Single-Cell Chemical Proteomics with an Activity-Based Probe: Identification of Low-Copy Membrane Proteins on Primary Neurons

We propose a novel single‐cell chemical proteomics (SCCP) strategy to profile low‐abundance membrane proteins in single cells. In this approach, the membrane protein GB1 and its splicing variants were targeted on cultured cell lines and primary neurons using a specifically designed activity‐based probe. The functionally labeled single cells were encapsulated in individual buffer droplets on a PDMS microwell array, and were further picked up one at a time and loaded into a capillary electrophoresis system for cell lysis, separation, and laser‐induced fluorescence detection of the targeted proteins. The results revealed the expression of GB1 splicing variants in HEK and MEF cells, which was previously only suggested at the transcriptional level. We further applied this method to investigate single primary cells and observed significant heterogeneity among individual mouse cerebellar granule neurons. Interference experiments with GB1 antagonist and agonist validated this observation. Singles only: A single‐cell chemical proteomics strategy with an activity‐based probe (ABP) enables observation of low‐abundance membrane receptors at the single‐cell level by discriminating them from the whole proteome. The strategy avoids problems associated with cytometry‐based techniques, and could be used to identify low‐copy receptors on the primary neurons of animals.