Saha-Shah A , Esmaeili M , Sidoli S , Hwang H , Yang J , Klein PS , Garcia BA .

R01 AI118891 NIAID NIH HHS , R01 GM110174 NIGMS NIH HHS , P01 CA196539 NCI NIH HHS , R01 GM111816 NIGMS NIH HHS , R35 GM131810 NIGMS NIH HHS , R01 HL141759 NHLBI NIH HHS , R01 GM115517 NIGMS NIH HHS

	Figure 1 (A) Schematic showing the work-flow for single cell proteomics study, (B) scanning electron micrograph of a micropipette showing end-on view, pipette sputter coated with 4 nm iridium for image quality. (C) Optical micrograph of a micropipette showing sideon view, (D) optical micrograph showing a micropipette puncturing the embryo. (E) A plot showing number of proteins detected from single Xenopus laevis blastomeres at various stages of development. The cell sizes decrease progressively from stage 1 (single cell stage) to 7 (128 cell stage). n=3replicates were used for the study (F) A correlation plot showing log2 transformed and normalized protein quantification data from blastomeres (ventral, 8 cell stage) sampled with pipette (in y-axis) and physically separated by dissection (x-axis),
	Figure 2. (A) A violin plot depicting the relative standard deviation in protein measurements (normalized iBAQ) across replicates belonging to same female and various females. Dorsal 1 (D1) and dorsal 2 (D2) cells from three females were used for the study. Median of each violin plot is listed in the figure. A time course plot showing decrease in abundance of vitellogenin A2 (B) and tubulin (C) with embryonic development. We see a sharp decrease in abundance of both proteins from 4 to 8 cell stage. Each trace in the plot represents a particular cell during 8 cell stage and its precursors at earlier stages. Each point in the plot (b and c) depicts one measurement, biological replicates are plotted separately (n=3 for 1- and 2 cell stage, n=4 for 4- and 8-cell stage).
	Figure 3. (A) Optical micrograph of an embryo at the 4-cell stage showing pigment asymmetry; the least pigmented cell is labeled dorsal 1 (D1) and the most pigmented cell is labeled ventral 2 (V2), scale: 0.5 mm (B) Significant differences in protein expression between two ventral cells and dorsal-ventral cells at the 4-cell stage is depicted in the volcano plot. (C) Volcano plots showing differences in protein composition between dorsal2 and ventral1 cell at both 4- and 8-cell stage. (D) A histogram depicting the number of differentially expressed proteins between cell types. Principal component analysis of proteome data at 4 (E) and 8 (F) cell stage, respectively.
	Figure 4. (A) A volcano plot showing differential localization of proteins across animal and vegetal hemispheres of embryos at the 8-cell stage (n=3). Marked in red are components of the 20S core particle that were found to be enriched in the animal hemisphere. In yellow are proteins enriched in the vegetal hemisphere and in green (also marked by arrow) is protein Hsc70 which is equally distributed across the animal and vegetal hemispheres. Enrichment of 20S CP in the animal hemisphere (B) and DDX4/VASA in the vegetal hemisphere (C) was further validated by immunofluorescence staining in sections of 8-cell embryos. Scale: 250 µm Equal distribution of Hsc70 protein was further validated by western blotting of protein extracted from individual animal vs. vegetal halves dissected from intact embryos (D). Western blot image from 6 embryos (lower panel) was scanned and quantitated by ImageJ, and fraction in each animal hemisphere relative to total for each embryo is plotted in the upper panel. l. The mean was found to be 0.52 with an error of 0.09 as depicted in the figure.

Aerts, Patch clamp electrophysiology and capillary electrophoresis-mass spectrometry metabolomics for single cell characterization. 2014, Pubmed

Aerts, Patch clamp electrophysiology and capillary electrophoresis-mass spectrometry metabolomics for single cell characterization. 2014, Pubmed
Bauer, Evaluation of data-dependent and -independent mass spectrometric workflows for sensitive quantification of proteins and phosphorylation sites. 2014, Pubmed
Baxi, Proteomic Characterization of the Neural Ectoderm Fated Cell Clones in the Xenopus laevis Embryo by High-Resolution Mass Spectrometry. 2018, Pubmed , Xenbase
Budnik, SCoPE-MS: mass spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation. 2018, Pubmed
Cox, MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. 2008, Pubmed
Cox, Andromeda: a peptide search engine integrated into the MaxQuant environment. 2011, Pubmed
Danilchik, Differentiation of the animal-vegetal axis in Xenopus laevis oocytes. I. Polarized intracellular translocation of platelets establishes the yolk gradient. 1987, Pubmed , Xenbase
De Robertis, Dorsal-ventral patterning and neural induction in Xenopus embryos. 2004, Pubmed , Xenbase
Flachsova, Single blastomere expression profiling of Xenopus laevis embryos of 8 to 32-cells reveals developmental asymmetry. 2013, Pubmed , Xenbase
Gawad, Single-cell genome sequencing: current state of the science. 2016, Pubmed
Gillet, Targeted data extraction of the MS/MS spectra generated by data-independent acquisition: a new concept for consistent and accurate proteome analysis. 2012, Pubmed
Gong, Single cell analysis with probe ESI-mass spectrometry: detection of metabolites at cellular and subcellular levels. 2014, Pubmed
Guillaume-Gentil, Single-Cell Mass Spectrometry of Metabolites Extracted from Live Cells by Fluidic Force Microscopy. 2017, Pubmed
Gupta, Quantitative Proteomics of Xenopus Embryos I, Sample Preparation. 2018, Pubmed , Xenbase
Gustafson, Vasa genes: emerging roles in the germ line and in multipotent cells. 2010, Pubmed
Heath, Single-cell analysis tools for drug discovery and development. 2016, Pubmed
Hwang, Novel functions of the ubiquitin-independent proteasome system in regulating Xenopus germline development. 2019, Pubmed , Xenbase
Jorgensen, The mechanism and pattern of yolk consumption provide insight into embryonic nutrition in Xenopus. 2009, Pubmed , Xenbase
King, Putting RNAs in the right place at the right time: RNA localization in the frog oocyte. 2005, Pubmed , Xenbase
Komiya, Isolation and characterization of a novel gene of the DEAD box protein family which is specifically expressed in germ cells of Xenopus laevis. 1994, Pubmed , Xenbase
Larabell, Establishment of the dorso-ventral axis in Xenopus embryos is presaged by early asymmetries in beta-catenin that are modulated by the Wnt signaling pathway. 1997, Pubmed , Xenbase
Li, Single-Cell Proteomics for Cancer Immunotherapy. 2018, Pubmed
Lombard-Banek, Single-Cell Mass Spectrometry for Discovery Proteomics: Quantifying Translational Cell Heterogeneity in the 16-Cell Frog (Xenopus) Embryo. 2016, Pubmed , Xenbase
Lombard-Banek, Label-free Quantification of Proteins in Single Embryonic Cells with Neural Fate in the Cleavage-Stage Frog (Xenopus laevis) Embryo using Capillary Electrophoresis Electrospray Ionization High-Resolution Mass Spectrometry (CE-ESI-HRMS). 2016, Pubmed , Xenbase
Mowry, RNA sorting in Xenopus oocytes and embryos. 1999, Pubmed , Xenbase
Onjiko, Single-cell mass spectrometry reveals small molecules that affect cell fates in the 16-cell embryo. 2015, Pubmed , Xenbase
Onjiko, In Situ Microprobe Single-Cell Capillary Electrophoresis Mass Spectrometry: Metabolic Reorganization in Single Differentiating Cells in the Live Vertebrate (Xenopus laevis) Embryo. 2017, Pubmed , Xenbase
Onjiko, Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog (Xenopus laevis) Embryos. 2017, Pubmed , Xenbase
Pan, The single-probe: a miniaturized multifunctional device for single cell mass spectrometry analysis. 2014, Pubmed
Portero, Dual cationic-anionic profiling of metabolites in a single identified cell in a live Xenopus laevis embryo by microprobe CE-ESI-MS. 2019, Pubmed , Xenbase
Qian, Detection of single cell heterogeneity in cancer. 2017, Pubmed
Saha-Shah, Segmented flow sampling with push-pull theta pipettes. 2016, Pubmed
Saha-Shah, Local collection, reaction and analysis with theta pipette emitters. 2017, Pubmed
Saha-Shah, Nanopipettes: probes for local sample analysis. 2015, Pubmed
Savage, Dynamics of germ plasm localization and its inhibition by ultraviolet irradiation in early cleavage Xenopus embryos. 1993, Pubmed , Xenbase
Schmidt, Absolute quantification of microbial proteomes at different states by directed mass spectrometry. 2011, Pubmed
Schwanhäusser, Global quantification of mammalian gene expression control. 2011, Pubmed
Searle, Chromatogram libraries improve peptide detection and quantification by data independent acquisition mass spectrometry. 2018, Pubmed
Shao, Integrated Proteome Analysis Device for Fast Single-Cell Protein Profiling. 2018, Pubmed
Sidova, Intracellular microRNA profiles form in the Xenopus laevis oocyte that may contribute to asymmetric cell division. 2015, Pubmed , Xenbase
Sindelka, Asymmetric distribution of biomolecules of maternal origin in the Xenopus laevis egg and their impact on the developmental plan. 2018, Pubmed , Xenbase
Spitzer, Mass Cytometry: Single Cells, Many Features. 2016, Pubmed
Sun, Quantitative proteomics of Xenopus laevis embryos: expression kinetics of nearly 4000 proteins during early development. 2014, Pubmed , Xenbase
Sun, Single Cell Proteomics Using Frog (Xenopus laevis) Blastomeres Isolated from Early Stage Embryos, Which Form a Geometric Progression in Protein Content. 2016, Pubmed , Xenbase
Vogel, Insights into the regulation of protein abundance from proteomic and transcriptomic analyses. 2012, Pubmed
Wühr, Deep proteomics of the Xenopus laevis egg using an mRNA-derived reference database. 2014, Pubmed , Xenbase
Yarger, Probe-Substrate Distance Control in Desorption Electrospray Ionization. 2018, Pubmed
Yin, Quantitative Extraction and Mass Spectrometry Analysis at a Single-Cell Level. 2018, Pubmed
Zhu, Nanodroplet processing platform for deep and quantitative proteome profiling of 10-100 mammalian cells. 2018, Pubmed