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 numbers of identified peptides
and proteins were markedly increased, while lower standard deviations
between replicates were observed. We repeatedly identified up to 5000
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, highlighting the
potential of this technology for low-input proteomics studies. LC-MS
data have been deposited to the ProteomeXchange Consortium with the
data set identifier PXD033143.