Ultrasensitive Proteome Profiling for 100 Living Cells by Direct Cell Injection, Online Digestion and Nano-LC-MS/MS Analysis

Single-cell proteome analysis has always been an exciting goal because it provides crucial information about cellular heterogeneity and dynamic change. Here we presented an integrated proteome analysis device (iPAD) for 100 living cells (iPAD-100) that might be suitable for single-cell analysis. Once cells were cultured, the iPAD-100 could be applied to inject 100 living cells, to transform the living cells into peptides, and to produce protein identification results with total automation. Due to the major obstacle for detection limit of mass spectrometry, we applied the iPAD-100 to analyze the proteome of 100 cells. In total, 813 proteins were identified in a DLD-cell proteome by three duplicate runs. Gene Ontology analysis revealed that proteins from different cellular compartments were well-represented, including membrane proteins. The iPAD-100 greatly simplified the sampling process, reduced sample loss, and prevented contamination. As a result, proteins whose copy numbers were lower than 1000 were identified from 100-cell samples with the iPAD-100, showing that a detection limit of 200 zmol was achieved. With increased sensitivity of mass spectrometry, the iPAD-100 may be able to reveal bountiful proteome information from a single cell in the near future.