In Vivo Subcellular Mass Spectrometry Enables Proteo‐Metabolomic Single‐Cell Systems Biology in a Chordate Embryo Developing to a Normally Behaving Tadpole (X. laevis)**

Abstract

We report the development of in vivo subcellular high-resolution mass spectrometry (HRMS) for proteo-metabolomic molecular systems biology in complex tissues.W ith light microscopy, we identified the left-dorsal and left-ventral animal cells in cleavage-stage non-sentient Xenopus laevis embryos.U sing precision-translated fabricated microcapillaries,t he subcellular content of each cell was double-probed, each time swiftly (< 5s/event) aspirating < 5% of cell volume ( % 10 nL). The proteins and metabolites were analyzed by home-built ultrasensitive capillary electrophoresis electrospray ionization employing orbitrap or time-of-flight HRMS.Labelfree detection of % 150 metabolites (57 identified) and 738 proteins found proteo-metabolomic networks with differential quantitative activities between the cell types.With spatially and temporally scalable sampling, the technology preserved the integrity of the analyzed cells,t he neighboring cells,a nd 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 abackground color preference assay.

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

“…Our recent work adapted in situ 27,34 and in vivo 33 capillary microsampling to microanalytical CE-ESI-MS platforms to enable direct proteomics and metabolomics in large, identified cells (~500to-250 µm diameter) in developing chordate embryos. These instruments preserved cell and embryonic viability, allowing for evaluations of anatomy and whole-organismal behavior.…”

A Data-Dependent Acquisition Ladder for Ultrasensitive (Neuro)Proteomics

“…CE-HRMS enabled the analysis of hundreds to thousands of different molecules in single cells and limited protein digests in exceptional sensitivity, in cases ~200-1,000-times higher than nanoLC. 4,[14][15][16][17][18] Biomolecules in these experiments migrated in a considerably short time frame, typically with an effective separation window lasting ~15-30 min. For complex 'omes, particularly an expanded peptidome in bottomup proteomics, compressed separation taxes identification due to limitations in HRMS-MS/MS sensitivity and speed.…”

“…Continuous advances at multiple stages of the proteomic workflow raise the possibility of further sensitivity improvements. Technologies enabling the isolation and handling of miniscule amounts of proteins with reduced loss, for example, by in vivo microsampling 16 , nanoPOTS 9 , and ScoPE 10 offer viable solutions for biopsies and limited populations of cells, including single cells. Detection of 428 proteins from protein amounts estimating ~10 neurons in this study, including many with important biological functions in homeostasis and disease, marks another technological leap in ultrasensitive proteomics, expanding the bioanalytical toolbox of neuroscience.…”

“…We and others developed robust methodologies and custom-built CE-ESI instruments capable of zeptomole-femtomole sensitivity (reviewed in references [11][12] ). For example, ~200 protein groups were identified from 5 ng of Pyrococcus furiosys digest 13 and up to 1,209 proteins groups from single embryonic cells dissected from [14][15] or analyzed in situ/vivo [16][17] in chordate embryos of important biological models (Xenopus laevis, zebrafish). A self-built CE platform and a sheath-flow tapered-tip CE-ESI design equipped HRMS with ~260 zmol sensitivity (156,000 copies) and robust operation on a quadrupole time-of-flight (TOF) mass spectrometer, allowing us to identify ~217 protein groups from ~1 ng protein (approximating ca.…”

Exploring functional protein covariation across single cells using nPOP

“…The large volumes and cleanup procedures entail sample losses that may be prohibitive for small samples, such as single mammalian cells 1,5,17 . Thus, numerous methods have been developed for preparing sub-microgram protein samples [22][23][24][25] and single-cell samples [26][27][28][29][30][31][32][33][34][35] . To enable some degree of parallel processing, some methods have been automated using multiwell plates 32,35,36 .…”