New techniques for proteomics study: Instrumentation, separation, and application

Proteomics, which is the study of all the proteins expressed by a genome, requires highly efficient analytical tools such as mass spectrometric techniques and advanced analytical protein separation methods. The common challenges of modern proteomics include efficient proteome separation techniques to reduce sample complexity, good quantitation strategies for comparative proteomics, new ionization sources for sampling sample at different environments, high sensitivity and resolving power of the MS instruments, and well-understanding of MS data.; My research work is focused on developing new strategies for several of the challenges mentioned above. First, to reduce the proteome complexity, we have developed a hybrid HPLC-Gel-MS method and applied it to the protein expression profiling. The application of step-gradient fractionation in a strong anion exchange column provided excellent reproducibility. Quantitative comparisons of protein abundance were done by side-by-side vertical comparison of band intensity in one gel. This separation strategy has been applied to the BACE and BACE2 over-expressed systems.; Second, to increase the mass measurement accuracy and protein identification confidence in FTICR-MS, we have developed a new calibration method, Calibration Optimization on Fragment Ions (COFI), for fragmentation data to correct the effects of space charge in FTICR-MS. There is a post-data processing strategy, and no need to include an internal calibrant or to apply ion population control. COFI has been successfully applied to different types of LC-FTICR fragmentation data, including LC-MS/MS data and multiplexed LC-CID data.; A number of newly developed atmospheric pressure ionization methods, such as ESI, ESSI, DESI, AP-MALDI, and ELDI, have been set up. Different types of atmospheric pressure interfaces have been investigated. By using the flared inlet tube, improved ion transmission and sensitivity were demonstrated. Increased spray tip positional tolerance was also observed with the implementation of the flared inlet tube. We have developed a hybrid atmospheric pressure interface which couples an air amplifier with a flared inlet tube. A two nano-electrosprayer device has also been coupled with the hybrid interface, which can easily be used to introduce the internal calibrant. Finally, we reported our study of coupling on-target enzymatic digestion with ELDI-FTICR-MS.