This dissertation introduces the development and the applications of liquid sample desorption electrospray ionization mass spectrometry (DESI-MS), a new ambient ionization method with focus on the direct analysis of liquid samples. A variety of new applications of liquid sample DESI are discussed in details including protein conformation study, fast reaction kinetics study, and the coupling with LC for enhancing protein signal and charge. In liquid sample DESI, direct ionization of continuous-flow liquid samples was achieved without the sample drying. In this way, biological samples can be directly analyzed in their native environment buffer to preserve the original conformation. Because of this unique feature, liquid sample DESI provides a complementary interface to couple MS to other techniques, such as liquid chromatography (LC), and electrochemistry (EC).
To evaluate the performance of liquid sample DESI, a variety of samples have been tested. For example, a large protein (BSA, MW 66 kDa) was successfully analyzed compared to a traditional DESI that was reported to be limited to examine smaller proteins (less than kDa). Protein enzymatic digestion can also be directly ionized without any sample pre-treatment such as LC separation, sample desalting, or sample clean-up processes, generating good sequence coverage of 97% and 52% for ubiquitin and BSA digestion, respectively. Also, biofluids such as untreated urine can be directly analyzed, demonstrating the high salt tolerance feature of liquid sample DESI. Furthermore, the online coupling of DESI with EC was also first demonstrated by the detection of perylene radical cations due to the online electrophoresis in a home-built electrochemical cell which served as the DESI desorption surface. The development of liquid sample DESI extended the applications of traditional DESI, which would have potential applications in the biological and forensic fields.
Due to the direct sampling feature of liquid sample DESI, liquid samples in pure aqueous solutions without the addition of acid or organic solvent can be directly ionized with high sensitivity over ESI. In this way, a new approach to study protein conformation was generated. By using liquid sample DESI, native protein ions were generated from the solution using pure water or ammonia acetate buffer. The sensitivity of DESI is about 60-70 times higher than that of ESI in terms of the aqueous protein sample analysis. Also, liquid sample DESI was used to monitor protein conformations with respect to pH or temperature changes. These results show that liquid sample DESI is a potential powerful tool for the study of protein conformation.
Based on the unique characteristics of fast and direct sampling and ionizing analyte from solution phase, fast "flying" liquid jet stream containing analyte can also be analyzed by DESI spray, from small amino acids to large proteins. Therefore, the concept of microsecond time-resolved DESI was developed to study the fast reaction kinetics by ionizing the jet stream after the fast mixing of two reagents. In this work, a time resolution of 60 μs was achieved, as demonstrated in the rate constant measurement for the reaction of 2, 6-dichlorophenol indophenol (DCIP) and L -ascorbic acid (L-AA). The pseudo-first order reaction rate constant measured by DESI is in agreement with the stopped-flow kinetics data as well as the reported value. Unlike classical spectroscopic techniques that require either chromophoric substrates or labeling, MS is a general detector with high chemical specificity. Therefore, this microsecond time-resolved DESI-MS method should find wide applications in fast (bio)chemical reaction investigations.
By utilizing the direct derivatization capability reactive DESI during ionization, the analytes separated by LC can be simultaneously derivatized and ionized without introducing an additional Tee for post-column derivatization, avoiding potential peak broadening. By spraying supercharging reagent, a specific compound to supercharge proteins, in the reactive DESI, protein eluent after LC can be supercharged. Interestingly, supercharging reagent can also reduce the notorious ion signal suppression effect caused by trifluoroacetic acid (TFA) present in the LC mobile phase, providing a novel protocol for LC-MS analysis of protein mixtures. The supercharging capability and the reduction of TFA signal suppression suggest that LC-reactive DESI-MS is a valuable method for proteomics research.