Protein and Lantibiotic Sequencing by Gas Phase Dissociation Involving Vibrational Excitation and Ion Electron Reactions.

Fourier Transform Ion Cyclotron Mass Spectrmetry;Ion-Electron Reactions;Peptide Sequencing;Lantibiotics;Chemistry;Science;Chemistry

Kalli, AnastasiaWalter, Nils G. ;

University of Michigan

关键词: Fourier Transform Ion Cyclotron Mass Spectrmetry;    Ion-Electron Reactions;    Peptide Sequencing;    Lantibiotics;    Chemistry;    Science;    Chemistry;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/62221/kalliana_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

In proteomics, protein identification and characterization are largely performed by tandem mass spectrometry (MS/MS), in which sequence specific product ions are generated from precursor peptide or protein ions.Such MS/MS data can reveal the protein identity either by database search, or via de novo sequencing.However, successful and confident protein identification, either by database searching or by de novo sequencing, relies heavily on the extent and quality of the obtained sequence information generated by MS/MS.In addition, the higher the extent of fragmentation, the higher the probability of localizing post-translational modifications (PTMs). Electron based reactions, electron capture dissociation (ECD) and electron detachment dissociation (EDD), have shown great promise for PTM analysis, and for improved peptide sequence coverage.In this thesis, ion electron reactions, ECD, EDD, and electron induced dissociation (EID), are explored for peptide sequencing, and for PTM analysis.Disulfide bond formation is a PTM present in extracellular proteins.We demonstrate that EDD and infrared multiphoton dissociation (IRMPD) of peptide anions containing disulfide linkages result in preferential cleavage of S-S and C-S bonds and, therefore, both techniques can be used for probing disulfide bonds in peptide anions.Factors such as precursor ion charge state and m/z value, peptide mass, and protease selection that may influence the dissociation outcome in ECD were investigated, aiming to improve peptide sequence coverage.We show that doubly protonated peptides do not fragment efficiently in ECD, and that precursor ion m/z value is the main factor determining a successful ECD outcome.Highly charged precursor ions at m/z < ~960 fragment efficiently in ECD and yield high peptide sequence coverage.The utility of EID for dissociation of singly deprotonated species was also explored.We show that EID results in extensive fragmentation, providing structural information for peptide anions.For modified peptides, EID results in retention of sulfation and phosphorylation allowing localization of the modification site.Vibrational excitation, collision induced dissociation and IRMPD, were explored for structural characterization of native and oxidized lantibiotics.These experiments provided insights into the fragmentation behavior of native and oxidized lantibiotics, allowing prediction of their fragmentation pathways.