UBC Theses and Dissertations
Method development and proteomics applications of nonaqueous capillary electrophoresis – mass spectrometry Cheng, Jianhui
Capillary electrophoresis (CE) is known for its low sample consumption, high-resolution efficiency as a separation technique, while mass spectrometry (MS) provides unprecedented selectivity and sensitivity as a detector, while adding another dimension in separation. The development of a robust CE-MS interface makes it possible to combine these two technologies for the analysis of many types of compounds. However, the coupling of CE to MS reduces its usable electrolyte ingredients substantially, and many of the popular buffer systems based on phosphate or borate, the MEKC methods dependent on the use of non-volatile surfactants, have to be excluded. As a result, formic acid and acetate buffers are the most widely used background electrolytes (BGEs) in CE-MS. In this work, we used mainly organic solvent systems to expand the choices of BGEs for CE-MS. In chapters 2 and 3, we presented two quantitative 100 % nonaqueous CE-MS methods for the separation and determination of small hydrophobic molecules. Two different BGEs were developed in this part: a basic buffer system for the analysis of negatively charged compounds and an acidic buffer system for the analysis of positively charged compounds. The compounds studied are three anthraquinones extracted from traditional Chinese medicine (TCM), Rhubarb, and six synthesized hydrophobic peptides, respectively. A high-organic-content CE-MS (HOCE) method was developed for the proteomics analysis of envelope proteins. A field amplified sample stacking technique was optimized to improve the concentration sensitivity of CE-MS for samples containing a large number of different analytes. The introduction of methanol into the buffer increased the performance of CE-MS for the detection of hydrophobic peptides in complex proteins digest. A half organic CE-MS method was used for the sequencing of novel mAbs. It was demonstrated that CE-MS/MS provided highly complementary information to LC-MS/MS with much less sample consumption. The last part of this thesis describes the application of a new generation mass spectrometry, timsTOF Pro connected with LC for the site-specific O-glycomics. In TIMS, charged compounds were separated based on their differential sizes and charges, similar to CE. The results suggest that combining CE-MS for PTMs analysis can be even more productive in the future.
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