Functional Nanomaterials-Based Pretreatment Approaches In Proteomic Analysis

Biological mass spectrometry (MS) is considered as the main technology for protein identification and quantification in proteomic research today. In the MS-based proteomics, sample pretreatment before MS analysis is one of the most essential procedures, generating detected objects for mass spectrometer and then directly affecting the resulting data. Therefore, effective proteolysis, separation and enrichment are the precondition of high throughput, resolution and confidence of MS analysis. For a long time, optimization of sample pretreatment has been a focused issue for the proteomic researchers.In this thesis, based on the platform of nanotechnology, we synthesized a series of functional nanomaterials with various and morphology, and applied them in protein digestion and peptide enrichment for further MS analysis. Besides, with the aid of the concept of microchip, fast analysis for minute sample was eventually realized. The detail contents were described as follows:1. Size-Selective Proteolysis on Mesoporous Silica-Based Trypsin Nanoreactor for Low-MW Proteome AnalysisIn this study, the concept of size-selective proteolysis was first described by using the mesoporous silica-based trypsin nanoreactor. For analysis of complex protein sample, low-MW proteins were preferential to be digested for identification, while high-molecular weight (MW) proteins were excluded from digestion for size-exclusive effect. We employed a protein mixture consisted of Cytochrome c/lysozyme/myoglobin as low-MW protein and BSA as high-MW protein to evaluate this proteolysis model. Furthermore, we applied this trypsin reactor to human serum sample, followed by spectral counting analysis, demonstrating more low-MW proteins were digested and identified in a biological sample than in-solution digestion.2. Novel Magnetic Mesoporous Silica Packed S-Shaped Microfluidic Reactor for Online Proteolysis of Low-MW ProteomeMagnetic mesoporous silica Fe3O4-SBA-15with magnetic cover and mesoporous core was successfully synthesized, employed as enzyme carrier and located in an S-shaped microfluidic reactor. Thanks to the size-selective effect of mesoporous silica, high-MW proteins were split to waste by fractionation, whilst low-MW proteins were retained on chip to be digested. In on-chip proteolysis of model proteins and human serum, spectral count percentage of low-MW proteins was dramatically enhanced than that by conventional approach. More importantly, the microchip could be to integrated into nano-LC-MS system for online proteolysis and analysis.3. Synthesis of Fe3O4-Graphene-TiO2Ternary Composite Networks for Enhanced Capture of PhosphopeptidesFe3O4-graphene-TiO2ternary composite networks were first preprared by electrostatic assembly of Fe3O4nanoparticles and in situ synthesis of TiO2nanoparticles on graphene nanosheets. The ternary composites exhibited high selectivity and capacity in the capture of phosphopeptides, due to the enhanced contact to phosphopeptides given by graphene scaffold. Besides, ascribed to magnetic separability from Fe3O4nanoparticles, Fe304-graphene-TiO2could be conveniently separated from the sample solution, fascinating the pretreatment process. We further applied this material in a complex sample, human serum for enrichment of endogenous phosphopeptides, demonstrating its high selectivity and good anti-interference.4. TiO2nanotube arrays/PDMS hybrid microdevice for on-chip enrichment of phosphopeptidesBy means of electrochemical anodic corrosion, TiO2nanotube arrays were successfully prepared in a patterned domain on glass slide. Employing TiO2nanotube arrays domain as separation bed and patterned PDMS as cover, we constructed TiO2nanotube arrays/PDMS microdevice, and applied it in on-chip enrichment of phosphopeptides. Without any instrument, gravity driven injection was employed here for sample processing, realizing fast analysis for minute sample. This is the first time for TiO2nanotube arrays to be integrated into a microdevice for phosphopeptide separation and enrichment.