Degree
Doctor of Philosophy (PhD)
Department
Chemistry
Document Type
Dissertation
Abstract
In the research described in this dissertation, a high-precision infrared laser ablation microsampling method was utilized for spatially resolved proteomic and lipidomic analyses of formalin fixed paraffin embedded (FFPE) tissue, a widely used archival sample in biomedical research. Despite its extensive use, FFPE tissue presents challenges for proteomic analysis due to protein cross-linking and modifications introduced during fixation, which can hinder extraction and identification. Developing a streamlined high-resolution method to recover biomolecules from FFPE samples is critical for advancing biomarker discovery, retrospective studies, and multi-omics research. To address these challenges, this study employs an infrared (IR) laser ablation microdissection system to enable precise, localized protein extraction from FFPE tissues without requiring antigen retrieval or cell lysis steps. A mid-IR laser with a wavelength of 3 µm was used to ablate tissue sections, achieving an ablation spot size of approximately 50 µm and precise sampling from mm² areas of thin FFPE tissue sections. The system enabled the identification of more proteins from FFPE tissue by laser ablation sampling compared to fresh frozen (FF) tissue and significantly outperformed manual dissection of FFPE tissue. Notably, no loss of hydrophilic proteins due to residual cross-linking was observed, highlighting the efficiency of the approach. The laser ablation system was further applied to Alzheimer’s disease (AD) mouse brain tissue. Hippocampal and cortical regions were ablated and analyzed for differential protein expression. Bioinformatics analyses revealed enrichment of Alzheimer’s disease pathways, along with regional differences in molecular responses, suggesting that mechanisms of disease progression vary across brain regions. Additionally, IR laser ablation was employed to sample lung tissues from COVID-19-infected mice. Despite challenges posed by the fibrotic nature of these tissues, the method enabled targeted protein analysis, demonstrating its versatility across various tissue types. A lipidomics workflow was also developed and utilized for sampling the cortical region of a rat brain FFPE tissue, identifying and comparing lipid classes to those in FF tissue. This highlights the adaptability of the laser ablation system for multi-omics studies. This study highlights the potential of IR laser ablation microsampling for spatially resolved proteomics and its utility in biomarker discovery for diseases such as Alzheimer’s and COVID-19 while also serving as a resource for retrospective studies. Furthermore, it allows for the analysis of biomolecules from FFPE tissue without requiring antigen retrieval or cell lysis steps. Future advancements, such as the use of nanosecond UV lasers, are anticipated to enhance precision and expand the method’s applications in spatial proteomics. Additionally, integrating detergent cleanup steps into the bottom-up proteomics workflow can improve protein identification, particularly in fibrotic tissue such as lung tissue.
Date
3-6-2025
Recommended Citation
Egbejiogu, Blessing Chisom, "Infrared Laser Ablation of Biomolecules from Formalin-Fixed Tissue" (2025). LSU Doctoral Dissertations. 6686.
https://repository.lsu.edu/gradschool_dissertations/6686
Committee Chair
Murray, Kermit K.
