Solid-Phase Extraction Capture (SPEC) in nanoliter volumes for fast, robust and ultra-sensitive proteomics

Le résumé fourni par la source

Abstract Sample preparation remains a critical bottleneck in mass spectrometry (MS)-based proteomics, particularly for limited sample amounts where surface adsorption and dilution cause substantial losses. Here, we present Solid-Phase Extraction Capture (SPEC), a workflow that confines protein processing to nanoliter volumes within ion-exchange or C18 matrix inside a pipette tip. This achieves near-complete proteolysis within 5 minutes instead of hours and maintains full compatibility with strong detergents without cleanup steps, enabling effective lysis of challenging samples. From 200 ng FFPE tissue, SPEC achieves proteome depth and reproducibility exceeding conventional bulk protocols using 100 µg, critical when sample is irreplaceable. The modular two-tip configuration enables on-tip chemical modifications for mTRAQ labeling and fractionation, while integration with enrichment workflows yields 2-fold improved glycopeptide identifications from plasma and 3-fold enhanced ubiquitin remnant identification at low amounts. SPEC enables nanoPhos for cell-type resolved tissue phosphoproteomics and provides a universal platform for proteomics sample preparation.

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