Microproteomics workflows for quantitative proteome analysis of single Caenorhabditis elegans nematodes

Miniaturization of sample preparation including omissible manual sample handling steps is key for reproducible nanoproteomics as material is often restricted to only hundreds of cells or single model organisms. Here, we demonstrate a highly sensitive digital microfluidics (DMF)-based sample preparation workflow making use of single-pot solid-phase enhanced sample preparation (SP3) in combination with high-field asymmetric-waveform ion mobility spectrometry (FAIMS), and fast and sensitive ion trap detection on an Orbitrap tribrid MS system. Compared to a manual in-tube SP3-supported sample preparation, the number of peptides identified, the achievable standard deviations between replicates, and the number of proteins identified as differentially abundant were markedly increased. We identified up to 5,000 proteins from single nematodes. Moreover, label-free quantification of protein changes in single Caenorhabditis elegans treated with a heat stimulus yielded 45 differentially abundant proteins when compared to the untreated control, demonstrating the potential of this technology for low-input proteomics studies.