ADVANCES IN PROTEOMIC WORKFLOW FROM CELL LYSIS TO PROTEIN PURIFICATION FOR HIGH-THROUGHPUT MASS SPECTROMETRY ANALYSIS

Abstract

Proteomics aims to characterize the complete set of proteins in a cell, tissue, or organism at a specific state or time, playing a significant role in various fields, notably molecular medicine for biomarker discovery and clinical profiling. Proteomics primarily relies on mass spectrometry (MS) for detection, though the complex, multistep sample processing workflow required to manipulate proteins ahead of MS is tedious, time consuming, and may lead to sample loss. The goal of this work is to develop novel technologies to improve the proteomics workflow, with emphasis on improving protein extraction efficiency, optimizing sample purity, and maximizing the throughput of sample processing, particularly for small scale samples.

The method introduced in Chapter 2 for automated protein extraction significantly enhances efficiency by utilizing water at subcritical temperatures alongside the surfactant sodium dodecyl sulfate (SDS). This approach has proven effective for extracting intact proteins from both S. cerevisiae and hemp seeds with only 5 minutes of extraction time, achieving approximately 80% extraction efficiency. When applied to hemp seeds, this method allowed for the isolation and MS analysis of a proteome, revealing the detection of 6824 proteins.

The third chapter aims to enhance a previous SDS depletion technology to make it applicable for low solubility proteins. The SDS depletion device, transmembrane electrophoresis (TME), is an electrokinetic device that permits rapid SDS removal from proteins within 5 minutes, generally retaining high protein recovery. It was observed that the addition of 40% methanol improved the recovery of membrane proteins collected from TME by a factor of 1.7. Furthermore, methanol accelerated the rate of SDS depletion in TME, reducing SDS concentration to below 100 ppm in less than 3 minutes. MS analysis on an enriched membrane proteome fraction processed by methanol-assisted TME revealed enhanced detection of low solubility proteins possessing low net charge in aqueous solutions.

The final project introduces cap-TME, a fully automated TME system based on a modified capillary electrophoresis platform. Cap-TME employs an inline filter to capture proteins and deplete SDS, achieving 81% protein recovery while reducing SDS concentration from 5000 ppm to less than 10 ppm in just 30 seconds. Future research suggests that both subcritical water extraction and cap-TME could significantly advance the development of a fully automated online proteomic sample preparation workflow, which is crucial for enhancing throughput and reproducibility in clinical research, especially in biomarker-related studies.