| Popis: |
Understanding the biochemistry of the cell requires measurement of all the molecules it produces. Single-cell proteomics recently became possible through advances in microanalytical sample preparation, separation by nano-flow liquid chromatography (nanoLC) and capillary electrophoresis (CE), and detection using electrospray ionization (ESI) high-resolution mass spectrometry (HRMS). Here, we demonstrate capillary microsampling CE-ESI-HRMS to be scalable to proteomics across broad cellular dimensions. This study established proof-of-principle using giant, ∼250-µm-diameter cells from embryos of the frog Xenopuslaevis and small, ∼35-µm-diameter neurons in culture from the mouse hippocampus. From ∼18 ng, or ∼0.2% of the total cellular proteome, subcellular analysis of the ventral-animal midline (V11) and equatorial (V12) cells identified 1,133 different proteins in a 16-cell embryo. CE-HRMS achieved ∼20-times higher sensitivity and doubled the speed of instrumental measurements compared to nanoLC, the closest neighboring single-cell technology of choice. Microanalysis was scalable to 722 proteins groups from ∼5 ng of cellular protein digest from identified left dorsal-animal midline cell (D11), supporting sensitivity for smaller cells. Capillary microsampling enabled the isolation and transfer of individual neurons from the culture, identifying 37 proteins between three different cells. A total of 224 proteins were detected from 500 pg of neuronal protein digest, which estimates to a single neuron. Serial dilution returned 157 proteins from sample amounts estimating to about half a cell (250 pg protein) and 70 proteins from ca. a quarter of a neuron (125 pg protein), suggesting sufficient sensitivity for subcellular proteomics. CE-ESI-HRMS complements nanoLC proteomics with scalability, sensitivity, and speed across broad cellular dimensions. |
