New sample pre-treatment method enables efficient protein recovery

The cells’ proteins contain a huge world of information that, when unlocked, can shed light on the causes of many fundamental biological occurrences. To observe the properties of individual cells at the protein level, a single-cell analysis method known as “single-cell proteomics” is used.

Single-cell proteomics has been applied by researchers in the disciplines of cancer genomics, cell differentiation, and tissue formation over the years. However, the present proteomics methods are limited in their variety, throughput, and protein sample recovery rates.

A group of scientists from Japan and the USA headed by Assistant Professor Takeshi Masuda from Kumamoto University in Japan identified a solution to these problems. The researchers introduced the “water droplet-in-oil method,” (WinO) a straightforward but incredibly effective sample preparation technique for single-cell proteomics.

The method takes advantage of the fact that water and oil/organic solvent cannot mix to create protein samples with less loss and higher chances of sample recovery.

The study was published on July 26^th, 2022 in the Analytical Chemistry journal.

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.”

Takeshi Masuda, Assistant Professor, Kumamoto University

Masuda adds, “The WinO technique not only reduces sample loss through adsorption but also provides better throughput when compared with conventional methods.”

The scientists first made an extraction buffer for the WinO procedure by combining 1 μl of water with phase transfer surfactants (which boost hydrophobic proteins’ solubility) and hydrophobic carboxyl-coated nanomagnetic beads. Then, 50 μl of ethyl acetate was added to the mixture.

The following stage involved extracting the protein, which was accomplished by combining cell droplets from the cell sorter with the ethyl acetate–water mixture and centrifuging it to cause the protein to collect within the water droplet.

After extraction, the sample was labeled with a “tandem mass tag” reagent and digested using the protein enzyme Lys-C. The extracted, digested, and tagged sample was subsequently recovered, purified, and used for proteomic profiling and single-cell analysis.

The scientists also generated samples using the typical in-solution digestion (ISD) approach and performed proteome analysis to assess the efficacy of the WinO method over conventional techniques.

When compared to ISD, they discovered that the WinO approach increased protein and peptide recovery by ten-fold. The considerable improvement was ascribed to less contact between the sample container and the extraction fluid.

The scientists also compared the derived proteome profiles to assess the sensitivity of both approaches. They discovered a strong association between the proteomic profiles produced using WinO for 100 cells and ISD for 10,000 cells. Additionally, the group measured 462 proteins using WinO, proving that it had a significantly greater throughput and extraction efficiency than traditional methods.

With WinO’s improved protein recovery and identification capabilities, it may be possible to examine cancer cells’ protein expression in greater detail and gain a deeper knowledge of the mechanisms underlying drug resistance to treat cancer. Additionally, WinO may be semi-automated utilizing a liquid handling robot, making it appropriate for processing samples at high speeds and in huge volumes.

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.”

Takeshi Masuda, Assistant Professor, Kumamoto University