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

ABSTRACT: Top-down proteomics of human serum is hindered by extreme dynamic range and cumbersome prefractionation. Here we establish a fully solution-based protein precipitation–solid-phase extraction (PP–SPE) workflow for deep, low-molecular-weight-biased proteoform profiling. Using systematically optimized combinations of five precipitants and three sorbents, the workflow enabled identification of 4,470 proteoforms from 480 proteins with excellent reproducibility (R > 0.96). Compared with the gel-based PEPPI method, PP–SPE provided substantially greater depth, including >10-fold more <5 kDa proteoforms, while shortening the overall sample preparation workflow and offering a format that is readily adaptable to plate-based platforms, thus providing a robust front end for high-throughput serum top-down analyses.

ORGANISM(S): Homo Sapiens

SUBMITTER: Fangjun Wang

PROVIDER: PXD073228 | iProX | Sat May 23 00:00:00 BST 2026

REPOSITORIES: iProX

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

Liu Ning N Zhang Meng M Qin Huiwen H Zhao Heng H Zhu Yinlong Y Xie Yuanzhi Y Liu Jing J Wang Fangjun F Liu Zheyi Z Chang Cheng C

Journal of proteome research 20260603

Top-down proteomics of human serum is hindered by extreme dynamic range and cumbersome prefractionation. Here, we establish a fully solution-based protein precipitation-solid-phase extraction (PP-SPE) workflow for deep, low-molecular-weight-biased proteoform profiling. Using systematically optimized combinations of five precipitants and three sorbents, the workflow enabled identification of 4470 proteoforms from 480 proteins with excellent reproducibility (<i>R</i> > 0.96). Compared with the gel ...[more]

PMID: 42236160

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