Vacuum ultraviolet photodissociation mass spectrometry: Mechanistic studies and applications in proteomics

Tandem mass spectrometry is widely used to characterize proteins in biological systems. Proteins are typically identified by sequence-specific mass spectra resulting from fragmentation of proteolytic peptides in the gas phase. Unfortunately, most conventional excitation processes generate limited numbers of fragments that are insufficient to de novo reconstruct peptide sequences. Photodissociation with 157 nm light provides a great alternative to conventional methods by generating abundant high-energy fragments with more than 90% sequence coverage. In this dissertation, photodissociation mechanisms are investigated and applications for biological sample analyses are also pursued.;Photodissociation has first been implemented in a linear ion trap mass spectrometer to investigate photolytic processes. Peptides with N-terminal arginine yield abundant a+1 radical ions, confirming that photodissociation is initiated by homolytic cleavage of peptide backbone C alpha-C(O) bonds. The resulting radical fragments then undergo radical-driven fragmentation that is directed by amino acid side chains, leading to highly predictable secondary fragments. Since 157 nm photodissociation does not mobilize the ionizing proton in singly-charged arginine-containing peptide ions, it has been employed to probe protonation sites in singly-charged peptide fragment ions produced in collision-induced dissociation (CID). The ionizing proton in these ions is shown to be located on the most basic residue(s), suggesting that the commonly used ion structures need to be revised.;Photodissociation has also been implemented in a commercial tandem time-off-light (TOF) mass spectrometer for peptide analysis. A peptide de novo sequencing algorithm has been developed to interpret peptide sequences directly from photodissociation spectra. These developments allow photodissociation to characterize peptide mixtures following liquid chromatography separation. In addition, photodissociation of glycopeptides in this apparatus enables both peptide sequences and glycan structures to be elucidated. With modest improvements, 157 nm photodissociation mass spectrometry could be applied to characterize complex biological samples.