Scientists Develop Novel Sample Pretreatment Method Based on Digestion of Water Droplets in Oil to Enable Efficient Protein Recovery

Single-cell proteomics provides information about a cell at its protein level, which can be useful for research on cancer drug resistance and cell differentiation. However, current proteomics methods are not versatile and often result in high sample losses. To overcome this problem, researchers have now developed a new method of sample preparation called “water droplet-in-oil digestion” which minimizes sample loss, maximizes protein identification and provides better sensitivity by compared to conventional methods.

The proteins that make up our cells contain a whole world of information that, once unlocked, can give us insight into origins of many essential biological phenomena. This information is gathered using an analytical strategy known as “single-cell proteomics,” in which single-cell analysis is performed to observe the characteristics of individual cells at their protein level. Over the years, scientists have used single-cell proteomics in the areas of cancer genomics, cell differentiation, and tissue development. However, current proteomics procedures suffer from low protein sample recovery, low throughput and lack of versatility.

Fortunately, a team of Researchers from Japan and the United States led by Assistant Professor Takeshi Masuda of Kumamoto University in Japan found an option to these problems. In a recent study posted online on 11 July 2022 and published in the quantity 94, number 29 of Analytical Chemistry on 26 July 2022, the team introduced an uncomplicated but highly efficient method of sample preparation for single-cell proteomics called the “water droplets-in-oil method” (WinO). The method uses the immiscibility of water with oil/organic solvent to its advantage to prepare protein samples with minimal loss and increased chances of sample recovery.

“To make single-cell proteomics more efficient, we either need to amplify the protein sample or ensure that no part is lost during sample preparation. Since we couldn’t afford to do the former, it was very important that we minimize absorption losses during sample preparation steps like sample transfer,” says Dr. Masuda. “The WinO approach not only reduces sample loss by adsorption, but also offers better throughput compared to conventional methods.”

For the WinO process, the team has first prepared an extraction buffer by mixing one microliter of water with section transfer surfactants (which increase the solubility of hydrophobic proteins) and carboxyl-coated hydrophobic nanomagnetic beads. This mixture was then poured dropwise into 29 microliters of ethyl acetate.

The step next was protein extraction, which was 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 in the water droplet. After extraction, the sample was digested using a protein enzyme, Lys-C, and labeled using a “tandem mass tag” reagent. The extracted-digested-labeled sample was then purified and recovered for single-cell analysis and proteomic profiles.

To compare the efficiency of the WinO method versus conventional methods , the team also prepared samples using the common method of optional digestion (ISD) and performed proteomic analysis. They found that the WinO method resulted in a 10 increase in protein and peptide recovery compared to ISD. This remarkable improvement was attributed to a reduced get in touch with area between the extraction remedy and the sample container.

To analyze the sensitivity of the two methods, the team also compared the proteomic profiles obtained. They observed a strong correlation between the proteomic profiles obtained for 100 cells with WinO and that for 10 cells with ISD. Additionally, the team successfully quantified 462 proteins using WinO, demonstrating that it provides much higher throughput and extraction efficiency than the methods

The enhanced protein retrieval and identification capability provided by WinO could allow closer examination of cancer cell protein expression and better understanding of the mechanisms underlying resistance to anticancer drugs. Additionally, WinO can be semi-automated using a liquid handling robot, making it suitable for high-speed, high-capacity sample processing. “Our research could allow scientists to perform proteomics on rare and limited sample quantities as well as provide new insight into protein expression, opening up possibilities for discovering new biological phenomena,” concludes Dr. Masuda.

2022 2022

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