Graduation Year

2023

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Chemistry

Major Professor

David Merkler, Ph.D.

Committee Member

James Leahy, Ph.D.

Committee Member

Theresa Evans-Nguyen, Ph.D.

Committee Member

John Koomen, Ph.D.

Keywords

Fatty acid amides, High-resolution mass spectrometry, Insect models, Phospholipids

Abstract

Mass spectrometry-based omics research involves global identification and quantification of molecules in the biological system, which can form the basis of the multi-omics approach integrating multiple layers of information to provide a comprehensive overview of systems biology. The investigation of fatty acid amides (FAA) is one such research avenue that benefits significantly from the high-throughput, multi-level, and interdisciplinary approach of integrative omics. Currently, FAA research is dominated by lipidomic-based studies that have successfully identified and quantified >100 different FAAs in different model systems. Few studies are credited with exploring the biological function and evolution of FAA-dependent signaling pathways. Previous literature includes studies on notable FAAs such as anandamide and oleamide. The promising results from these have opened avenues for studies to be done on other FAAs. In this dissertation, we have explored novel tools like binding-based proteomics profiling (BBPP) and mass spectrometry imaging (MSI)-based spatial omics techniques to follow a multi-omics approach to research various lipid classes, including FAAs.

With the knowledgebase from earlier quantitative lipidomic studies that profiled multiple FAAs in model organisms, we developed specific FAA-based BBPP probes to identify FAA-binding proteins, be it receptors, enzymes, transporters, or any proteins allosterically regulated by these lipids and used the information to assess their functions. The process involved designing and synthesizing specific FAA analogs with important structural modifications like the incorporation of a diazirine ring and a terminal alkyne to facilitate photo-crosslinking with probe-bound protein and appending reporter tags (fluorescent dye or biotin), respectively. The identification and quantification of the labeled and subsequently enriched proteome was based on liquid chromatography with tandem mass spectrometry (LC-MS/MS) label-free quantitative proteomics. A modest panel of differentially expressed proteins was identified for each FAA-based probe.

In addition, spatial mapping of lipids was performed with matrix-assisted laser desorption and ionization (MALDI)-MSI technique. This strategy was aimed at introducing a spatial dimension to the lipidomic and proteomics-based profiling studies. The preliminary studies performed here were successful in spatially profiling phospholipids in two model organisms. The results suggest that with some optimization of the sample preparation and data acquisition methods, FAA imaging can be attempted with MALDI-MSI.

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