work.Pyrene-1-butyric acid was first assembled onto Mo S2 nanosheets via the strong hydrophobic stacking.And then trypsin was covalently immobilized onto the nanocomposite supports through amidation reaction.Compared with traditional in-solution digestion,higher sequence coverage(84%)and shorter time(5 min)could be achieved by the novel trypsin reactor during the digestion of BSA.The excellent performances of as-prepared trypsin reactor can be mainly attributed to the designed novel structure of the composites with high surface area resulting in high enzyme loading.In addition,strong reusability,good reproducibility and long storage of the trypsin reactor were also obtained.The novel immobilized trypsin reactor was further applied in large-scale proteomics research.The proteins extracted from He La cells and Amygdalus Pedunculata Pall.kernels were chosen to evaluate the digestion performance for the novel Mo S2-based immobilized trypsin reactor,and the experimental results showed that the number of identified proteins from complex real bio-samples with 1 h immobilized tryptic digestion was slightly more than that obtained by 12 h in-solution digestion.The above results demonstrated that the protein digestion with our novel Mo S2-based immobilized trypsin reactor is superior to the conventional protein digestion with free trypsin.Also,results of Gene Ontology analysis give an explanation for the good survival of Amygdalus Pedunculata Pall.in harsh desert environments from proteomics points of view.Therefore,the novel 2D-Mo S2-based immobilized trypsin is potentially suitable for the high throughput proteome analysis.Secondly,in phosphoproteomics research,although a variety of materials with different affinity and enrichment selectivity have been developed for the selective enrichment of phosphoprotein/phosphopeptide in phosphoproteomics,the cumbersome and complicated preparation methods of many materials need to be further improved.In this work,we take full advantage of the large specific surface area and strong negative electronegativity of two-dimensional ultra-thin planar structure of molybdenum disulfide,a large amount of titanium ions are directly fixed on surface of a thin two-dimensional molybdenum disulfide nanomaterial without any additional coating and chelating ligand.The titanium(IV)ion-modified two-dimensional molybdenum disulfide nanomaterials showed high binding capacity(100 ?g mg-1),low limit of detection(5 fmol)and high selectivity(?-casein: BSA= 1: 100)for the enrichment of phosphopeptide from a ?-casein tryptic digest.In addition,the titanium(IV)ion-modified two-dimensional molybdenum disulfide nanomaterials showed good universality in phosphopeptide enrichmenttested by an enrichment experiment with a ?-casein tryptic digest.For a bio-complex sample,non-fat milk,twelve phosphopeptides were enriched and identified from its tryptic digests.In all,the titanium(IV)ion-modified two-dimensional molybdenum disulfide nanomaterials could be used as a promising tool for the in-depth and comprehensive characterization of phosphoproteomes.Finally,a novel Mo S2/Au-NP-L-cysteine nanocomposite was prepared for glycopeptide enrichment.The two-dimensional structured Mo S2 nanosheets served as a matrix that could provide a large surface area for immobilizing hydrophilic groups such as L-cysteine with low steric hindrance between the materials and the glycopeptides which endowed the novel nanomaterials with an excellent ability to capture glycopeptide.Compared to a commercial ZIC-HILIC material,the novel nanomaterials exhibited excellent enrichment performance with ultra-high selectivity and sensitivity(approximately 10 fmol),high binding capacity(120 ?g mg-1),high enrichment recovery(more than 93%),high batch-to-batch reproducibility and good universality for glycopeptide enrichment.In addition,its outstanding specificity and efficiency for glycopeptide enrichment was confirmed by the detection of glycopeptides from a human serum immunoglobulin G(Ig G)tryptic digest in quantities as low as a molar ratio(1:1250)of Ig G to bovine serum albumin.The novel nanocomposites were further used for the analysis of complex samples,and 2502 glycopeptides from 851 glycoproteins were identified in three replicate analyses of 50 ?g of proteins extracted from He La cell exosomes.