New Proteomics Sample Prep Method Boasts Minimum Loss

Researchers from Japan and the United States have proposed a new sample pretreatment method for single-cell proteomics that achieves a ten-fold increase in protein and peptide recovery. The breakthrough could advance understanding of protein expression of cancer cells and the mechanisms underlying anticancer drug resistance as well as enable analysis of rare or limited samples.

The proteins that make up cells contain a wealth of information on the origins of many essential biological processes. Single-cell analysis helps uncover these insights, and for years researchers have used single-cell proteomics to study cancer genomics, cell differentiation, and tissue development. However, current proteomics techniques suffer from low recovery rate of protein samples, low throughput, and lack of versatility.

In a recent issue of the journal Analytical Chemistry, a research team led by Assistant Professor Takeshi Masuda from Kumamoto University in Japan report a new technique that overcomes these issues. Dubbed water droplet-in-oil (WinO), the simple and efficient sample preparation method uses the immiscibility of water with oil/organic solvent to its advantage to prepare protein samples with minimum loss and increased chances of sample recovery.

“To make single cell-proteomics more efficient, we either need to amplify the protein sample or make sure none of it is lost during sample preparation. Since we didn’t have the means to do the former, it was crucial that we reduced absorption losses during sample preparation steps like sample transfer,” says Dr. Masuda. “The WinO technique not only reduces sample loss through adsorption but also provides better throughput when compared with conventional methods.”

The WinO process involves preparing an extraction buffer by mixing one microliter of water with phase transfer surfactants (to increase the solubility of hydrophobic proteins) and hydrophobic carboxyl-coated nanomagnetic beads. This mixture is then dropped into 50 microliters of ethyl acetate.

The next step is protein extraction, which is performed by adding cell droplets from the cell sorter to the ethyl acetate-water droplet combo and spinning it in a centrifuge to allow the protein to accumulate within the water droplet. After the extraction, the sample is digested using a protein enzyme, Lys-C, and labeled using a tandem mass tag reagent. The extracted-digested-labeled sample is then purified and recovered for single-cell analysis and proteomic profiles.

To compare the efficacy of the WinO method against conventional methods, the team prepared samples using the standard in-solution digestion (ISD) method and carried out proteomic analysis. They found that the WinO method led to a 10-fold increase in protein and peptide recovery compared to ISD. This remarkable improvement was attributed to a reduced contact area between the extraction solution and the sample container.

To analyze the sensitivity of both methods, the team also compared the obtained proteomic profiles. They observed a high correlation between proteomic profiles obtained for 100 cells using WinO and that for 10,000 cells using ISD. Furthermore, the team successfully quantified 462 proteins using WinO, demonstrating that it provided a much higher throughput and extraction efficiency than conventional techniques.

The enhanced protein recovery and identification ability provided by WinO could enable a closer look at the protein expression of cancer cells and a better understanding of the mechanisms underlying anticancer drug resistance. Further, WinO can be semi-automated using a liquid handling robot, making it suitable for high-speed, large-capacity processing of samples. “Our research could allow scientists to perform proteomics on rare and limited sample amounts as well as provide a novel perspective on protein expression, opening up possibilities for discovering new biological phenomena,” concludes Dr. Masuda.