Simplifying low-input archival formalin-fixed paraffin-embedded proteomics: a detergent-free workflow in archival...

Archival formalin-fixed paraffin-embedded (FFPE) tissue is the largest clinically annotated human biospecimen resource, yet proteomic analysis of low-input microdissected regions remains limited. We performed a streamlined workflow enabling deep proteome coverage from laser-microdissected archival FFPE kidney tissue using standard laboratory infrastructure. We systematically compared extraction with n-dodecyl-β-D-maltoside followed by single-pot, solid-phase-enhanced sample preparation (DDM + SP3), detergent-free triethylammonium bicarbonate followed by SP3 cleanup (TEAB + SP3), and direct TEAB processing without offline bead-based cleanup. Across the experimental conditions evaluated, elimination of detergent-based cleanup was associated with increased proteome depth in low-input FFPE samples. A direct TEAB workflow reproducibly yielded up to 5500 proteins from 27000 μm² microdissected regions of decade-old biopsies. Hydrophobicity profiling and functional enrichment analyses showed similar physicochemical and cellular-compartment coverage across workflows, with no evidence of major workflow-associated bias under the conditions evaluated. Comparable proteome coverage was observed across platforms, although not directly controlled due to differences in liquid chromatography-mass spectrometry (LC-MS) configuration. The required input corresponds to common microanatomical units and does not rely on automation or specialized microfluidics. This workflow provides an accessible framework for compartment-resolved proteomics in kidney tissue, enabling retrospective and rare-disease proteomic research from long-term FFPE repositories. Further studies are needed to assess our workflow's applicability to other scarce archival specimens.