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Front Chem
2022 Jan 01;10:863979. doi: 10.3389/fchem.2022.863979.
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Capillary Electrophoresis Mass Spectrometry for Scalable Single-Cell Proteomics.
Shen B
,
Pade LR
,
Choi SB
,
Muñoz-LLancao P
,
Manzini MC
,
Nemes P
.
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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. This study established proof-of-principle using giant, ∼250-µm-diameter cells from embryos of the frog Xenopus laevis and small, ∼35-µm-diameter neurons in culture from the mouse hippocampus. From ∼18 ng, or ∼0.2% of the total cellular proteome, subcellular analysis of the ventral-animal midline (V11) and equatorial (V12) cells identified 1,133 different proteins in a 16-cell embryo. CE-HRMS achieved ∼20-times higher sensitivity and doubled the speed of instrumental measurements compared to nanoLC, the closest neighboring single-cell technology of choice. Microanalysis was scalable to 722 proteins groups from ∼5 ng of cellular protein digest from identified left dorsal-animal midline cell (D11), supporting sensitivity for smaller cells. Capillary microsampling enabled the isolation and transfer of individual neurons from the culture, identifying 37 proteins between three different cells. A total of 224 proteins were detected from 500 pg of neuronal protein digest, which estimates to a single neuron. Serial dilution returned 157 proteins from sample amounts estimating to about half a cell (250 pg protein) and 70 proteins from ca. a quarter of a neuron (125 pg protein), suggesting sufficient sensitivity for subcellular proteomics. CE-ESI-HRMS complements nanoLC proteomics with scalability, sensitivity, and speed across broad cellular dimensions.
FIGURE 1. CE-HRMS for scalable single-cell proteomics. The protein content was collected from identified cells in Xenopus laevis embryos and neurons cultured from the mouse hippocampus. The example labels the normally neural-tissue fated dorsal-animal midline cell (D11) and the normally epidermally fated ventral-animal equatorial cell (V12). The proteins were detected on a custom-built microanalytical capillary electrophoresis (CE) electrospray ionization (ESI) platform using orbitrap (OT) high-resolution tandem mass spectrometry (HRMS-MS/MS).
FIGURE 2. Scalable collection of trace amounts of proteins from single cells for HRMS detection. (A) Example showing microaspiration of cell contents into a microfabricated glass pipette from a D, D1, and D11 blastomere in a 4, 8-, and 16-cell X. laevis embryo. Scale, 250 µm. (B) Collection of a single cultured mouse neuron into a fabricated microcapillary. Scale, 20 µm. (C) Representative base-peak ion signal traces recorded between m/z 350–1,500 from 400 ng of digest using nanoLC and 10 ng of protein digest using CE, revealing enhancement in sensitivity and speed by electrophoresis.
FIGURE 3. Benchmarking the analytical figures of merit. (A) Protein identifications in the V12 X. laevis cell from ∼400 ng of protein digest by nanoLC and ∼18 ng of protein digest by CE HRMS. (B) Single-shot analyses identifying (ID) complementary types of proteins. The CE experiment quantified more proteins than nanoLC. (C) Pearson correlation analysis of proteins that were quantifiable by both technologies. (D) Statistical comparison of protein quantification (Wilcoxon signed tests) revealing indistinguishable abundance distribution for commonly quantified proteins. Proteins that were only quantifiable by CE were detected in statistically higher abundance.
FIGURE 4. Single-to-subcellular neuroproteomics by CE-HRMS. (A) Comparison of the linear dynamic range and depth of the quantified neuroproteome from diluted proteome digests estimating to a single neuron (∼500 pg protein digest), a half neuron (∼250 pg), and quarter of a neuron (∼125 pg). (B) Comparison of proteins identified from the diluted digests.
FIGURE 5. Reactome analysis of protein-protein interactions from a single-neuron equivalent protein digest (500 pg proteome). More than 700 pathways were represented (Supplementary Table S7). Detected nodes are highlighted in yellow. Pathway key: (A), muscle contraction; (B), digestion and absorption; (C), developmental biology; (D), neuronal system; (E), immune system; (F), signal transduction; (G), hemostasis; (H), gene expression; (I), metabolism of RNA; (J), metabolism; (K), chromatin organization; (L), DNA repair; (M), DNA replication; (N), transport of small molecules; (O), sensory perception; (P), cell cycle; (Q), reproduction; (R), organelle biogenesis and maintenance; (S), metabolism of proteins; (T), programmed cell death; (U), cellular responses to stimuli; (V), autophagy; (W), protein localization; (X), vesicle-mediated transport; (Y), extracellular matrix organization; (Z), cell-cell communication.
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