A New Approach For High Through-put Single Cell Proteomics

The advent of post-genomic era entails the study of protein expression and protein-protein interaction networks.Recently,researchers have discovered that heterogeneity is inherent in many cellular processes including stem cell differentiation,development,oncogenesis,and immune response.Therefore,to fully understand the diverse and often rare behaviors in complex cell populations,development of analytical approaches for protein analysis at single-cell resolution is essential for detecting target proteins with high specificity and throughput.Traditional methods for the analysis of single cell proteomics,including flow cytometry and mass spectrometry,have shown advantages of high throughput and sensitivity,facilitating the development of single cell proteomics research.However,these techniques fail to obtain dynamic analysis from individual cells.Recently,microfluidic chip has becoming an attractive tool for the analysis of single cell proteomics thanks to its high-throughput and good biological compatibility.Moreover,the size of microfluidic chip microchannels can perfectly fit with a single cell.Herein,we developed a high-throughput microfluidic chip based on polyacrylamide gel electrophoresis to fast separate proteins of a single cell with laser induced real time fluorescence as the high sensitive detection system.To optimize the system for single cell proteomics research,we investigated the size of microchannels,the time for cell lysis and the applied separation voltage.Results demonstrated that the developed system could realize real time detection of single cell proteomics.Further work focused on the analysis of cell heterogeneity and cysteine cathepsins inside the lysosome at the single-cell resolution.Cysteine cathepsins were covalently labeled,employing the membrane permeability of the ABP.We compared the expression level of the cysteine cathepsins derived from a single cell and a cell population to investigate the heterogeneity of protein expression.In addition,we analyzed the cellular heterogeneity by using the genetic and fusion protein probe CD3.Results showed that only one minute was needed to separate the protein from a single cell and the analysis of single cell proteomics could be completed in two hours.As a result,cysteine protease expression was heterogeneous.In summary,a new PAGE-based microfluidic device was developed to enable the automatic formation of single cell array for rapid and high-throughput analysis the protein expression.We expect the new method to be a useful tool for fast analysis of single-cell proteomics.