Abstract

Proteins are responsible for important cellular functions and processes including regulation, structural integrity, signaling, transport, and immune functions. Proteomics directly identifies these proteins and enables deeper characterization of the dynamic cellular environment beyond the capabilities of genomics and transcriptomics. Single-cell proteomics further enhances this approach by capturing cellular heterogeneity, which bulk proteomics obscures due to averaging protein expression across large cell populations. Despite significant advances in single-cell proteomics, continued innovation is necessary to increase workflow accessibility and throughput, minimize peptide losses, and achieve comprehensive coverage of the proteome. This dissertation addresses these challenges by developing improved sample preparation methods, two-dimensional liquid chromatography separations, and optimized mass spectrometry acquisition strategies specifically tailored for single-cell proteomic analysis. Specifically, we enhanced sample preparation throughput by creating a one-step digestion mixture that accomplishes protein denaturation and digestion within a single hour. We also evaluated an offline two-dimensional liquid chromatography platform for low-input samples. Lastly, we optimized mass spectrometry acquisition parameters for data-independent acquisition-based gas-phase fractionation. Collectively, these improvements broaden access to in-depth proteomic analyses and facilitate comprehensive biological characterization across diverse cellular environments.

Degree

PhD

College and Department

Computational, Mathematical, and Physical Sciences; Chemistry and Biochemistry

Rights

https://lib.byu.edu/about/copyright/

Date Submitted

2025-04-23

Document Type

Dissertation

Handle

http://hdl.lib.byu.edu/1877/etd13623

Keywords

single-cell proteomics, two-dimensional liquid chromatography, gas-phase fractionation

Language

english

Download

Share

COinS