Enhanced Peptide Detection Toward Single-Neuron Proteomics by Reversed-Phase Fractionation Capillary Electrophoresis Mass Spectrometry

The ability to detect peptides and proteins in single cells is vital for understanding cell heterogeneity in the nervous system. Capillary electrophoresis (CE) nanoelectrospray ionization (nanoESI) provides high-resolution mass spectrometry (HRMS) with trace-level sensitivity, but compressed separation during CE challenges protein identification by tandem HRMS with limited MS/MS duty cycle. Here, we supplemented ultrasensitive CE-nanoESI-HRMSwith reversed-phase (RP) fractionation to enhance identifications from protein digest amounts that approximate to a few mammalian neurons. An similar to 1 to 20 mu g neuronal protein digest was fractionated on a RP column (ZipTip), and 1 ng to 500 pg of peptides were analyzed by a custombuilt CE-HRMS system. Compared with the control (no fractionation), RP fractionation improved CE separation (theoretical plates similar to 274,000 versus 412,000 maximum, resp.), which enhanced detection sensitivity (2.5-fold higher signal-to-noise ratio), minimized co-isolation spectral interferences during MS/MS, and increased the temporal rate of peptide identification by up to similar to 57%. From 1 ng of protein digest (< 5 neurons), CE withRP fractionation identified 737 protein groups (1,753 peptides), or similar to 480 protein groups (similar to 1,650 peptides) on average per analysis. The approach was scalable to 500 pg of protein digest (similar to a single neuron), identifying 225 protein groups (623 peptides) in technical triplicates, or 141 protein groups on average per analysis. Among identified proteins, 101 proteins were products of genes that are known to be transcriptionally active in single neurons during early development of the brain, including those involved in synaptic transmission and plasticity and cytoskeletal organization.