Single cell proteomics breakthrough

Professor Fang Qun of the Department of Chemistry of Zhejiang University’s Institute of Microanalysis Systems teamed up with Prof. Huang Chaolan, the director of the Precision Medicine Multigroup Research Center of the Department of Medicine of Peking University, and achieved breakthroughs in the field of single-cell proteomics analysis. Research papers were recently published online in the Analytical Chemistry magazine.

According to Huang Chaolan, in recent years, research on proteomics in cell populations has become increasingly difficult to meet the need for in-depth exploration of life functions. From the single-cell level to understand the characteristics of cells and their interactions with each other, it can provide more valuable information for intercellular heterogeneity in biological systems. However, the protein content in a single cell is very small, and intracellular proteins usually require complicated multi-step pretreatment operations in a centrifuge tube. Due to the contact between the sample and the centrifuge tube, multi-step sample transfer, etc., it is not possible to enter the mass spectrometer before entering the mass spectrometer. Loss of protein has been avoided.

For this reason, this research has been collaborating on the combination of microfluidic droplet technology and proteome analysis technology since 2014, and developed a miniaturized oil-gas-liquid "sandwich" chip. The corresponding nanoscale liquid manipulation and injection methods enable multistep sample preparation operations necessary for small-scale proteomic profiling in in situ static nanoliter droplets, and achieve direct and efficient droplet sampling. Into the chromatographic column to complete the subsequent liquid chromatography and mass spectrometry detection. The chip system successfully identified 1,360, 612, 192 and 51 proteins from 100, 50, 10 and 1 HeLa cells, respectively. For the first time, the researchers achieved a proteomic analysis using a single mouse oocyte as the initial sample. A total of 355 proteins were identified, including a series of genes related to reproductive development and disease. Moreover, the microfluidic droplet system has lower sample adsorption loss, higher enzyme digestion efficiency and more efficient identification of hydrophobic proteins, and is more suitable for proteomic analysis of trace protein samples.

Huang Chaolan introduced that this research first successfully developed a proteomic analysis sample preparation chip and method suitable for single-cell and similar micro-samples, which significantly reduced the loss of contact between the sample and the reactor; second, developed a The direct injection method for nanoliter droplets completely avoids the loss of the sample transfer and the intricate piping in the liquid-phase instrument; the third is the introduction of a new droplet that uses the gas phase to separate the droplet phase and the oil phase. The chip structure, while successfully preventing the droplets from evaporating, also avoids the loss of fat-soluble samples due to direct contact between the oil phase and the droplet phase, and also allows the system to more easily perform subsequent separation column injections and chromatographic separations. And mass spectrometry detection.

The first author of this paper was Li Ziyi, a doctoral student at Zhejiang University. (Reporter Fu Donghong correspondent Li Ziyi)

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