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

Abstract

AbstractWhile 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.