Mass spectrometry (MS) has been growing as one of the most widely used tools in the field of analytical chemistry. Various applications have been developed to harness the high sensitivity and specificity of mass spectrometric analysis. In this dissertation, two major challenges are addressed. By developing mass spectrometric-based methods, absolute quantitation of proteins/peptides have been achieved. Elucidation of various reaction mechanisms are also enabled. These are the focuses of this dissertation. In Chapters 2 to 4, a novel quantitation method is developed, titled as coulometric mass spectrometry (CMS). The strength of this method is that no reference standard or isotope-labeled compound is required for absolute quantitation. The method relies on electrochemical oxidation of an electrochemically active target compound to determine the amount of the oxidized compound using Faraday's Law. On the other hand, the oxidation reaction yield can be determined based on the MS signal change following electrolysis. Therefore, the absolute amount of the analyte can be calculated. In the project for quantifying the mixture of dopamine and serotonin, this method is optimized and proved to quantify the compounds in a mixture after the chromatographic separation. Gradient elution is used for separation and each compound can be quantified using the electrochemical mass spectrometry method. Furthermore, the tyrosine-containing peptides are targeted and electrochemically oxidized to generate electric current for successful quantitation by CMS method. In addition, the CMS method is further applied to absolute quantitation of proteins, as proteins can be digested into peptides. The results for surrogate peptide quantity measured by our method and by traditional isotope dilution method are in excellent agreement, with the discrepancy of 0.3-3%, validating our CMS method for absolute protein quantitation. Due to the high specificity and sensitivity of MS and no need to use isotope-labeled peptide standards, the CMS method would be of high value for the absolute proteomic quantification. In Chapter 5, elucidation of ion dissociation patterns for structural analysis is presented by using an atmospheric pressure thermal dissociation mass spectrometry (APTD-MS) technique. By using this technique, neutral CO resulting from amino acid and peptide ion dissociation is detected. In the future, more meaningful analytes can be investigated by APTD-MS to study dissociation mechanisms at the ambient environment. In Chapter 6, a gold-catalyzed oxidative coupling reaction is reported via electrochemical approach. Oxidation of Au(I) to Au(III) can be achieved through anode oxidation, which facilitates facile access to conjugated diynes via homo-coupling or cross-coupling. Besides, transient reaction intermediates are detected and confirmed by mass spectrometry which provides evidence to mechanistic studies. In Chapter 7, a novel and rapid method is developed for antibody characterization. By which, multiple reactions (e.g., reduction, digestion and deglycosylation) can take place on antibodies in microseconds in the microdroplets. The resulting antibody fragments can be either collected or online analyzed by mass spectrometry. It suggests that microdroplet environment is a powerful reactor for both exploring large molecule reactions and speeding their analysis.
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