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Integrated Protein Precipitation-SPE Workflow for Deep Top-Down Proteomic Analysis of Human Serum

ABSTRACT: Deep top-down proteomic analysis of human serum is challenged by the extreme dynamic range of protein abundances and significant matrix interference. Here, we establish an integrated protein precipitation and solid-phase extraction (PP-SPE) workflow to overcome these challenges. We systematically investigated how precipitant chemistry and sorbent selection influence proteoform recovery. Our findings reveal that organic solvents (e.g., ACN, acetone) reduce the dielectric constant, preferentially precipitating large, acidic, and hydrophobic proteoforms, whereas inorganic salts (e.g., ammonium sulfate) function via ion hydration and dehydration, leading to a distinct size-selective precipitation. The optimized workflow, utilizing Oasis PRiME HLB sorbent, identified an average of 1,138 proteoforms and 155 proteins per condition, demonstrating superior sensitivity compared to conventional C18- or silica-based methods. In total, 5,728 proteoforms were identified, establishing one of the most extensive serum top-down proteomic datasets reported to date, with a high proportion of confident identifications (1,197 proteoforms with E-value < 10⁻³). Our study establishes PP-SPE as a robust, tunable, and highly effective strategy for deep serum proteoform profiling, providing a practical and accessible foundation for biomarker discovery at proteoform level.

ORGANISM(S): Homo Sapiens

SUBMITTER: Fangjun Wang

PROVIDER: PXD073228 | iProX | Sat Jan 17 00:00:00 GMT 2026

REPOSITORIES: iProX

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