Digital Microfluidics and Magnetic Bead‐Based Intact Proteoform Elution for Quantitative Top‐down Nanoproteomics of Single C. elegans Nematodes

While most nanoproteomics approaches for the analysis of low-input samples are based on bottom-up proteomics workflows, top-down approaches enabling proteoform characterization are still underrepresented. Using mammalian cell proteomes, we established a facile one-pot sample preparation protocol based on protein aggregation on magnetic beads and intact proteoform elution using 40 % formic acid. Performed on a digital microfluidics device, the workflow enabled sensitive analyses of single Caenorhabditis elegans nematodes, thereby increasing the number of proteoform identifications compared to in-tube sample preparation by 46 %. Label-free quantification of single nematodes grown under different conditions allowed to identify changes in the abundance of proteoforms not distinguishable by bottom-up proteomics. The presented workflow will facilitate proteoform-directed analysis on samples of limited availability.

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Leipert, J., Kaulich, P.T., Steinbach, M.K., Steer, B., Winkels, K., Blurton, C.L., Leippe, M., Tholey, A., 2023. Digital Microfluidics and Magnetic Bead‐Based Intact Proteoform Elution for Quantitative Top‐down Nanoproteomics of Single C. elegans Nematodes. Angew Chem Int Ed 62, e202301969. https://doi.org/10.1002/anie.202301969
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