Novel Fourier Transform Ion Cyclotron Resonance Mass Spectrometric Strategies for Peptide Nucleic Acids and Acidic Biomacromolecules: Qualitative Characterization and Quantification.

Abstract

Mass spectrometry is widely applied to characterize oligo- and poly-nucleotides, peptides, and proteins. In tandem mass spectrometry (MS/MS), analytes are dissociated in the gas phase and their fragments are analyzed to generate structural information. Among the various MS/MS techniques, few are well suited for acidic biomolecules, particularly nucleic acids and acidic peptides/proteins. In this dissertation emerging MS/MS methods are applied to new problems and to molecules not previously studied by such techniques. Gas-phase fragmentation of peptide nucleic acid (PNA) cations and anions was examined in collision activated dissociation (CAD), infrared multiphoton dissociation (IRMPD), electron capture dissociation (ECD), and electron detachment dissociation (EDD). Negative-ion CAD and IRMPD provided the most sequence information and were simplest to interpret. Positive-ion mode MS3 and double resonance experiments revealed mechanistic details on the water loss pathway. N-terminal acetylation blocked this pathway and simplified spectrum interpretation. A 27-mer ribonucleic acid (RNA) and its complexes with aminoglycoside antibiotics were characterized by ion-mobility MS, negative-ion CAD, and EDD. Higher binding specificity and lower gas-phase charge state were correlated with a more compact RNA structure. EDD fragmentation efficiency was higher for higher charge states. Negative-ion IRMPD and EDD were compared to positive-ion IRMPD and ECD for dissociating intact proteins. For acidic proteins, negative ion mode techniques provided complementary sequence information to positive ion mode. Negative-ion ECD (niECD) was investigated for oligonucleotide characterization. Activated ion-niECD and niECD-IRMPD MS3 provided similar fragmentation patterns as EDD, possibly due to similar gas-phase precursor ion structures. Characteristic “ECD-like” radical fragments also suggested similarities to ECD, which may be due to DNA/RNA zwitterionic structures in negative ion mode. Proton transfer reaction (PTR) was performed prior to niECD of phosphopeptides. For peptides larger than 1700 Da, PTR reduced the ion accumulation time and increased the detection efficiency of niECD fragments. niECD was also coupled to offline nano-LC by segmented flow fraction collection, which allowed more time for signal averaging, compared with online LC coupling. Overall this dissertation presents method development for improved MS/MS analysis in systems biology, i.e., the study of organisms as a whole rather than focusing on individual biochemical pathways.