Spatially Resolved Micro/Nano Chromatography Analysis

The identification of proteins from tissue samples is a classical challenge in biological research.Although LC-MS/MS-based proteomics can realize deep-coverage of proteins and provide comprehensive information,it fails in retaining proteins' spatial information during the sample preparation process.In recent years,proteomics demands spatial resolution in analysis of tissue samples.Imaging mass spectrometry solves the problem to some extent,however,due to the limitation of mass spectrometry,understanding the underlying biology in the imaging data remains challenging.Therefore,there is an urgent need for the spatially resolved proteomic analysis.In this thesis,we developed polyacrylamide cryogel-based in-situ digestion technology and L-lysine silica stick-based immobilized enzyme probe technology,in order to realize spatially resolved proteomics.The thesis includes the following contents:In the chapter one,we introduced the challenges in modern proteomics and reviewed the sample preparation methods of the classic protein workflow.We also introduced some current spatially resolved proteomics analytical technologies.The purpose of this research is achieving spatially resolved proteomic analysis and realizing deep coverage of proteins.In the chapter two,we used polyacrylamide cryogel to digest proteins and sample in situ at the same time.This new technology achieved spatially resolved proteomic analysis.Experiments showed that the cryogel has a better size precision and operatability by preparing in the capillary.6%polyacrylamide cryogel has a larger pore size,which has a higher mass transfer efficiency and loading capacity and performed better in in-situ digestion.The minimum resolution of cryogel was 92 ?m,and identified 450 proteins.The spatially resolved proteomic analysis of cerebral cortex and striatum in rat are successfully achieved.In the chapter three,we developed L-lysine silica stick immobilized enzyme probe to achieve spatially resolved proteome analysis.L-lysine was used as a monomer,GPTMS and TMOS were used as co-precursors,in the preparation of hybrid monolithic columns.A trypsin reactor based on the hybrid monolith was obtained by covalent bonding of trypsin with glutaraldehyde as bridging reagent.Experiments showed that this trypsin reactor has good permeability,high crosslinking degree and good mechanical strength.This trypsin reactor obtained sequence coverage of 94.4%for bovine serum albumin,which shows great potential for analysis of complex proteins.L-lysine silica stick immobilized enzyme probe was prepared by the droplet microfluidics platforms and immobilized in the capillaries.However,due to the small pore size and limited surface as well as the steric effects,the performance of sampling and in-situ digestion is not good enough.In the chapter four,we summarized the research work and discussed the advantages and limitations,and also outlines the future work.