We advance mass spectrometry from a cell population‐averaging tool to one capable of quantifying the expression of diverse proteins in single embryonic cells. Our instrument combines capillary electrophoresis (CE), electrospray ionization, and a tribrid ultrahigh‐resolution mass spectrometer (HRMS) to enable untargeted (discovery) proteomics with ca. 25 amol lower limit of detection. CE‐μESI‐HRMS enabled the identification of 500–800 nonredundant protein groups by measuring 20 ng, or <0.2% of the total protein content in single blastomeres that were isolated from the 16‐cell frog (Xenopus laevis) embryo, amounting to a total of 1709 protein groups identified between n=3 biological replicates. By quantifying ≈150 nonredundant protein groups between all blastomeres and replicate measurements, we found significant translational cell heterogeneity along multiple axes of the embryo at this very early stage of development when the transcriptional program of the embryo has yet to begin.

Label-free single-cell proteomics by mass spectrometry (MS) is currently incompatible with complex tissues without requiring cell culturing, single-cell dissection, or tissue dissociation. We here report the first example of label-free single-cell MS-based proteomics directly in single cells in live vertebrate embryos. Our approach integrates optically guided in situ subcellular capillary microsampling, one-pot extraction/digestion of the collected proteins, peptide separation by capillary electrophoresis, ionization by an ultrasensitive electrokinetically pumped nanoelectrospray, and detection by high-resolution MS (Orbitrap). With a 700-zmol (420,000 copies) lower limit of detection, this trace-sensitive technology confidently identified and quantified ~750-800 protein groups (<1% false discovery rate) by analyzing just ~5 ng of protein digest, viz. <0.05% of the total protein content from individual cells in the 16-cell Xenopus laevis (frog) embryo. After validating the approach by recovering animal-vegetal pole proteomic asymmetry in the frog zygote, the technology was used to uncover proteomic reorganization as the dorsal-animal (D11) cell of the 16-cell embryo gave rise to its neural-tissue fated clone in the embryo developing to the 32-, 64-, and 128-cell stage. In addition to enabling proteomics on smaller cells in X. laevis, we also demonstrated this technology to be scalable to single cells in live zebrafish embryos. Microsampling single-cell MS-based proteomics raises exciting opportunities to study cell and developmental processes directly in complex tissues and whole organisms at the (sub)level and of the building block of life: the cell.

Quantification of protein expression in single cells promises to advance a systems-level understanding of normal development. Using a bottom-up proteomic workflow and multiplexing quantification by tandem mass tags, we recently demonstrated relative quantification between single embryonic cells (blastomeres) in the frog (Xenopus laevis) embryo. In this study, we minimize derivatization steps to enhance analytical sensitivity and use label-free quantification (LFQ) for single Xenopus cells. The technology builds on a custom-designed capillary electrophoresis microflow-electrospray ionization high-resolution mass spectrometry platform and LFQ by MaxLFQ (MaxQuant). By judiciously tailoring performance to peptide separation, ionization, and data-dependent acquisition, we demonstrate an ϳ75-amol (ϳ11 nM) lower limit of detection and quantification for proteins in complex cell digests. The platform enabled the identification of 438 nonredundant protein groups by measuring 16 ng of protein digest, or <0.2% of the total protein contained in a blastomere in the 16-cell embryo. LFQ intensity was validated as a quantitative proxy for protein abundance. Correlation analysis was performed to compare protein quantities between the embryo and n ‫؍‬ 3 different single D11 blastomeres, which are fated to develop into the nervous system. A total of 335 nonredundant protein groups were quantified in union between the single D11 cells spanning a 4 log-order concentration range. LFQ and correlation analysis detected expected proteomic differences between the whole embryo and blastomeres, and also found translational differences between individual D11 cells. LFQ on single cells raises exciting possibilities to study gene expression in other cells and models to help better understand cell processes on a systems biology level. Molecular & Cellular

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. An ~1 to 20 μg neuronal protein digest was fractionated on a RP column (ZipTip), and 1 ng to 500 pg of peptides were analyzed by a custom-built CE-HRMS system. Compared with the control (no fractionation), RP fractionation improved CE separation (theoretical plates ~274,000 versus 412,000 maximum, resp.), which enhanced detection sensitivity (2.5-fold higher signal-to-noise ratio), minimized co-isolation spectral interferences during MS/MS, and increased the temporal rate of peptide identification by up to ~57%. From 1 ng of protein digest (<5 neurons), CE with RP fractionation identified 737 protein groups (1,753 peptides), or ~480 protein groups (~1,650 peptides) on average per analysis. The approach was scalable to 500 pg of protein digest (~a single neuron), identifying 225 protein groups (623 peptides) in technical triplicates, or 141 protein groups on average per analysis. Among identified proteins, 101 proteins were products of genes that are known to be transcriptionally active in single neurons during early development of the brain, including those involved in synaptic transmission and plasticity and cytoskeletal organization. Graphical abstract ᅟ.

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.

The renin angiotensin system (RAS) of the brain produces a series of biologically active angiotensinogen derived peptides involved in physiological homeostasis and pathophysiology of disease. Despite significant research efforts to date, a comprehensive understanding of brain RAS physiology is lacking. A significant challenge has been the limited set of bioanalytical assays capable of detecting angiotensin (Ang) peptides at physiologically low concentrations (2-15 fmol/g of wet tissue) and sufficient chemical specificity for unambiguous molecular identifications. Additionally, a complex brain anatomy calls for microanalysis of specific tissue regions, thus further taxing sensitivity requirements for identification and quantification in studies of the RAS. To fill this technology gap, we here developed a microanalytical assay by coupling a laboratory-built capillary electrophoresis (CE) nanoelectrospray ionization (nanoESI) platform to a high-resolution mass spectrometer (HRMS). Using parallel reaction monitoring, we demonstrated that this technology achieved confident identification and quantification of the Ang peptides at approx. 10 amol to hundreds of zmol sensitivity. This microanalytical assay revealed differential Ang peptide profiles between tissues that were micro-sampled from the subfornical organ and the paraventricular nucleus of the hypothalamus, important brain regions involved in thirst and water homeostasis and neuroendocrine regulation to stress. Microanalytical CE-nanoESI-HRMS extends the analytical toolbox of neuroscience to help better understand the RAS.

We advance mass spectrometry from ac ell population-averaging tool to one capable of quantifying the expression of diverse proteins in single embryonic cells.O ur instrument combines capillary electrophoresis (CE), electrospray ionization, and at ribrid ultrahigh-resolution mass spectrometer (HRMS) to enable untargeted (discovery) proteomics with ca. 25 amol lower limit of detection. CE-mESI-HRMS enabled the identification of 500-800 nonredundant protein groups by measuring 20 ng,o r< 0.2% of the total protein content in single blastomeres that were isolated from the 16-cell frog (Xenopus laevis) embryo,amounting to atotal of 1709 protein groups identified between n = 3b iological replicates.B y quantifying % 150 nonredundant protein groups between all blastomeres and replicate measurements,w ef ound significant translational cell heterogeneity along multiple axes of the embryo at this very early stage of development when the transcriptional program of the embryo has yet to begin.

The molecular program by which embryonic ectoderm is induced to form neural tissue is essential to understanding normal and impaired development of the central nervous system. Xenopus has been a powerful vertebrate model in which to elucidate this process. However, abundant vitellogenin (yolk) proteins in cells of the early Xenopus embryo interfere with protein detection by high-resolution mass spectrometry (HRMS), the technology of choice for identifying these gene products. Here, we systematically evaluated strategies of bottom-up proteomics to enhance proteomic detection from the neural ectoderm (NE) of X. laevis using nanoflow high-performance liquid chromatography (nanoLC) HRMS. From whole embryos, high-pH fractionation prior to nanoLC-HRMS yielded 1319 protein groups vs 762 proteins without fractionation (control). Compared to 702 proteins from dorsal halves of embryos (control), 1881 proteins were identified after yolk platelets were depleted via sucrose-gradient centrifugation. We combined these approaches to characterize protein expression in the NE of the early embryo. To guide microdissection of the NE tissues from the gastrula (stage 10), their precursor (midline dorsal-animal, or D111) cells were fate-mapped from the 32-cell embryo using a fluorescent lineage tracer. HRMS of the cell clones identified 2363 proteins, including 147 phosphoproteins (without phosphoprotein enrichment), transcription factors, and members from pathways of cellular signaling. In reference to transcriptomic maps of the developing X. laevis, 76 proteins involved in signaling pathways were gene matched to transcripts with known enrichment in the neural plate. Besides a protocol, this work provides qualitative proteomic data on the early developing NE.