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June 3rd, 2025
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Max-Planck Institute for Biochemistry
systems biology
biorxiv

A Solid-Phase Extraction Capture (SPEC) workflow in nanoliter volumes for fast, robust and ultrasensitive proteomics

Heymann, T.Open in Google Scholar•Oliinyk, D.Open in Google Scholar•Henneberg, L.Open in Google Scholar•Rodriguez, E.Open in Google Scholar•Bardziukova, A.Open in Google Scholar•Oeller, M.Open in Google Scholar•Murgia, M.Open in Google Scholar•Olsen, J. V.Open in Google Scholar•Stoychev, S.Open in Google Scholar•Bache, N.Open in Google Scholaret al.

Despite great progress, sample preparation remains an area for improvement in proteomics, particularly for low-input samples where conventional protocols lead to losses and incomplete digestion. We present Solid-Phase Extraction Capture (SPEC), a modular workflow that confines protein processing to nanoliter-scale volumes within C18 or ion-exchange tips. SPEC enables highly efficient proteolysis directly on the solid phase, minimizing surface losses and reagent dilution. A two-tip configuration further allows orthogonal phase combinations for optimal digestion and on-tip manipulation. Compared to alternative protocols, SPEC improves protein recovery while accelerating protein digestion which completes in minutes. The method is compatible with a diverse set of sample types and lysis conditions, including strong detergents, FFPE tissue, and muscle fibers. SPEC nanoliter digestion volume, high recovery, and flexible design make it a powerful tool for ultrasensitive and high-throughput proteomics, facilitating applications from spatial biology to clinical profiling. SPEC represents a universal proteome sample preparation protocol that spans all sample types, sensitivity levels, and throughput requirements.

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