Integrated Protein Precipitation-Solid-Phase Extraction Workflow for Deep Top-Down Proteomic Analysis of Human Serum

Area of Science:

• Proteomics
• Biochemistry
• Analytical Chemistry

Background:

• Top-down proteomics of human serum faces challenges due to extreme dynamic range and complex prefractionation steps.
• Existing methods can be time-consuming and may not efficiently capture low-molecular-weight proteoforms.

Purpose of the Study:

• To establish a fully solution-based workflow for deep proteoform profiling of human serum.
• To develop a method biased towards identifying low-molecular-weight proteoforms.
• To create a robust and high-throughput front-end for serum top-down proteomic analyses.

Main Methods:

• Development and optimization of a protein precipitation-solid-phase extraction (PP-SPE) workflow.
• Systematic optimization of five precipitants and three sorbents for sample preparation.
• Application of the PP-SPE workflow to human serum samples for proteoform analysis.

Main Results:

• Identification of 4470 proteoforms from 480 proteins with high reproducibility (R > 0.96).
• The PP-SPE method demonstrated a >10-fold increase in the identification of proteoforms <5 kDa compared to gel-based methods.
• Significant reduction in sample preparation time and a format adaptable to plate-based platforms.

Conclusions:

• The developed PP-SPE workflow offers a deep and low-molecular-weight-biased proteoform profiling capability for human serum.
• This method provides a more efficient and reproducible alternative to existing techniques for top-down serum proteomics.
• The PP-SPE workflow serves as a robust front-end for high-throughput serum proteomic studies.