Mass spectrometry is a versatile analytical tool for chemical and biomolecule identification, quantitation, and structural analysis. Tandem mass spectrometry further expands the applications of mass spectrometry, making it more than a mere detector. With tandem mass spectrometry, the mass spectrometer is capable of probing reaction mechanisms, monitoring reaction processes, and performing fast analysis on complex samples. In tandem mass spectrometry, after activation the precursor ions fragment into small fragment ions through one or more pathways, which are affected by the ion’s inherit property, the ion type, and the activation method. To obtain complementary information, one can alter the fragmentation pathway by changing the ion via ion charge manipulation and covalent modification to the ion. Gas-phase ion/ion reactions provide an easy approach to changing ion type and facile modification to the analyte ions. It has been extensively used for spectrum simplification and analyte structural studies. In this dissertation, ion/ion reaction facilitated mass spectrometry methods are studied, and explorations into the method development involving front-end mass spectrometer are discussed.<br>The first work demonstrates a special rearrangement reaction for gas-phase Schiff-base-modified peptides. Gas-phase Schiff-base modification of peptides has been applied to facilitate the primary structural characterization via tandem mass spectrometry. A major or minor fragment pathway related to the novel rearrangement reaction was observed upon in-trap collisional activation of the gas-phase Schiff-base-modified peptides. The rearrangement reaction involves the imine of the Schiff base and a nucleophile present in the polypeptide. The occurrence of the rearrangement reaction is affected by several factors, such as ion polarity, identity of the nucleophile in the peptide (e.g., side chains of lysine, histidine, and arginine), and the position of the nucleophile relative to the imine. The rearrangement reaction does not affect the amount of structural information that can be obtained by collisional activation of the Schiff-base-modified peptide, but when the rearrangement reaction is dominant, it can siphon away signal from the structurally diagnostic processes.<br>Efforts have also been put into the method development of peptide and protein aggregation detection via electrospray ionization mass spectrometry (ESI-MS). People have studied peptide and protein aggregation processes to understand the mechanism of amyloid-related diseases and to control the quality of the peptide and protein pharmaceuticals. ESI-MS is suitable for solution aggregation studies because of its compatibility with solution samples and the straightforward result of the analyte’s oligomeric state on the mass spectrum. However, peak overlap issue and nonspecific aggregation in the ESI process can obscure the result. Here, the application of proton transfer ion/ion reaction to the analyte has been found useful to reduce or eliminate the peak overlap issue. A statistical model based on Poisson statistics has been proposed to deal with the ESI-induced nonspecific aggregation in the droplet and to differentiate the solution-phase aggregation from the droplet-induced aggregation. Factors that affect the accuracy of the statistical model have been discussed with MATLAB simulations.<br>In the era of biological system studies, sample complexity is a challenge every analytical chemist has to face. The analysis of complex sample can be facilitated by the combination of separation techniques outside the mass spectrometer (such as differential mobility spectrometry (DMS)) and ion structure probing techniques inside the mass spectrometer (such as tandem mass spectrometry and gas-phase ion/ion reactions). Here the coupling method between DMS and ion/ion reaction is developed and tested with model peptide systems to demonstrate its possible application in complex sample characterization such as isomer identification.<br>

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Identification of trace unknown analytes in complex samples remains a significant challenge for analytical chemistry. Mass spectrometry (MS) and analytical separations techniques can now be used to develop and support a new analytical strategy called non-target analysis which aims to provide comprehensive identification and quantification of all detectable chemical species in a complex sample. This thesis addresses challenges currently limiting the utility of this non-target approach by developing analytical methods for acquiring MS data suitable for identification of trace unknowns and investigating current tools available for unknown identification from MS spectral data. Liquid chromatography (LC) - MS, a widely used technique in trace analysis, was used to develop an analytical method capable of simultaneously acquiring high resolution MS and tandem mass spectrometry (MS/MS) data for hundreds of metabolites in urine. An emerging separation technique called high field asymmetric waveform ion mobility spectrometry (FAIMS) was also investigated, as an alternative to LC, for the identification of non-target analytes in urine. Modifications were carried out to the FAIMS-MS source interface allowing for transmission of small metabolite ions from FAIMS to MS. The challenge of direct electrospray (ESI) in urine analysis using ESI-FAIMS-MS was addressed by using sample dilution and extending MS data acquisition time using FAIMS. This allowed for higher quality MS data to be acquired for low abundance urinary metabolites than was possible by LC-MS and the complete elimination of ionization suppression in dilute urine samples. Insight gained into ESI suppression in complex samples allowed for two methods of semi-quantification to be proposed for non-target analytes in complex samples without using unavailable chemical standards. To address the challenge of unknown identification, faced throughout this thesis, an integrated approach was implemented to identify metabolites based only on spectral data without the usual requirement of availability of chemical standards. This approach combined spectral libraries, literature reports on ion chemistry and de novo identification based on gas phase ion chemistry with a detailed fragmentation study on nucleic acid bases, notably protonated uracil. Together, the instrumental methods and approaches to data analysis described allowed for the identification of 110 abundant chemical species detected in urine. / Natural Sciences and Engineering Research Council of Canada, Ontario Ministry of Training, Colleges and Universities, Canadian Foundation for Innovation