Date Awarded


Document Type


Degree Name

Master of Science (M.Sc.)




John C Poutsma

Committee Member

Christopher J Abelt

Committee Member

Nathan Kidwell


Mass spectrometry (MS) is one of the most used techniques in proteomics because it allows for both high-throughput and quantitative analyses. Bottom-up MS-based proteomics involves breaking down proteins into smaller chains of amino acids called peptides, ionizing and fragmenting the peptides, and identifying the fragments using sequencing databases. These databases depend on the random fragmentation at the backbone peptide bond of the peptides, as predicted by the mobile proton model. Research has shown that peptides containing proline or pipecolic acid have selective fragmentations that could lead to incorrect identification in the sequencing algorithms. These selective cleavages are called “the proline effect” and “the pipecolic effect,” respectively. However, more research is needed to fully understand the mechanisms and factors that influence these effects. This study aims to understand how the identity of different amino acids adjacent to proline and pipecolic acid can enhance or inhibit these selective fragmentation effects. Tandem mass spectrometry and the extended kinetic method were used to measure the proton affinities of XxxPro and XxxPip dipeptides. In addition, computational work using hybrid density functional theory (B3LYP) and a triple zeta basis set was performed to calculate the theoretical proton affinities and provide structural information for each dipeptide. While ArgPro, ArgPip, LysPro, LysPip, and HisPip could not be quantified due to their high PAs, the remaining dipeptides had proton affinities ranging from 960.1 ± 5.3 kJ/mol for GlyPro to 1046.5 ± 6.8 kJ/mol for HisPro and 962.5 ± 6.5 kJ/mol for GlyPip to 1005.2 ± 5.7 kJ/mol for ProPip. There was general agreement between the computational and experimental results, but more work is needed to fully understand the "proline effect" and "pipecolic acid effect."



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