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

Autor: Leipert J; Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, 24105, Kiel, Germany., Kaulich PT; Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, 24105, Kiel, Germany., Steinbach MK; Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, 24105, Kiel, Germany., Steer B; Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, 24105, Kiel, Germany., Winkels K; Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, 24105, Kiel, Germany., Blurton C; Comparative Immunobiology, Zoological Institute, Christian-Albrechts-Universität zu Kiel, 24118, Kiel, Germany., Leippe M; Comparative Immunobiology, Zoological Institute, Christian-Albrechts-Universität zu Kiel, 24118, Kiel, Germany., Tholey A; Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, 24105, Kiel, Germany.
Jazyk: angličtina
Zdroj: Angewandte Chemie (International ed. in English) [Angew Chem Int Ed Engl] 2023 Jul 10; Vol. 62 (28), pp. e202301969. Date of Electronic Publication: 2023 May 31.
DOI: 10.1002/anie.202301969
Abstrakt: 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.
(© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)
Databáze: MEDLINE
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