In VivoSubcellular Mass Spectrometry Enables Proteo-Metabolomic Single-cell Systems Biology in a Chordate Embryo Developing to a Normally Behaving Tadpole (X. laevis)

Abstract

We present the first example of in vivo high-resolution mass spectrometry (HRMS) for subcellular molecular systems biology of proteins and metabolites. With light microscopy, we identified the left-dorsal and left-ventral animal cells in cleavage-stage non-sentient Xenopus laevis embryos. Using precision-translated fabricated microcapillaries, the subcellular content of each cell was double-probed, each time collecting <5% of cell volume (~10 nL) swiftly (<5 s/event). The proteins and metabolites were analyzed by custom-built ultrasensitive capillary electrophoresis electrospray ionization employing Orbitrap and time-of-flight HRMS. Label-free detection of ~150 metabolites (57 identified) and 738 proteins found proteo-metabolomic networks with differential quantitative activities between the cell types. Spatially and temporally scalable sampling the technology preserved the integrity of the analyzed cells, the neighboring cells, and the embryo. 95% of the analyzed embryos developed into sentient tadpoles that were indistinguishable from their wild-type siblings based on anatomy and visual function in a background color preference assay.

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

“…The goal of this study was to integrate electrophysiology with single-cell HRMS proteomics. Our recent work adapted in situ , and in vivo capillary microsampling to microanalytical CE-ESI-HRMS platforms to enable direct proteomics and metabolomics in large, identified cells (∼500 to 250 μm diameter) in developing chordate embryos. These instruments preserved cell and embryonic viability, allowing for evaluations of anatomy and whole-organismal behavior .…”

“…Our recent work adapted in situ , and in vivo capillary microsampling to microanalytical CE-ESI-HRMS platforms to enable direct proteomics and metabolomics in large, identified cells (∼500 to 250 μm diameter) in developing chordate embryos. These instruments preserved cell and embryonic viability, allowing for evaluations of anatomy and whole-organismal behavior . In a series of studies, we also found the technology scalable to smaller cells and other functional measurements, including electrical recording of neuronal activity. − This report captures the development, testing, and validation of this technology following the systematic approach outlined in Figure .…”

“…Standard bottom-up proteomics requires extraction and digestion of the proteins, often also calling for chemical reduction/alkylation to deepen the coverage of the detectable proteome. These starting protein amounts, however, are ∼1–10 million times smaller than traditionally processed in MS-based bottom-up proteomics, ∼1–100 thousand times less than handled from single dissected cells, ,, and ∼100-times smaller than recently collected from individual cells using capillary microsampling. ,, As a simplified alternative to automated droplet processing, − we opted to perform shot-gun proteomics in Lo-Bind vials; these consumables are readily available in laboratories, thus supporting adoption of our approach (see the Experimental Section). While this platform ensured simplicity and facilitates methodology adoption in other laboratories, it inherently leads to protein losses on the surfaces of pipette tips and the vial.…”

“…CE aids HRMS detection with exquisite separation power to tackle complexomes and various methods of in-column enrichment to enhance sensitivity. , An international study spanning 16 CE–MS platforms between 13 laboratories found CE-HRMS a robust and viable technology . We − and others , custom-built microanalytical CE platforms and built ultrasensitive CE electrospray ionization (ESI) interfaces (reviewed in ref ) to quantify transcripts, proteins, peptides, and metabolites in single cells ex vivo, , in situ, , or in vivo . These studies yielded new tools for cell, developmental, and neurobiology and led to the discovery of molecules capable of altering normal tissue developmental trajectories in Xenopus laevis embryos …”

“…26 We 27−29 and others 30,31 custom-built microanalytical CE platforms and built ultrasensitive CE electrospray ionization (ESI) interfaces (reviewed in ref 23) to quantify transcripts, proteins, peptides, and metabolites in single cells ex vivo, 29,32 in situ, 33,34 or in vivo. 33 These studies yielded new tools for cell, developmental, and neurobiology and led to the discovery of molecules capable of altering normal tissue developmental trajectories in Xenopus laevis embryos. 28 These performance metrics make CE−HRMS proteomics attractive for integration with patch-clamp electrophysiology.…”