Water Droplet-in-Oil Digestion Method for Single-Cell Proteomics

Abstract

Recent advances in single-cell proteomics highlight the promise of sensitive analyses in limited cell populations. However, technical challenges remain for sample recovery, throughput, and versatility. Here, we first report a water droplet-in-oil digestion (WinO) method based on carboxyl-coated beads and phase transfer surfactants for proteomic analysis using limited sample amounts. This method was developed to minimize the contact area between the sample solution and the container to reduce the loss of proteins and peptides by adsorption. This method increased protein and peptide recovery 10-fold. The proteome profiles obtained from 100 cells using the WinO method highly correlated with those from 10,000 cells using the in-solution digestion method. We successfully applied the WinO method to single-cell proteomics and quantified 462 proteins. Using the WinO method, samples can be easily prepared in a multi-well plate, making it a widely applicable and suitable method for single-cell proteomics.

Recent advances in microfluidics for single-cell functional proteomics

“…196 Recently, Masuda and colleagues introduced a water droplet-in-oil digestion (WinO) approach. 197 This method uses immiscibility of solvents (water and oil) to reduce sample loss during sample preparation and to increase protein recovery. Briefly, an extraction buffer containing 1 μL of water droplet is prepared with phase transfer surfactants and hydrophobic carboxyl-coated nanomagnetic beads, and then cells are loaded in ethyl acetate to mix with the extraction buffer.…”

Recent methodological advances towards single-cell proteomics

“…Inspired by these single-cell MS studies, many innovative methods to prepare samples with minimal volume have been reported, for example, sub-nanoliter volume droplet samples on a hydrophobic polymer film (Fig. 2 C), 23 ) preparing a lysate water droplet-in-oil, 24 ) usage of a commercial picoliter dispenser, 25 ) and microfluidic chip, 26 ) and performing seamless sample preparation and nano-LC separation. 27 )…”

Enhanced feature matching in single-cell proteomics characterizes response to IFN-γ and reveals co-existence of different cell states

“…Yet, SCP still suffers from several limitations in proteomic depth and throughput. Since the material from individual cells is scarce, several studies focused on reducing losses during sample preparation by process miniaturization (13, 14) and avoiding surface adsorption (15, 16). As one important example, the SCoPE- MS (single-cell proteomics by mass spectrometry) methodology uses sample multiplexing via TMT- labeling to concomitantly increase analyte concentration and throughput (17, 18).…”