Applications Of Graphite Dots-assisted MALDI Mass Spectrometry In Small Molecular Analysis/Imaging And Enzyme Kinetics Research

Since matrix assisted laser desorption ionization mass spectrometry(MALDI-MS)appeared for the first time in 1980s,this technology has been widely used in a variety of research fields.As a soft ionization technology,it has many advantages such as high precision(0.1%-1%),rapid detection,high tolerance to environmental factors,and detection for bio-macromolecules without any fragments.These advantages boost the research progress on nucleic acids,proteins and polypeptides.However,MALDI MS is still very limited in the detection of small molecules due to the high background noise by the traditional organic matrices in the LDI process.Those organic matrices also tend to form random crystals after deposition on the MALDI plate,terribly compromising the reproducibility of the MS signals.This thesis mainly describes my research efforts in developing graphite dots(GDs)-assisted MALDI mass spectrometry for different applications.First,I developed an electrochemical corrosion method to prepare high crystalline GDs and tune the surface chemical groups subsequently.The as-prepared GDs are featured with ultra-small size(less than 10 nm),high crystallinity,excellent solubility,high chemical inertness,desirable biocompatibility,strong UV-absorption,and unique photochemical properties.I characterized the properties of GD series carefully.Then I found that GDs can serve as a great matrix for MALDI MS,due to their extremely low background noise in the range of low molecule weight(m/z<700)in a comparison to the traditional matrices.The efficiency of small molecule ionization by GDs was demonstrated to be very high,thus allowing for sensitive detection of a broad spectrum of different molecules below m/z 700.A family of oligosaccharides as bioactive components in Chinese herbal medicine were successfully detected,and then analyzed by tandem MS.Our approach was applied to study the metabolic behaviors of puerarin in mice by directly measuring the small molecular metabolites in the serum samples without additional purification procedures.The distribution of puerarin and its metabolites in the tissue slices of mouse kidney were visualized by MALDI MS imaging for the first time.In my second project,I developed a new approach to modulate the enzyme activities and investigate enzyme kinetics simultaneously using GDs.I studied how the enzyme activities of pancreatic lipase(PL)can be tuned by adjusting the surface functional groups of GDs.The results suggested that the species and percentages of the functional groups(hydroxyl or carbonyl groups)may influence the PL enzyme conformation in complexes with GDs,leading to changes in biocatalytic abilities.As a unique advantage,GDs in the enzyme reaction can also function as a versatile matrix for MALDI MS.Therefore,sensitive detection of the residual enzyme substrate,the intermediate or final product of lipolytic digest by PL enzyme was enable by GDs-asssited MALDI MS,providing a real-time,high-throughput format to collect enzyme kinetic data.My work provides a powerful new method for enzymological research in hybrid biocatalytic processes using high quality graphite dots.My thesis combines the synthesis of GDs containing tunable chemical groups,characterization of nanomaterials,development of the GDs-assisted MALDI MS technique,and several well-designed applications in detection/imaging of small molecules or enzyme kinetic research.Due to the high performance of GDs matrix in MALDI MS,our new approach has the potential to transform metabolic analysis,multiplexed tissue imaging,and enzyme kinetic studies,which shall spur further development in this interdisciplinary research area.