The development and implementation of novel peptide fragmentation methods for proteomics.

摘要

The work described herein can be broadly classified as 1) The development of techniques to improve the efficacy of electron transfer dissociation (ETD) in proteomics applications, 2) The extension of infrared multiphoton dissociation (IRMPD) to a variety of mass spectrometry instrumentation, with evaluation of the potential utility, and 3) The development of an MS/MS multiplexing technique.;Chapters two through four outline the progression of activated-ion ETD. During this technique, precursor peptides are bombarded with IR photons during electron transfer dissociation; in so doing the gas phase secondary structure is continuously disrupted, leading to considerable improvement in the dissociation efficiency. This proof of concept for this idea was performed on stand-alone ion trap instrumentation, but as the utility became apparent, the technique was extended to a hybrid mass spectrometer comprising both ion trap and orbitrap mass analyzers.;In chapters five and six, I describe the first implementation of IRMPD on an orbitrap-containing mass spectrometer. I similarly describe the implementation of IRMPD on a dual-cell ion trap mass spectrometer, undertaking a large study and concluding that IRMPD represents an excellent option for the interrogation of isobaric-tagged peptides.;Finally, I present a novel spectral multiplexing scheme. This technique involves the fragmentation of peptide precursors using resonant-excitation collisional activation (CAD). The fragments of several CAD events from different peptide precursors are stored in an external RF device, and analyzed in a single orbitrap m/z analysis step, resulting in considerable duty cycle improvement.