Enhanced Peptide Detection Toward Single-Neuron Proteomics by Reversed-Phase Fractionation Capillary Electrophoresis Mass Spectrometry
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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-HRMS with reversed-phase (RP) fractionation to enhance identifications from protein digest amounts that approximate to a few mammalian neurons. An ~1 to 20 μg neuronal protein digest was fractionated on a RP column (ZipTip), and 1 ng to 500 pg of peptides were analyzed by a custom-built CE-HRMS system. Compared with the control (no fractionation), RP fractionation improved CE separation (theoretical plates ~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 ~57%. From 1 ng of protein digest (<5 neurons), CE with RP fractionation identified 737 protein groups (1,753 peptides), or ~480 protein groups (~1,650 peptides) on average per analysis. The approach was scalable to 500 pg of protein digest (~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.
KeywordsCapillary electrophoresis Mass spectrometry Fractionation Bottom-up proteomics Neurons Hippocampus Mouse Central nervous system
This work was supported by the Arnold and Mabel Beckman Foundation Young Investigator Grant (to P.N.), the American Society for Mass Spectrometry Research Award (to P.N.), the DuPont Young Professor Award (to P.N.), and Cosmos Club Foundation Fellowships (to S.B.C. and C.L-B.).
P.N., S.B.C., and C.L-B. designed the research; P.M-L. and M.C.M. provided the neuron cultures; S.B.C. and C.L-B. processed the samples; S.B.C. measured the samples; P.N. and S.B.C. analyzed the data; P.N. and S.B.C. wrote the manuscript. All authors commented on the manuscript.
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