Free-flow zone electrophoresis facilitated proteomics analysis of heterogeneous subpopulations in H1299 lung cancer cells

Cancer cells are usually heterogeneous, whose subpopulations contain distinct biological information that related to their unique phenotype. However, preparative separating heterogeneous cells into subpopulations is very challenging, especially based on their surface charge status. Here, free-flow zone electrophoresis (FFZE) was employed to separate and fractionate lung cells. The experimental parameters for FFZE, including voltage, running speed, and buffer pH, were optimized and the optimized method was then implemented to separate MRC-5 and H1299 cells. Normal lung cells MRC-5 could be separated into one major peak and few minor peaks. For lung cancer cell line H1299, two major peaks and a few minor peaks were fractionated. Those prepared major peaks were subjected to quantitative proteomics analysis for heterogeneous subpopulation. For H1299 cells, 2076 proteins were identified in the first major peak, and 1398 proteins were revealed in the second major peak, which increased by 13% on average (n = 3) when compared to that of before FFZE fractionation. Furthermore, we found that H1299 cells in the first major peak have higher mobility than the cells in the second one. Many cell mobility and metastasis related proteins, such as RHBDF2, TRIM59, and ADAM17, were solely expressed in the first major peak of H1299 after FFZE, indicating our method can distinguish cancer cell subpopulations with higher metastatic phenotype. Our results showed that FFZE can effectively fractionate heterogeneous cell sub-populations, which might be used for the comprehensive analysis of unique phenotypes of different cancers.

Automated summary

Cancer cells are heterogeneous, and their subpopulations contain distinct biological information. In this study, free-flow zone electrophoresis was used to separate lung cells, and quantitative proteomics analysis was performed. The results showed that our method can distinguish cancer cell subpopulations with different metastatic phenotypes.