Quantification of Low Copy Number Proteins in Single Cells.

We have developed an ultrasensitive and highly selective method to quantify low copy number intracellular proteins in a single cell using a low-cost laser-induced fluorescence (LIF) detector and a BV605 fluorescent probe. Active caspase3 proteins in cells were labeled by corresponding antibody-BV605 fluorescent binding, and a cell was injected into a 20 cm × 50 μm i.d. capillary column, followed by in situ lysis and capillary electrophoresis (CE)-LIF analysis. About seven active caspase3 protein molecules in a detection volume of 91 pL could be detected. In our method, cross-bounding proteins other than active caspase3 could be separated and distinguished by differences of retention time. By using Si photodiode assembly as a fluorescent detector instead of PMT, the dynamic range of the LIF is over 4 orders of magnitude. In this experiment, we found that the number of active caspase3 molecules in 98 single Jurkat cells were from 629 to 12171, reflecting significant heterogeneity among the cells although they were from the same batch. For extended application, it could also be applied to quantify other types of low copy number proteins in a single cell as long as the corresponding antibodies are provided. This high-sensitive method could also be a promising tool for earlier cancer diagnosis and related disease pathway research which is relevant to low copy number proteins. In addition, this low-cost system could also be easily expanded to an array system for high-throughput quantitation of low copy proteins in single cells.