Deep proteomic characterization is informative of the molecular state as neurons mature to establish vast cell heterogeneity during development of the nervous system. However, detection of proteins in small populations of neurons requires improvements in mass spectrometry (MS) sensitivity. Here, we present a data-dependent acquisition (DDA) methodology that enables ultrasensitive characterization of proteins from limited populations of neurons in the mouse hippocampus using a custom-built microanalytical capillary electrophoresis (CE) nanoelectrospray ionization (nanoESI) MS. The purpose of the CE-nanoESI system is to electrophoretically separate and ionize peptides that are generated by digesting neuronal proteins with trypsin protease. The system enabled a ~260 zmol (156,000 copies) of limit of detection for angiotensin II, quantification over a 5-log order concentration range, and high separation efficiency (~350,000/m theoretical plates). These performance metrics allowed the identification of 15 amol (1 pg) of digested bovine serum albumin with 7.4% sequence coverage, raising sufficient sensitivity to measure proteins from limited neuron populations. To evaluate this, we utilized the CE-nanoESI-MS platform to measure ~500 pg of protein digest from hippocampal neurons, which estimates the total amount of protein contained in a ca. a handful of neurons. Our approach enabled the identification of 361 different protein groups with a 4-log order dynamic range, corresponding to 30% improvement compared to the control. Ultrasensitive CE-nanoESI-MS with DDA ladder raises new potentials to study how differential gene expression underlies neuron heterogeneity during early brain development.

Bottom-up proteomics for small neuron populations by ultrasensitive mass spectrometry

254th National Meeting (2017)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.