Bioinformatic methods were developed for improved identification of endogenous peptides using mass spectrometry. As a framework for these methods, a database for endogenous peptides, SwePep, was created. It was designed for storing information about endogenous peptides including tandem mass spectra. SwePep can be used for identification and validation of endogenous peptides by comparing experimentally derived masses of peptides and their fragments with information in the database. To improve automatic peptide identification of neuropeptides, targeted sequence collections that better mimic the peptidomic sample was derived from the SwePep database. Three sequence collections were created: SwePep precursors, SwePep peptides, and SwePep predicted. The searches for neuropeptides performed against these three sequence collections were compared with searches performed against the entire mouse proteome, and it was observed that three times as many peptides were identified with the targeted SwePep sequence collections. Applying the targeted SwePep sequence collections to identification of previously uncharacterized peptides yielded 27 novel potentially bioactive neuropeptides. Two fragmentations studies were performed using high mass accuracy tandem mass spectra of tryptic peptides. For this purpose, two databases were created: SwedCAD and SwedECD for CID and ECD tandem mass spectra, respectively. In the first study, fragmentation pattern of peptides with missed cleaved sites was studied using SwedCAD. It was observed that peptides with two arginines positioned next to each other have the same ability to immobilize two protons as peptides with two distant arginines. In the second study, SwedECD was used for studying small neutral losses from the reduced species in ECD fragmentation. The neutral losses were characterized with regard to their specificity and sensitivity to function as reporter ions for revealing the presence of specific amino acids in the peptide sequence. The results from these two studies can be used to improve identification of both tryptic and endogenous peptides. In summary, a collection of methods was developed that greatly improved the sensitivity of mass spectrometry peptide identification.

This dissertation elucidates a greater understanding of protein folding and unfolding processes during the lifetimes of electrospray and nano-spray droplets in laser electrospray mass spectrometry (LEMS) and nano-laser electrospray mass spectrometry (nano-LEMS) measurements, respectively. The similarity in mass spectral features obtained from conventional electrospray measurements for supercharged proteins with those of LEMS measurements suggested that supercharging phenomena occurs in the electrospray droplets during the droplet desolvation process. It was observed that the laser vaporization of protein from condensed phase into the electrospray droplets containing denaturing electrospray solution and a supercharging reagent resulted in the increase in ion abundance of higher charge states in comparison with electrospray measurements. Conversely, the addition of solution additives with varying gas phase basicity in the electrospray solvent resulted in charge reduction for unfolded protein upon laser vaporization from condensed phase into the charged electrospray droplets. The extent of charge reduction and the fraction of folded protein within the electrospray droplets was found to be dependent upon both the extent of protein denaturation in the solution prior to laser vaporization and the gas phase basicity of solution additives. The ability of the LEMS technique to analyze molecules from solution with high matrix effects was established by the successful detection of protein molecules from solution with high salt concentration. Experiments with LEMS enabled the detection of a protonated protein feature as the dominating peak in the mass spectra for up to 250 mM sodium chloride while conventional electrospray resulted in predominantly salt-adducted features, with suppression of the protonated protein ions for the salt concentration of 5 mM. This dissertation also expanded upon the use of a reaction system to measure the lifetimes of laser vaporized liquid droplets coupled with electrospray and nano-spray postionization mass spectrometry. Electrospray and nanospray droplet lifetimes were measured to be 4.5±0.6 ms and 1.4±0.3 ms using LEMS and nano-LEMS measurements, respectively. Time dependent protein folding measurements using LEMS revealed intermediate states during protein folding processes which are often limited in conventional electrospray measurements where bulk solution in manipulated (change in pH) to achieve protein folding. / Chemistry