Cell lineage-guided mass spectrometry reveals increased energy metabolism and reactive oxygen species in the vertebrate organizer.

Molecular understanding of the vertebrate Organizer, a tissue center critical for inductive signaling during gastrulation, has so far been mostly limited to transcripts and a few proteins, the latter due to limitations in detection and sensitivity. The Spemann-Mangold Organizer (SMO) in the South African Clawed Frog (X. laevis), a popular model of development, has long been known to be the origin of signals that pattern the mesoderm and central nervous system. Molecular screens of the SMO have identified several genes responsible for the ability of the SMO to establish the body axis. Nonetheless, a comprehensive study of proteins and metabolites produced specifically in the SMO and their functional roles has been lacking. Here, we pioneer a deep discovery proteomic and targeted metabolomic screen of the SMO in comparison to the remainder of the embryo using high-resolution mass spectrometry (HRMS). Quantification of ~4,600 proteins and a panel of targeted metabolites documented differential expression for 460 proteins and multiple intermediates of energy metabolism in the SMO. Upregulation of oxidative phosphorylation and redox regulatory proteins gave rise to elevated oxidative stress and an accumulation of reactive oxygen species in the SMO. Imaging experiments corroborated these findings, discovering enrichment of hydrogen peroxide in the SMO. Chemical perturbation of the redox gradient perturbed mesoderm involution during early gastrulation. HRMS expands the bioanalytical toolbox of cell and developmental biology, providing previously unavailable information on molecular classes to challenge and refine our classical understanding of the Organizer and its function during early patterning of the embryo.

Initial recommendations for performing, benchmarking and reporting single-cell proteomics experiments.

Analyzing proteins from single cells by tandem mass spectrometry (MS) has recently become technically feasible. While such analysis has the potential to accurately quantify thousands of proteins across thousands of single cells, the accuracy and reproducibility of the results may be undermined by numerous factors affecting experimental design, sample preparation, data acquisition and data analysis. We expect that broadly accepted community guidelines and standardized metrics will enhance rigor, data quality and alignment between laboratories. Here we propose best practices, quality controls and data-reporting recommendations to assist in the broad adoption of reliable quantitative workflows for single-cell proteomics. Resources and discussion forums are available at https://single-cell.net/guidelines .

Biological mass spectrometry enables spatiotemporal 'omics: From tissues to cells to organelles.

Biological processes unfold across broad spatial and temporal dimensions, and measurement of the underlying molecular world is essential to their understanding. Interdisciplinary efforts advanced mass spectrometry (MS) into a tour de force for assessing virtually all levels of the molecular architecture, some in exquisite detection sensitivity and scalability in space-time. In this review, we offer vignettes of milestones in technology innovations that ushered sample collection and processing, chemical separation, ionization, and 'omics analyses to progressively finer resolutions in the realms of tissue biopsies and limited cell populations, single cells, and subcellular organelles. Also highlighted are methodologies that empowered the acquisition and analysis of multidimensional MS data sets to reveal proteomes, peptidomes, and metabolomes in ever-deepening coverage in these limited and dynamic specimens. In pursuit of richer knowledge of biological processes, we discuss efforts pioneering the integration of orthogonal approaches from molecular and functional studies, both within and beyond MS. With established and emerging community-wide efforts ensuring scientific rigor and reproducibility, spatiotemporal MS emerged as an exciting and powerful resource to study biological systems in space-time.

Dilute to Enrich for Deeper Proteomics: A Yolk-Depleted Carrier for Limited Populations of Embryonic (Frog) Cells.

Abundant proteins challenge deep mass spectrometry (MS) analysis of the proteome. Yolk, the source of food in many developing vertebrate embryos, complicates chemical separation and interferes with detection. We report here a strategy that enhances bottom-up proteomics in yolk-laden specimens by diluting the interferences using a yolk-depleted carrier (YODEC) proteome via isobaric multiplexing quantification. This method was tested on embryos of the South African Clawed Frog (Xenopus laevis), where a >90% yolk proteome content challenges deep proteomics. As a proof of concept, we isolated neural and epidermal fated cell clones from the embryo by dissection or fluorescence-activated cell sorting. Compared with the standard multiplexing carrier approach, YODEC more than doubled the detectable X. laevis proteome, identifying 5,218 proteins from D11 cell clones dissected from the embryo. Ca. ∼80% of the proteins were quantified without dropouts in any of the analytical channels. YODEC with high-pH fractionation quantified 3,133 proteins from ∼8,000 V11 cells that were sorted from ca. 2 embryos (1.5 µg total, or 150 ng yolk-free proteome), marking a 15-fold improvement in proteome coverage vs the standard proteomics approach. About 60% of these proteins were only quantifiable by YODEC, including molecular adaptors, transporters, translation, and transcription factors. While this study was tailored to limited populations of Xenopus cells, we anticipate the approach of "dilute to enrich" using a depleted carrier proteome to be adaptable to other biological models in which abundant proteins challenge deep MS proteomics.

Deep Proteome of the Developing Chick Midbrain.

The epithelial-to-mesenchymal transition (EMT) and migration of cranial neural crest cells within the midbrain are critical processes that permit proper craniofacial patterning in the early embryo. Disruptions in these processes not only impair development but also lead to various diseases, underscoring the need for their detailed understanding at the molecular level. The chick embryo has served historically as an excellent model for human embryonic development, including cranial neural crest cell EMT and migration. While these developmental events have been characterized transcriptionally, studies at the protein level have not been undertaken to date. Here, we applied mass spectrometry (MS)-based proteomics to establish a deep proteomics profile of the chick midbrain region during early embryonic development. Our proteomics method combines optimal lysis conditions, offline fractionation, separation on a nanopatterned stationary phase (µPAC) using nanoflow liquid chromatography, and detection using quadrupole-ion trap-Orbitrap tribrid high-resolution tandem MS. Identification of >5900 proteins and >450 phosphoproteins in this study marks the deepest coverage of the chick midbrain proteome to date. These proteins have known roles in pathways related to neural crest cell EMT and migration such as signaling, proteolysis/extracellular matrix remodeling, and transcriptional regulation. This study offers valuable insight into important developmental processes occurring in the midbrain region and demonstrates the utility of proteomics for characterization of tissue microenvironments during chick embryogenesis.

Microanalytical Mass Spectrometry with Super-Resolution Microscopy Reveals a Proteome Transition During Development of the Brain's Circadian Pacemaker.

During brain development, neuronal proteomes are regulated in part by changes in spontaneous and sensory-driven activity in immature neural circuits. A longstanding model for studying activity-dependent circuit refinement is the developing mouse visual system where the formation of axonal projections from the eyes to the brain is influenced by spontaneous retinal activity prior to the onset of vision and by visual experience after eye-opening. The precise proteomic changes in retinorecipient targets that occur during this developmental transition are unknown. Here, we developed a microanalytical proteomics pipeline using capillary electrophoresis (CE) electrospray ionization (ESI) mass spectrometry (MS) in the discovery setting to quantify developmental changes in the chief circadian pacemaker, the suprachiasmatic nucleus (SCN), before and after the onset of photoreceptor-dependent visual function. Nesting CE-ESI with trapped ion mobility spectrometry time-of-flight (TOF) mass spectrometry (TimsTOF PRO) doubled the number of identified and quantified proteins compared to the TOF-only control on the same analytical platform. From 10 ng of peptide input, corresponding to <∼0.5% of the total local tissue proteome, technical triplicate analyses identified 1894 proteins and quantified 1066 proteins, including many with important canonical functions in axon guidance, synapse function, glial cell maturation, and extracellular matrix refinement. Label-free quantification revealed differential regulation for 166 proteins over development, with enrichment of axon guidance-associated proteins prior to eye-opening and synapse-associated protein enrichment after eye-opening. Super-resolution imaging of select proteins using STochastic Optical Reconstruction Microscopy (STORM) corroborated the MS results and showed that increased presynaptic protein abundance pre/post eye-opening in the SCN reflects a developmental increase in synapse number, but not presynaptic size or extrasynaptic protein expression. This work marks the first development and systematic application of TimsTOF PRO for CE-ESI-based microproteomics and the first integration of microanalytical CE-ESI TimsTOF PRO with volumetric super-resolution STORM imaging to expand the repertoire of technologies supporting analytical neuroscience.

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

Understanding of the relationship between cellular function and molecular composition holds a key to next-generation therapeutics but requires measurement of all types of molecules in cells. Developments in sequencing enabled semiroutine measurement of single-cell genomes and transcriptomes, but analytical tools are scarce for detecting diverse proteins in tissue-embedded cells. To bridge this gap for neuroscience research, we report the integration of patch-clamp electrophysiology with subcellular shot-gun proteomics by high-resolution mass spectrometry (HRMS). Recording of electrical activity permitted identification of dopaminergic neurons in the substantia nigra pars compacta. Ca. 20-50% of the neuronal soma content, containing an estimated 100 pg of total protein, was aspirated into the patch pipette filled with ammonium bicarbonate. About 1 pg of somal protein, or ∼0.25% of the total cellular proteome, was analyzed on a custom-built capillary electrophoresis (CE) electrospray ionization platform using orbitrap HRMS for detection. A series of experiments were conducted to systematically enhance detection sensitivity through refinements in sample processing and detection, allowing us to quantify ∼275 different proteins from somal aspirate-equivalent protein digests from cultured neurons. From single neurons, patch-clamp proteomics of the soma quantified 91, 80, and 95 different proteins from three different dopaminergic neurons or 157 proteins in total. Quantification revealed detectable proteomic differences between the somal protein samples. Analysis of canonical knowledge predicted rich interaction networks between the observed proteins. The integration of patch-clamp electrophysiology with subcellular CE-HRMS proteomics expands the analytical toolbox of neuroscience.

Microanalysis of Brain Angiotensin Peptides Using Ultrasensitive Capillary Electrophoresis Trapped Ion Mobility Mass Spectrometry.

While the role of the renin-angiotensin system (RAS) in peripheral circulation is well characterized, we still lack an in-depth understanding of its role within the brain. This knowledge gap is sustained by lacking technologies for trace-level angiotensin detection throughout tissues, such as the brain. To provide a bridging solution, we enhanced capillary electrophoresis (CE) nanoflow electrospray ionization (ESI) with large-volume sample stacking and employed trapped ion mobility time-of-flight (timsTOF) tandem HRMS detection. A dynamic pH junction helped stack approximately 10 times more of the sample than optimal using the field-amplified reference. In conjunction, the efficiency of ion generation was maximized by a cone-jet nanospray on a low sheath-flow tapered-tip nano-electrospray emitter. The platform provided additional peptide-dependent information, the collision cross section, to filter chemical noise and improve sequence identification and detection limits. The lower limit of detection reached sub-picomolar or ∼30 zmol (∼18,000 copies) level. All nine targeted angiotensin peptides in mouse tissue samples were detectable and quantifiable from the paraventricular nucleus (PVN) of the hypothalamus even after removal of circulatory blood components (perfusion). We anticipate CE-ESI with timsTOF HRMS to be broadly applicable for the ultrasensitive detection of brain peptidomes in pursuit of a better understanding of the brain.

Altering metabolite distribution at Xenopus cleavage stages affects left-right gene expression asymmetries.

The left-right (L-R) axis of most bilateral animals is established during gastrulation when a transient ciliated structure creates a directional flow of signaling molecules that establish asymmetric gene expression in the lateral plate mesoderm. However, in some animals, an earlier differential distribution of molecules and cell division patterns initiate or at least influence L-R patterning. Using single-cell high-resolution mass spectrometry, we previously reported a limited number of small molecule (metabolite) concentration differences between left and right dorsal-animal blastomeres of the eight-cell Xenopus embryo. Herein, we examined whether altering the distribution of some of these molecules influenced early events in L-R patterning. Using lineage tracing, we found that injecting right-enriched metabolites into the left cell caused its descendant cells to disperse in patterns that varied from those in control gastrulae; this did not occur when left-enriched metabolites were injected into the right cell. At later stages, injecting left-enriched metabolites into the right cell perturbed the expression of genes known to: (a) be required for the formation of the gastrocoel roof plate (foxj1); (b) lead to the asymmetric expression of Nodal (dand5/coco); or (c) result from asymmetrical nodal expression (pitx2). Despite these perturbations in gene expression, we did not observe heterotaxy in heart or gut looping at tadpole stages. These studies indicate that altering metabolite distribution at cleavage stages at the concentrations tested in this study impacts the earliest steps of L-R gene expression that then can be compensated for during organogenesis.

Data-Dependent Acquisition Ladder for Capillary Electrophoresis Mass Spectrometry-Based Ultrasensitive (Neuro)Proteomics.

Measurement of broad types of proteins from a small number of cells to single cells would help to better understand the nervous system but requires significant leaps in sensitivity in high-resolution mass spectrometry (HRMS). Microanalytical capillary electrophoresis electrospray ionization (CE-ESI) offers a path to ultrasensitive proteomics by integrating scalability with sensitivity. Here, we systematically evaluate performance limitations in this technology to develop a data acquisition strategy with deeper coverage of the neuroproteome from trace amounts of starting materials than traditional dynamic exclusion. During standard data-dependent acquisition (DDA), compact migration challenged the duty cycle of second-stage transitions and redundant targeting of abundant peptide signals lowered their identification success rate. DDA was programmed to progressively exclude a static set of high-intensity peptide signals throughout replicate measurements, essentially forming rungs of a "DDA ladder." The method was tested for ∼500 pg portions of a protein digest from cultured hippocampal (primary) neurons (mouse), which estimated the total amount of protein from a single neuron. The analysis of ∼5 ng of protein digest over all replicates, approximating ∼10 neurons, identified 428 nonredundant proteins (415 quantified), an ∼35% increase over traditional DDA. The identified proteins were enriched in neuronal marker genes and molecular pathways of neurobiological importance. The DDA ladder enhances CE-HRMS sensitivity to single-neuron equivalent amounts of proteins, thus expanding the analytical toolbox of neuroscience.

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

We report the development of in vivo subcellular high-resolution mass spectrometry (HRMS) for proteo-metabolomic molecular systems biology in complex tissues. 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 swiftly (<5 s/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. Label-free 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, 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.

Capillary Electrophoresis-Mass Spectrometry at Trial by Metabo-Ring: Effective Electrophoretic Mobility for Reproducible and Robust Compound Annotation.

Capillary zone electrophoresis-mass spectrometry (CE-MS) is a mature analytical tool for the efficient profiling of (highly) polar and ionizable compounds. However, the use of CE-MS in comparison to other separation techniques remains underrepresented in metabolomics, as this analytical approach is still perceived as technically challenging and less reproducible, notably for migration time. The latter is key for a reliable comparison of metabolic profiles and for unknown biomarker identification that is complementary to high resolution MS/MS. In this work, we present the results of a Metabo-ring trial involving 16 CE-MS platforms among 13 different laboratories spanning two continents. The goal was to assess the reproducibility and identification capability of CE-MS by employing effective electrophoretic mobility (µeff) as the key parameter in comparison to the relative migration time (RMT) approach. For this purpose, a representative cationic metabolite mixture in water, pretreated human plasma, and urine samples spiked with the same metabolite mixture were used and distributed for analysis by all laboratories. The µeff was determined for all metabolites spiked into each sample. The background electrolyte (BGE) was prepared and employed by each participating lab following the same protocol. All other parameters (capillary, interface, injection volume, voltage ramp, temperature, capillary conditioning, and rinsing procedure, etc.) were left to the discretion of the contributing laboratories. The results revealed that the reproducibility of the µeff for 20 out of the 21 model compounds was below 3.1% vs 10.9% for RMT, regardless of the huge heterogeneity in experimental conditions and platforms across the 13 laboratories. Overall, this Metabo-ring trial demonstrated that CE-MS is a viable and reproducible approach for metabolomics.

Microsampling Capillary Electrophoresis Mass Spectrometry Enables Single-Cell Proteomics in Complex Tissues: Developing Cell Clones in Live Xenopus laevis and Zebrafish Embryos.

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 a 16-cell Xenopus laevis (frog) embryo. After validating the approach by recovering animal-vegetal-pole proteomic asymmetry in the frog zygote, the technology was applied to uncover proteomic reorganization as the animal-dorsal (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 stages. 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 level of the building block of life: the cell.

Deciphering Metabolic Heterogeneity by Single-Cell Analysis.

Single-cell analysis provides insights into cellular heterogeneity and dynamics of individual cells. This Feature highlights recent developments in key analytical techniques suited for single-cell metabolic analysis with a special focus on mass spectrometry-based analytical platforms and RNA-seq as well as imaging techniques that reveal stochasticity in metabolism.

Trace, Machine Learning of Signal Images for Trace-Sensitive Mass Spectrometry: A Case Study from Single-Cell Metabolomics.

Recent developments in high-resolution mass spectrometry (HRMS) technology enabled ultrasensitive detection of proteins, peptides, and metabolites in limited amounts of samples, even single cells. However, extraction of trace-abundance signals from complex data sets ( m/ z value, separation time, signal abundance) that result from ultrasensitive studies requires improved data processing algorithms. To bridge this gap, we here developed "Trace", a software framework that incorporates machine learning (ML) to automate feature selection and optimization for the extraction of trace-level signals from HRMS data. The method was validated using primary (raw) and manually curated data sets from single-cell metabolomic studies of the South African clawed frog ( Xenopus laevis) embryo using capillary electrophoresis electrospray ionization HRMS. We demonstrated that Trace combines sensitivity, accuracy, and robustness with high data processing throughput to recognize signals, including those previously identified as metabolites in single-cell capillary electrophoresis HRMS measurements that we conducted over several months. These performance metrics combined with a compatibility with MS data in open-source (mzML) format make Trace an attractive software resource to facilitate data analysis for studies employing ultrasensitive high-resolution MS.

Dual cationic-anionic profiling of metabolites in a single identified cell in a live Xenopus laevis embryo by microprobe CE-ESI-MS.

In situ capillary microsampling with capillary electrophoresis (CE) electrospray ionization (ESI) mass spectrometry (MS) enabled the characterization of cationic metabolites in single cells in complex tissues and organisms. For deeper coverage of the metabolome and metabolic networks, analytical approaches are needed that provide complementary detection for anionic metabolites, ideally using the same instrumentation. Described here is one such approach that enables sequential cationic and anionic (dual) analysis of metabolites in the same identified cell in a live vertebrate embryo. A calibrated volume was microaspirated from the animal-ventral cell in a live 8-cell embryo of Xenopus laevis, and cationic and anionic metabolites were one-pot microextracted from the aspirate, followed by CE-ESI-MS analysis of the same extract. A laboratory-built CE-ESI interface was reconfigured to enable dual cationic-anionic analysis with ∼5-10 nM (50-100 amol) lower limit of detection and a capability for quantification. To provide robust separation and efficient ion generation, the CE-ESI interface was enclosed in a nitrogen gas filled chamber, and the operational parameters were optimized for the cone-jet spraying regime in both the positive and negative ion mode. A total of ∼250 cationic and ∼200 anionic molecular features were detected from the cell between m/z 50-550, including 60 and 24 identified metabolites, respectively. With only 11 metabolites identified mutually, the duplexed approach yielded complementary information on metabolites produced in the cell, which in turn deepened network coverage for several metabolic pathways. With scalability to smaller cells and adaptability to other types of tissues and organisms, dual cationic-anionic detection with in situ microprobe CE-ESI-MS opens a door to better understand cell metabolism.

A microanalytical capillary electrophoresis mass spectrometry assay for quantifying angiotensin peptides in the brain.

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) nano-electrospray ionization (nano-ESI) 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. 5 amol to 300 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-nano-ESI-HRMS extends the analytical toolbox of neuroscience to help better understand the RAS.

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).

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 ∼75-amol (∼11 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.

Single-cell mass spectrometry reveals small molecules that affect cell fates in the 16-cell embryo.

Spatial and temporal changes in molecular expression are essential to embryonic development, and their characterization is critical to understand mechanisms by which cells acquire different phenotypes. Although technological advances have made it possible to quantify expression of large molecules during embryogenesis, little information is available on metabolites, the ultimate indicator of physiological activity of the cell. Here, we demonstrate that single-cell capillary electrophoresis-electrospray ionization mass spectrometry is able to test whether differential expression of the genome translates to the domain of metabolites between single embryonic cells. Dissection of three different cell types with distinct tissue fates from 16-cell embryos of the South African clawed frog (Xenopus laevis) and microextraction of their metabolomes enabled the identification of 40 metabolites that anchored interconnected central metabolic networks. Relative quantitation revealed that several metabolites were differentially active between the cell types in the wild-type, unperturbed embryos. Altering postfertilization cytoplasmic movements that perturb dorsal development confirmed that these three cells have characteristic small-molecular activity already at cleavage stages as a result of cell type and not differences in pigmentation, yolk content, cell size, or position in the embryo. Changing the metabolite concentration caused changes in cell movements at gastrulation that also altered the tissue fates of these cells, demonstrating that the metabolome affects cell phenotypes in the embryo.