EXPLORING THE FUNCTION OF PLASTOGLOBULES USING TOP-DOWN AND BOTTOM-UP PROTEOMICS BY CAPILLARY ZONE ELECTROPHORESIS – MASS SPECTROMETRY
Plastoglobules (PGs) are lipoprotein particles with dynamic morphology and composition responding to abiotic stress and senescence, and their functions are influenced by post-translational modifications (PTMs) of PG proteome. Capillary zone electrophoresis – tandem mass spectrometry (CZE-MS/MS) based bottom-up proteomics (BUP) and top-down proteomics (TDP) approaches study proteins with their unique PTMs status (i.e., proteoforms) in complex samples. Chapter 2 presents the first large-scale TDP analysis on Arabidopsis thaliana leaf and chloroplast samples, where 3198 and 1836 proteoforms were identified respectively. Notable 1024 and 363 proteoforms exhibited mass shifts from the theoretical mass. Among them, proteoforms with phosphorylation and acetylation were validated by their electrophoretic mobility shifts. Additionally, this analysis also provided direct evidence of N- and C- terminal sequencing that precisely delineated the true transit peptide cleavage sites, offering valuable insights to plant biologists. Despite these advancements, no PG-localized proteins were identified, likely due to challenges related to protein size and hydrophobicity. Thus, the subsequent chapters are dedicated to developing methods to address these issues. In Chapter 3, we developed the CZE-MS method for TDP analysis of integral membrane proteoforms (IMPs) enriched from mouse brains. We employed a sample buffer containing 30% (v/v) formic acid and 60% (v/v) methanol to solubilize IMPs and a separation buffer composed of 30% (v/v) acetic acid and 30% (v/) methanol to maintain solubility of IMPs during CZE separation. Single-shot CZE-MS/MS identified 51 IMPs. Coupling size-exclusion chromatography (SEC)-CZE-MS enabled the identification of 276 IMPs with 1-4 transmembrane domains. This proof-of-concept work demonstrates the high potential of CZE-MS/MS for the large-scale TDP of IMPs. In Chapter 4, we tackle the issues related to separation resolution and reproducibility in CZE for TDP, which stem from non-specific protein adsorption in linear polyacrylamide (LPA) coated capillaries. We developed a simple method for applying a cationic, poly(acrylamide-co-(3-acrylamidopropyl) trimethylammonium chloride [PAMAPTAC]) to the capillaries. This PAMAPTAC coating significantly improves the resolution of proteoform separation and achieves consistent measurements across both standard and complex samples, such as yeast cell lysates. The coating enables the detection of large proteoforms (≥30 kDa) without prefractionation and allows for accurate prediction of proteoform mobility, establishing PAMAPTAC for high-resolution and reproducible TDP analysis. In Chapter 5, we pioneered the native proteomics measurement of large proteoforms or protein complexes up to 400 kDa from a complex proteome via online coupling of native capillary zone electrophoresis (nCZE) to an ultra-high mass range Orbitrap mass spectrometer (UHMR). The nCZE-MS technique enabled the measurement of a 115-kDa standard protein complex while consuming only about 100 pg of protein material. nCZE-MS analysis of an E. coli cell lysate detected 76 proteoforms or protein complexes in a mass range of 30-400 kDa in a single run while consuming only 50-ng protein material. The mass distribution of detected proteoforms or protein complexes agreed well with that from mass photometry measurement. This work represents a technical breakthrough of native proteomics for measuring complex proteomes. In Chapter 6, we summarized the current challenges in TDP and discussed the advancements made in this dissertation. Additionally, we explore two future directions for advancing the field. The first direction involves cross-laboratory collaborations to enhance reproducibility and broaden the application of CZE-MS-based TDP techniques. The second direction proposes combining BUP and TDP to leverage the advantages of each method, integrating their strengths to yield more comprehensive information about proteoforms and their functions. For example, combining BUP and TDP in a PG shaving experiment aims to deepen the understanding of how proteins are localized and recruited on PGs, providing insights into their functional dynamics under various biological conditions.
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- In Collections
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Electronic Theses & Dissertations
- Copyright Status
- In Copyright
- Material Type
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Theses
- Authors
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Wang, Qianjie
- Thesis Advisors
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Sun, Liangliang
Lundquist, Peter K.
- Committee Members
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Hu, Jian
Spence, Dana M.
- Date Published
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2024
- Program of Study
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Chemistry - Doctor of Philosophy
- Degree Level
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Doctoral
- Language
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English
- Pages
- 136 pages
- Permalink
- https://doi.org/doi:10.25335/1k5d-ws13