Degree

Doctor of Philosophy (PhD)

Department

Chemistry

Document Type

Dissertation

Abstract

A high precision infrared laser ablation sampling system was developed for localized tissue proteomics. A Schwarzschild reflective objective with 10 cm working distance and 0.3 numerical aperture was used to focus a mid-IR laser at 3 µm wavelength to a beam diameter of approximately 6 µm. A laser ablation spot size of 10 µm was achieved on thin ink films and tissue sections. The sampling system was used to ablate and capture rat brain tissue sections in transmission geometry. Captured tissue protein was extracted, digested, and analyzed with offline liquid chromatography tandem mass spectrometry (LC-MS/MS) for peptide and protein identification. A microcentrifuge tube capture approach followed by a single-pot, solid-phase-enhanced sample preparation (SP3) method was used for sample collection and protein digestion. Proteins were identified from 50 µm thick tissue sections down to 0.04 mm2 ablated tissue area. Further, a small volume sample preparation method was adapted to the IR laser ablation sampling to improve sensitivity. A PTFE coated glass slide with 2 mm diameter well arrays was used to capture the ablated tissue and perform protein digestion in situ within a 1 µL processing volume. The small volume approach significantly improved the efficiency: approximately 600 proteins were identified from 0.01 mm2 area. Localized tissue proteomics was demonstrated with 10 µm sampling precision. The sampling system was also applied for localized protein analysis of blast-induced mild traumatic brain injury (mTBI) mice. Cerebral cortex and corpus callosum of injured and control mouse tissue sections were ablated and collected for bottom-up proteomics. Approximately 2450 proteins were identified in a 2 mm2 region. Quantitative proteomics showed that 5% of the proteins were differentially expressed in the corpus callosum and 2% of the proteins were altered in the cerebral cortex of blast brains compared to control brains. Distinctive KEGG pathways were identified in the two regions of injured brains and Alzheimer’s disease related pathways were associated in both regions.

Committee Chair

Murray, Kermit K.

Available for download on Saturday, October 26, 2024

Share

COinS