Microanalytical Mass Spectrometry with Super-Resolution Microscopy Reveals a Proteome Transition During Development of the Brain's Circadian Pacemaker.

During brain development, neuronal proteomes are regulated in part by changes in spontaneous and sensory-driven activity in immature neural circuits. A longstanding model for studying activity-dependent circuit refinement is the developing mouse visual system where the formation of axonal projections from the eyes to the brain is influenced by spontaneous retinal activity prior to the onset of vision and by visual experience after eye-opening. The precise proteomic changes in retinorecipient targets that occur during this developmental transition are unknown.

Single-Cell Mass Spectrometry of Metabolites and Proteins for Systems and Functional Biology.

Molecular composition is intricately intertwined with cellular function, and elucidation of this relationship is essential for understanding life processes and developing next-generational therapeutics. Technological innovations in capillary electrophoresis (CE) and liquid chromatography (LC) mass spectrometry (MS) provide previously unavailable insights into cellular biochemistry by allowing for the unbiased detection and quantification of molecules with high specificity. This chapter presents our validated protocols integrating ultrasensitive MS with classical tools of cell, developmental, and neurobiology to assess the biological function of important biomolecules.

Microanalysis of Brain Angiotensin Peptides Using Ultrasensitive Capillary Electrophoresis Trapped Ion Mobility Mass Spectrometry.

While the role of the renin-angiotensin system (RAS) in peripheral circulation is well characterized, we still lack an in-depth understanding of its role within the brain. This knowledge gap is sustained by lacking technologies for trace-level angiotensin detection throughout tissues, such as the brain. To provide a bridging solution, we enhanced capillary electrophoresis (CE) nanoflow electrospray ionization (ESI) with large-volume sample stacking and employed trapped ion mobility time-of-flight (timsTOF) tandem HRMS detection.

Capillary Electrophoresis Mass Spectrometry for Scalable Single-Cell Proteomics.

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.

Patch-Clamp Proteomics of Single Neurons in Tissue Using Electrophysiology and Subcellular Capillary Electrophoresis Mass Spectrometry.

Understanding of the relationship between cellular function and molecular composition holds a key to next-generation therapeutics but requires measurement of all types of molecules in cells. Developments in sequencing enabled semiroutine measurement of single-cell genomes and transcriptomes, but analytical tools are scarce for detecting diverse proteins in tissue-embedded cells. To bridge this gap for neuroscience research, we report the integration of patch-clamp electrophysiology with subcellular shot-gun proteomics by high-resolution mass spectrometry (HRMS).

Data-Dependent Acquisition Ladder for Capillary Electrophoresis Mass Spectrometry-Based Ultrasensitive (Neuro)Proteomics.

Measurement of broad types of proteins from a small number of cells to single cells would help to better understand the nervous system but requires significant leaps in sensitivity in high-resolution mass spectrometry (HRMS). Microanalytical capillary electrophoresis electrospray ionization (CE-ESI) offers a path to ultrasensitive proteomics by integrating scalability with sensitivity. Here, we systematically evaluate performance limitations in this technology to develop a data acquisition strategy with deeper coverage of the neuroproteome from trace amounts of starting materials than traditional dynamic exclusion.

Single-cell proteomics in complex tissues using microprobe capillary electrophoresis mass spectrometry.

Direct measurement of proteins produced by single cells promises to expand our understanding of molecular cell-to-cell differences (heterogeneity) and their contribution to normal and impaired development. High-resolution mass spectrometry (HRMS) is the modern technology of choice for the label-free identification and quantification of proteins, albeit usually in large populations of cells. Recent advances in microscale sample collection and processing, separation, and ionization have extended this powerful technology to single cells.

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-HRMS with reversed-phase (RP) fractionation to enhance identifications from protein digest amounts that approximate to a few mammalian neurons.

Tapered-Tip Capillary Electrophoresis Nano-Electrospray Ionization Mass Spectrometry for Ultrasensitive Proteomics: the Mouse Cortex.

Ultrasensitive characterization of the proteome raises the potential to understand how differential gene expression orchestrates cell heterogeneity in the brain. Here, we report a microanalytical capillary electrophoresis nano-flow electrospray ionization (CE-nanoESI) interface for mass spectrometry to enable the measurement of limited amounts of proteins in the mouse cortex. Our design integrates a custom-built CE system to a tapered-tip metal emitter in a co-axial sheath-flow configuration.