Development of Photoactivation and Data Independent Acquisition Mass Spectrometry Methods for Chemical Biology

Abstract

Mass spectrometry allows for the characterization of proteins, lipids, natural products, oligonucleotides, and other small molecules. As mass spectrometry tools are further developed, the rate at which data are acquired, the depth of information that can be attained from a single spectrum are both increased leading to novel approaches for interrogating biological questions. Photodissociation mass spectrometry techniques such as ultraviolet photodissociation and infrared multiphoton dissociation (IRMPD) allow for highly tunable and even highly selective methods. These two techniques have been coupled to almost every type of mass analyzer for every type of -omics problem. In Chapter 2 we utilize the selectivity of IRMPD for charactering phosphorylated peptides. We coupled a 10.6 µm IR laser to a high-resolution orbitrap-type instrument to push the boundaries for generating highly resolved, non-stochastic sampling of chromatographic data at 0.77 s at 800 m/z window. This allows for >10 MS1 spectra in a nanoLC chromatographic peak. This data independent acquisition (DIA) approach allows for the detection and characterization of peptides that have been modified with a chromophore, whether exogenous or endogenous, thus increasing dynamic range by allowing sampling of low abundance peptides. In Chapter 3 we extend this methodology to identify phosphopantetheinylated peptides from biosynthetic proteins. Infrared activation allows us to selectively dissociate peptides containing the phosphopantetheine (Ppant) modification in a mixture and further interrogate covalently bound natural product intermediates. DIA allows for generation of mass lists for targeted analysis of Ppant-containing peptides via MS3 to query the structure of the biosynthetic intermediates. The Ppant ejection ion at 261.1267 can be extracted selective identification of Ppantylated and Ppantylated bound intermediate peptides. This method can be coupled with MS3 dissociation of the Ppantylated bound intermediate ejection for characterization of biosynthetic bound intermediates. Multiple nominal mass neutral loss species at m/z 58, 88, 102, and 142 were determined indicating dehydration, C-C and C=C bond locations of a Ppantylated pentaketide. This developed method is complementary to current methods for Ppant site discovery and allows further insight into the order of biosynthetic transformations in natural product biosynthetic enzymes. Chapter 4 describes the development of a strategy for automated kinetic analysis of ultraviolet photodissociation (UVPD) at 213 nm. This approach allows detailed analysis of differences in fragmentation under different conditions to further understand and optimize this relatively recent commercially available fragmentation method. A program was built into ThermoFisher Scientific’s Tribrid series for mapping fragment ion abundances at multiple irradiation times. This program was verified with the small molecule caffeine, which showed a half-life of 76.19+/-1.18 ms and a rate constant of -9.1+/-0.14 s-1. We also used this program to evaluate ion trap pressure effects in UVPD of diacyl glycerol and determined that higher pressure regions generate more informative fragments for double bond localization due to rapid collisional cooling of vibrationally excited states. Chapter 5 discusses customized collisional activation of anionic gaseous oligonucleotides in a linear ion trap. Experiments were performed with both closed-shell ions and radical anions generated inside the trap. Reduced resonant excitation generated less complex spectra compared with conventional settings, which increases sensitivity and decreases false positive identifications. Overall, these methods will allow for rapid and comprehensive characterization of biomolecules in complex mixtures with a focus on health and medicine.