In Situ Microprobe Single-Cell Capillary Electrophoresis …, including 70 known ... cell dissection...

In Situ Microprobe Single-Cell Capillary Electrophoresis MassSpectrometry: Metabolic Reorganization in Single DifferentiatingCells in the Live Vertebrate (Xenopus laevis) EmbryoRosemary M. Onjiko,† Erika P. Portero,† Sally A. Moody,‡ and Peter Nemes*,†

†Department of Chemistry and ‡Department of Anatomy and Regenerative Biology, The George Washington University,Washington, D.C., 20052, United States

*S Supporting Information

ABSTRACT: Knowledge of single-cell metabolism would provide a powerful lookinto cell activity changes as cells differentiate to all the tissues of the vertebrate embryo.However, single-cell mass spectrometry technologies have not yet been madecompatible with complex three-dimensional changes and rapidly decreasing cell sizesduring early development of the embryo. Here, we bridge this technological gap byintegrating capillary microsampling, microscale metabolite extraction, and capillaryelectrophoresis electrospray ionization mass spectrometry (CE-ESI-MS) to enabledirect metabolic analysis of identified cells in the live frog embryo (Xenopus laevis).Microprobe CE-ESI-MS of <0.02% of the single-cell content allowed us to detect ∼230 different molecular features (positive ionmode), including 70 known metabolites, in single dorsal and ventral cells in 8-to-32-cell embryos. Relative quantification followedby multivariate and statistical analysis of the data found that microsampling enhanced detection sensitivity compared to whole-cell dissection by minimizing chemical interferences and ion suppression effects from the culture media. In addition, higherglutathione/oxidized glutathione ratios suggested that microprobed cells exhibited significantly lower oxidative stress than thosedissected from the embryo. Fast (5 s/cell) and scalable microsampling with minimal damage to cells in the 8-cell embryo enabledduplicate and triplicate metabolic analysis of the same cell, which surprisingly continued to divide to the 16-cell stage. Last, weused microprobe single-cell CE-ESI-MS to uncover previously unknown reorganization of the single-cell metabolome as thedorsal progenitor cell from the 8-cell embryo formed the neural tissue fated clone through divisions to the 32-cell embryo,peering, for the first time, into the formation of metabolic single-cell heterogeneity during early development of a vertebrateembryo.

In situ characterization of the metabolome as individual cellsdifferentiate is a major gap in mass spectrometry (MS)

technology that promises to empower a systems cell biologyunderstanding of normal and impaired development. Withdynamic response to both intrinsic and extrinsic effects, themetabolome provides a unique look into the activity state of thecell. Recent studies found metabolism to actively contribute tokey developmental processes, ranging from the viability of thepreimplantation embryo1 and establishment of cell fates,2,3

body patterning,4 and organs5,6 to transitioning between stemcell and cancer phenotypes.7−9 Contemporary MS enabled thedetection of gross metabolic changes in whole developingembryos of powerful vertebrate models in cell and devel-opmental biology and health studies, such as the zebrafish(Danio rerio)10 and the South African clawed frog (Xenopuslaevis).11 However, there is limited information available onhow the metabolome is reorganized as embryonic cells undergodifferentiation to establish cell heterogeneity and give rise to allthe different types of tissues in the body.The challenge has been mainly a lack of single-cell MS tools

capable of unbiased measurement of the single-cell metabolomedirectly in the live, morphologically complex vertebrate embryo.Current single-cell MS tools are confined to denaturingconditions or cells dissected free from other cells or cultured

or patterned into flat arrays (see reviews in refs 12−17). Forexample, metabolites have been measured in single cells inplant or animal tissues or cell cultures under vacuum usingMALDI,13,18,19 SIMS,19,20 NIMS,21 and NAPA22 and atambient conditions using DESI,23 LAESI,24 live single-cellvideo MS,25 and combinations of in situ microsampling withdirect electrospray ionization (ESI)26−30 or MALDI.31 Toenhance detection sensitivity and molecular identification, weand others have developed high-sensitivity capillary electro-phoresis (CE) ESI-MS platforms capable of separatingmolecules from dissected single neurons32−34 and embryoniccells.2,16,35−38 Using whole-cell dissection, we recently uncov-ered previously unknown metabolic cell heterogeneity in the 8-and 16-cell frog (Xenopus laevis) embryo along all three primarydevelopmental axes2,35 and discovered metabolites capable ofaltering dorsal−ventral specification.2 However, cell dissectionsacrifices live embryonic development, is too slow to track fastmetabolic changes, and becomes increasingly difficult forsmaller and more tightly adherent cells in the morphologicallycomplex developing embryo. To better understand the

Received: March 9, 2017Accepted: April 22, 2017Published: April 22, 2017

Article

pubs.acs.org/ac

© 2017 American Chemical Society 7069 DOI: 10.1021/acs.analchem.7b00880Anal. Chem. 2017, 89, 7069−7076

pubs.acs.org/ac

http://dx.doi.org/10.1021/acs.analchem.7b00880

molecular basis of cell differentiation, there is a high and yetunmet need for single-cell MS tools capable of scalable, fast, insitu, and preferably minimally invasive characterization of themetabolome in the live vertebrate embryo.Here, we address this technological gap by developing

“microprobe single-cell CE-ESI-MS” to enable, for the first time,the in situ mass spectrometric analysis of metabolites in singlecells in the freely developing vertebrate embryo. Afterestablishing technical performance metrics for single-cellanalysis of the X. laevis embryo, we validate microprobe CE-ESI-MS against whole-cell dissection, which is the closestneighboring single-cell MS technology for the vertebrateembryo. Additionally, we employ microprobe CE-ESI-MS todetermine how the metabolome is altered as a single dorsalembryonic cell forms a neural-fated clone in the 8- to 32-cellX. laevis embryo. The presented work demonstrates that in situsingle-cell CE-ESI-MS is sensitive, is scalable to broad spatialand temporal dimensions, is compatible with the complexthree-dimensional body of the vertebrate embryo, and enablesdiscovery or targeted analysis of the single-cell metabolome. Weexpect this technology to be also adaptable to other types ofcells and biological models, opening new potentials to advanceour systems cell biology understanding of normal and impaireddevelopment.

■ METHODSMaterials and Reagents. LC-MS-grade methanol, formic

acid, water, acetonitrile, sodium chloride, potassium chloride,and magnesium sulfate were from Fisher Scientific (Fair Lawn,NJ). Calcium nitrite, cysteine, Trizma hydrochloride, andTrizma base were from Sigma-Aldrich (Saint Louis, MO).Acetylcholine and amino acid standards were purchased atreagent grade or higher purity from Acros Organics (Fair Lawn,NJ).Solutions. Steinberg’s solution (100%) and fresh 2%

cysteine solution were prepared following established proto-cols.39 The “embryo culture solution” contained 50% Steinberg’ssolution. The metabolite “extraction solvent” contained 40%acetonitrile and 40% methanol in LC-MS-grade water, whichwe previously found to be able to efficiently extract small polarmetabolites from X. laevis cells.35 The CE “backgroundelectrolyte” consisted of 1% formic acid in LC-MS-gradewater. The CE-ESI “sheath solution” contained 50% methanoland 0.1% formic acid in LC-MS-grade water.Animals and Cell Identification. Adult male and female

X. laevis frogs were purchased from Nasco (Fort Atkinson, WI)and housed in a breeding colony at the George WashingtonUniversity (GWU). All protocols related to the handling andmanipulation of animals were approved by the GWUInstitutional Animal Care and Use Committee (IACUC#A311). Fertilized eggs were obtained by gonadotropin-induced natural mating of male and female adult frogs asdescribed elsewhere.39,40 The jelly coats surrounding theembryos were removed using 2% cysteine solution as describedelsewhere.41 Dejellied embryos were transferred to 100%Steinberg’s solution in a Petri dish and monitored until theyreached the two-cell stage. Two-cell stage embryos in whichasymmetric pigmentation marked the stereotypical dorsal−ventral axis with high accuracy (in reference to established cellfate maps42−47) were isolated into a separate Petri dish andmonitored; only these embryos were used in this study. On thebasis of pigmentation and location in the X. laevis embryo withregards to established cell fate maps,42−47 we identified the

right V1 (V1R) and right D1 (D1R) cell in the 8-cell embryo,the right D11 (D11R) and right D12 (D12R) cell in the 16-cellembryo, and the right D111 (D111R) and right D121 (D121R)cell in the 32-cell embryo. For microdissection studies, embryoswere collected at the 8-cell stage into a separate Petri dishcoated with 2% agarose gel and containing 50% Steinberg’ssolution at room temperature.

Dissection of Single Identified Cells and MetaboliteExtraction. For technology validation, the identified cells weredissected free of other cells using protocols reported else-where.41 To quench enzymatic reactions, each dissected cellwas immediately transferred into a separate microvialcontaining 20 μL of methanol chilled on ice. Contents of thevials were lyophilized at 4 °C, and metabolites were extracted in5 μL of extraction solvent at 4 °C for 3 min, facilitated byperiodic sonication and incubation on ice, following our recentprotocol.2,35 The single-cell extracts were then centrifuged at8000g for 5 min at 4 °C and stored together with the cell debrisin the same vial at −80 °C until measurement by CE-ESI-MS.

Microprobe Sampling of Single Identified Cells andMetabolite Extraction. We designed an optically guidedmicrosampling platform to enable the collection of a portion ofthe cell content from select identified cells in the live embryo.Embryos were immobilized in individual wells made of 2%agarose gel in a 50 mm Petri dish containing 50% Steinberg’ssolution at room temperature. Borosilicate capillaries (partnumber B100-50-10, 0.5/1 mm inner/outer diameter, SutterInstrument Co., Novato, CA) were pulled to a taper with abarrel length of ∼850 μm in a capillary puller (model P-1000,Sutter Instrument Co.) using the following settings: heat = 355;pull = 65, velocity = 80; time = 150. The pulled capillaries weremanually cleaved using sharp forceps and a high-resolutionstereomicroscope (model SMZ18, Nikon, Melville, NY) toproduce “micropipettes” with an ∼20 μm-diameter tapered-tip.To enable accurate positioning of the micropipette tip, themicropipette was mounted on a motorized three-axis micro-manipulator (model TransferMan 4r, Eppendorf, Hauppauge,NY) capable of <20 nm resolution and remote control with ajoystick. With guidance from the high-resolution stereo-microscope, the tip of the micropipette was fine-positionedinto individual identified single-cells in the live embryo thatwere selected for analysis. To aspirate a portion of the cellcytoplasm, −30 psi pressure was applied to the microcapillaryusing a microinjector (model PLI-100A, Warner Instrument,Handem, CT). Thereafter, the tip of the microcapillary wasretrieved from the cell, and the aspirated cell content wasexpelled using one second pressure surge (at 80 psi) into amicrovial (Fisher Scientific, Pittsburgh PA) containing 4 μL ofextraction solvent chilled to 4 °C on ice. Each sampling eventutilized a separate micropipette to eliminate potential samplecarryover between cells. These microvials were vortex-mixed for∼1 min at room temperature to facilitate metabolite extractionand centrifuged at 8000g for 5 min at 4 °C to pellet cell debrisand precipitates (Sorvall Legend X1R; Thermo Scientific,Waltham, MA). The resulting cell extracts were stored togetherwith the cell debris and precipitates in the same vial at −80 °Cuntil measurement by CE-ESI-MS.

CE-ESI-MS Instrument. The cell extracts were analyzedusing our custom-built CE-ESI-MS platform followingprotocols that we have reported elsewhere.2,32,35 Briefly, 10nL of each cell extract was hydrodynamically injected into afused silica capillary (40/105 μm inner/outer diameter, 1 mlength) filled with the background electrolyte. Compounds were

Analytical Chemistry Article

DOI: 10.1021/acs.analchem.7b00880Anal. Chem. 2017, 89, 7069−7076

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electrophoretically separated by applying 21 kV across thecapillary until glutathione was detected, which was used tomark the end of separation experiments (∼45 min). Thecompounds were ionized in a custom-built CE microflow ESIinterface, which coaxially supplied the sheath solution at 1 μL/min around the CE capillary in an earth-grounded metalcapillary to complete the CE electrical circuit. The ESI needlewas positioned ∼2 mm from the mass spectrometer’s orifice tomaintain the electrospray in the stable cone-jet regime forefficient ionization.48 Generated ions were analyzed in thepositive ion mode between m/z 50 and 500 at 40 000 (fwhm)resolution by a quadruple orthogonal acceleration time-of-flighttandem mass spectrometer (Impact HD, Bruker Daltonics,Billerica, MA). The mass spectrometer was tuned andcalibrated following vendor instructions and then furtherrefined for acetylcholine and S-adenosylmethionine to max-imize detection sensitivity. External mass calibration provided 5ppm mass accuracy. The CE-ESI-MS instrument provided a 60amol lower limit of detection and 4−5 log-order quantitativerange. To ensure robust instrumental performance, we requireda signal-to-noise (S/N) > 15 for sensitivity and <10% relativestandard deviation (RSD) in migration time and <25% RSD inpeak area for 300 amol of acetylcholine (6 nL of 50 nManalyzed) each day.Experimental Design. All embryos were collected from

natural mating of 2 pairs of parents to minimize genomicvariability in this study. Each cell type was identified and thensampled in a random order. To account for biological variancebetween embryos and to enhance statistical analysis, at least n =4−7 biological replicates were collected for each cell type.Additionally, single-cell extracts were measured in a randomizedorder to minimize the effect of potential instrumental biases onresults interpretation. Each cell extract was measured in 2−4interday technical replicates.Data Analysis. Raw mass spectrometric data files were

processed in Compass Data Analysis version 4.3 (BrukerDaltonics) using custom-written scripts. Each file was externallymass-calibrated using sodium formate clusters that were formedin situ in the CE-ESI interface as sodium ions were separated byCE. Molecular features (signals with unique m/z vs migrationtime values) were semiautomatically searched following aprotocol described elsewhere.2,32 To relatively quantif y metab-olite abundances, selected-ion electropherograms were gener-ated for the detected molecular features with ±5 mDa window,and the under-the-curve peak areas were integrated, whichserve as a quantitative proxy for metabolite concentration.2

Statistical and multivariate data analysis was performed usingANOVA and principal component analysis (PCA) inMetaboAnalyst ver. 3.049 with the following settings: normal-ization by sum; log transformation; data scaling, with meancentering. Data normalization allowed us to comparemetabolism in different portions of the total cell volume thatwere collected by whole-cell dissection and microprobe CE-ESI-MS. A p value ≤0.05 was chosen to mark statisticalsignificance (Student’s t-test). Cluster analysis was performed inGProX ver. 1.1.1650 with the following settings: cluster number,4; upper limit, 1.5; lower limit, 0.67; fuzzification value, 2;minimum membership for plot, 0.5; standardizations and 100iterations.Safety Considerations. Standard safety procedures were

followed when handling chemicals and biological samples.Capillary micropipettes, which pose a potential needle-stickhazard, were handled with gloves and safety goggles. High

voltage presents electrical shock hazard; therefore, to preventusers from exposure, all connective parts were earth-groundedor isolated in a Plexiglas enclosure equipped with a safetyinterlock-enabled door.

■ RESULTS AND DISCUSSIONOur single-cell analysis strategy integrates live imaging, in situmicrosampling, and high-sensitivity CE-ESI-MS. As shown inFigure 1, we began with raising the embryo to the desired

developmental stage, followed by the identification of individualcells using bright-field microscopy. Alternatively, epifluores-cence may be used to identify cells that express fluorescentmarkers. Figure 1 demonstrates how we used morphologicalcues and fate maps42−47 to identify the right ventral cell (calledV1R) in the 8-cell X. laevis embryo. To sample a desired cell insitu, the tip of a sharpened capillary was precision-positionedinto the cell under translational control by a multiaxismicromanipulator and guidance by the stereomicroscope, anda calibrated portion of the cell content was aspirated by anonline connected microinjector delivering negative pressurepulses to the capillary. Using a borosilicate capillary pulled to an∼20 μm tip, we were able to collect ∼10−15 nL, viz., 10% ofthe V1R cell in <5 s. After retracting the capillary tip, thecollected cell material was pressure-ejected into a microvial,

Figure 1. Microprobe single-cell capillary electrophoresis (CE) massspectrometry (MS) enabling in situ metabolic characterization of liveXenopus laevis embryos (1). A 10−15 nL portion of single cellsidentified under a stereomicroscope (2) were aspirated into a pulledcapillary (3) using a multiaxis translation stage (4) and a microinjector(5) delivering vacuum (−ΔP). The collected cell content (6) waspressure-injected (+ΔP) into a vial for metabolite extraction (7). Theextract was measured by a microloading CE platform (8) connected toa CE electrospray ionization (ESI) source (9) operated in the cone-jetmode (see Taylor-cone, Tc). Metabolite ions were identified andquantified using a high-resolution tandem mass spectrometer (10).Scale bars = 500 μm (dark/gray); 10 mm (white). Key: SP, syringepump.



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where proteins were denatured and small molecules wereextracted in 4−5 μL of aqueous organic solvent mixturefollowing our established protocols.2,35 The next stepsperformed metabolic analysis of the extract to infer themetabolic state of the cell. An ∼10 nL portion of the extract,corresponding to <0.02% of the cell metabolome, was injectedinto a fused silica capillary, where molecules were electro-phoretically separated by a custom-built CE system, efficientlyionized by a custom-designed CE-ESI interface operated in thecone-jet spraying regime, and generated ions were mass-analyzed by a high-resolution mass spectrometer. Metaboliteswere identified and quantified using stringent protocols that wedeveloped for CE-MS.2,35 While sharpened capillaries havebeen used with MS to analyze metabolites in single cells inculture26−30 or tissues, such as single neurons,32,51 orplants,25,30,51−53 microprobe CE-ESI-MS from this studyenabled, for the first time, the in situ characterization ofmetabolites in single identified cells in the live, freelydeveloping vertebrate embryo.Microprobe Single-Cell CE-ESI-MS versus Whole-Cell

Dissection. We compared microprobe CE-ESI-MS to whole-cell dissection (Figure 2), which is the closest neighboring

single-cell MS technology for the X. laevis embryo.2 Asreference, n = 5 different V1R cells were dissected over ∼5min/cell, each from a different embryo (biological replicates).In parallel, a 10−15 nL portion of the V1R cell was aspirated in<5 s in situ from n = 7 live embryos. Unlike whole-celldissection, 50−100-times faster sampling and minimal damageto the cell allowed us to repeatedly sample the same cell,including duplicate and triplicate analysis of the same cell. Forexample, as shown in Figure 2A, locations where the V1R cellwas probed twice are marked by spots that formed as the cellmembrane healed. Intriguingly, the singly or even doublyprobed V1R cell continued to divide in synchrony with othercells as the embryo progressed to the 16-cell stage over the next∼20 min, as expected for normal development. In theory,microprobe CE-ESI-MS also raises a technical capability tocharacterize multiple cells in the same embryo, thus

empowering statistics of single-cell analysis by reducingknown metabolic variability between embryos.11 Additionally,microprobe single-cell CE-ESI-MS is scalable to smaller cellsand compatible with the complex three-dimensional body ofthe embryo, which we also demonstrate using the 16- and 32-cell embryos in this study.Microprobe CE-ESI-MS enhanced the qualitative and

quantitative characterization of the single-cell metabolome incomparison to whole-cell dissection. Typically, we detected∼230 distinct molecular features in each single cell, ∼170 ofwhich were repeatedly detected in at least 50% of the biologicalreplicates. We identified ∼70 metabolites by comparing theaccurate mass, tandem mass spectrum, and migration time ofmolecular features that were detected in the cells to datameasured using authentic chemical standards, available inmetabolite MS databases (Metlin54 or HMDB55), or previouslyidentified in X. laevis using our CE-ESI-MS platform.2,35

Detected molecular features and metabolite identifications arelisted in Table S1. Representative separation of identifiedmetabolites is shown in Figure 2B. Surprisingly, despitesampling an ∼10−20-times smaller portion of the single-cellmetabolome, microprobe sampling yielded comparable levels ofsignals to whole-cell dissection (e.g., compare spermidine andputrescine) and higher separation performance (e.g., respectivetheoretical plate numbers were ∼71 800/m vs ∼3800/m forspermidine and 137 600/m vs 110 800/m for carnitine). Weattribute this improvement by microprobe CE-ESI-MS tocollecting substantially lowered amounts of salts, bufferingagents, and other additives from the embryo culture media(Table S1 and Figure S1), which are known to interfere withseparation (e.g., less efficient field amplified sample stacking inCE) and ion generation (e.g., ion suppression during ESI).Next, we evaluated the repeatability of quantification (Figure

3). To relatively quantify metabolite abundances, we generatedselected-ion electropherograms for the detected molecularfeatures with ±5 mDa window and integrated the under-the-curve peak areas, which serve as a quantitative proxy forconcentration.2 These metadata were used to calculate themean quantitative error (μ), expressed as relative standarddeviation (RSD), on the basis of technical (same extractanalyzed multiple times) and biological (same or different V1Rcells analyzed multiple times) replicates. The technicalrepeatability of the CE-ESI-MS platform was characterizedwith μ = 8.2% RSD by triplicate analysis of the same cell extract(see “CE-MS” in Figure 3A). Microprobe sample collectionfollowed by metabolite extraction before CE-ESI-MS slightlyincreased the quantitative error to μ = 13.9% RSD with thisdifference being significant (P = 7.4 × 10−5). In comparison,biological replicates yielded higher quantitative variability.Triplicate microsampling of the same V1R cell (consideredbiological replicate here) yielded μ = 21.5% RSD, which wassignificantly higher than the technical repeatability of microp-robe CE-ESI-MS (P = 2.1 × 10−3). Because microsampling wascompleted long before cell division to the 16-cell stage embryo(compare <5 s/cell vs 5 min/cell in Figure 2A), greaterbiological than technical repeatability may reflect highly activecell metabolism (even before cell division), rapid metabolicresponse to perturbation caused by the microcapillary (viz.,within seconds), and/or subcellular metabolic heterogeneity.Indeed, metabolism is known to be highly active during earlyembryonic development in many vertebrates, such as Xenopus,in which 50% of transcripts56 and 60% of proteins36 have beenassociated with metabolic processes. In contrast, metabolites in

Figure 2. Metabolite detection by microprobe CE-ESI-MS vs whole-cell dissection. (A) As reference, the whole right-ventral (V1R) cellwas dissected from the 8-cell X. laevis embryo. In comparison, the V1Rcell was analyzed once (1×) and twice (2×) in the live embryo usingthe microprobe (spots mark analysis locations; see white arrows).Intriguingly, microprobed cells divided to form the 16-cell embryo.(B) Electropherograms exemplifying metabolites identified in a V1Rcell using whole-cell dissection (top panel) and microsampling(bottom panel). Scale bars = 250 μm. Key: HPX, hypoxanthine;SAM, S-adenosylmethionine; AcCho, acetylcholine; AcCar, acetylcar-nitine. Peaks in gray correspond to compounds from the embryoculture media. See detected molecular features and metaboliteidentifications in Table S1.



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V1R cells were quantified with μ = 42.5% RSD using whole-celldissection (see “Diss”), and importantly, this error wasstatistically significantly higher than repeated microsamplingof the same cell (P = 5.7 × 10−5). Therefore, repeated analysisof the same cell by microprobe sampling eliminated metabolicvariability that exists between dissected cells as a result of, e.g.,different stages of the cell cycle or embryo-to-embryodifferences. With higher detection sensitivity and quantitativerepeatability than dissection, microprobe CE-ESI-MS raises thepotential to detect differences between cells that would bemasked by embryo-to-embryo differences, within even the samecell type.The single-cell MS data revealed differences in metabolic

activity between the dissected and in situ microprobed cells.For example, Figure 2B suggests a higher ratio of lysine/arginine, valine/serine, and glutathione (GSH)/oxidizedglutathione (GSSG) in the microprobed cells. To systematicallycompare the metabolomes, we performed unsupervisedprincipal component (PC) analysis of the metadata. The firstthree most significant principal components explained 51.9%,11.1%, and 8.2% variance in the data; therefore, PC1 and PC2were sufficient for the PCA model used in this study. Datacorresponding to the dissected versus microprobed cells formedseparate clusters in the scores plot (Figure 3B), revealingreproducible metabolic differences between the approaches. Inthe corresponding loadings plot, distant clustering of themolecular features from the origin revealed enrichment in thedissected (e.g., GSSG) or the microprobed cells (e.g., GSH),but not both. To further evaluate these apparent chemicaldifferences, we normalized the signal areas in each sample andthen calculated the average ratio of small molecule abundancebetween microsampled-to-dissected cells and the correspond-ing statistical significance. The results, presented in Figure 3C,exposed clear metabolic differences between these single-cellanalysis strategies. Molecular features with significantly differentabundances are tabulated in Table S2. Notably, the majority ofsmall molecules were relatively enriched during microprobesampling, including cysteine, methionine, and GSH, whereas

GSSG along with compounds originating from the embryoculture media (see filled circles) were significantly moreabundant in the dissected cells.Therefore, we proposed that dissection and microprobe

sampling differentially influenced the metabolic state of theV1R cells. To test this, we compared the oxidative status of themicroprobed versus dissected V1R cells. GSH is an abundantantioxidant that is known to provide defense against oxidativedamage by conversion to GSSG; thus, the GSH/GSSG ratio isan effective metric of oxidative stress. As noted earlier,microprobe-generated extracts contained systematically (Figure3B) and significantly (Figure 3C) higher amounts of GSH andlower levels of GSSG compared to whole-cell dissection. Thesedata suggested a higher level of oxidative stress in the dissectedcells. Indeed, the ratio between the average GSH/GSSG ionsignal was 45 ± 27 by dissection and 1045 ± 76 by microprobesampling with this difference being statistically significant (P =2.8 × 10−5). We credit significantly less oxidative stressimposed on the V1R cell by microprobe CE-ESI-MS to acombination of factors, including rapid sampling (<5 s bymicroprobe vs ∼5−10 min by dissection), minimal physicaldamage to the cell (membrane heals and the cell divides duringmicroprobe sampling), and no/undetectable perturbation toneighboring cells (compare with dissection, which sacrifices theremaining cells in the embryo) (see Figure 2A). These resultsrevealed that microprobe CE-ESI-MS provided a direct, in situsnapshot of innate single-cell metabolism in the live, freelydeveloping vertebrate embryo.

Reorganization of the Metabolome within NeuralCell-Clones. Last, we used microprobe CE-ESI-MS to quantify,for the first time, metabolic changes as an embryonic cell formsa neural cell lineage in the live X. laevis embryo (Figure 4).Using whole-cell dissection, we previously uncovered spatialmetabolic cell heterogeneity in the 8- and 16-cell embryo,2

suggesting that single-cell CE-ESI-MS is also able to provideinsights on cell differentiation in the broader spatial andtemporal domain. To test this hypothesis, we used microprobeCE-ESI-MS to determine how the metabolome is reorganized as

Figure 3. Microprobe quantification of metabolites in single cells. (A) Technical repeats by CE-ESI-MS (CE-MS) and microprobe CE-ESI-MS(“μP” techn.) versus biological repeats by microprobe CE-ESI-MS (μP on the same cell) and whole-cell dissection (Diss). Key: **P < 0.005 (B)Unsupervised principal component (PC) analysis scores plot (top panel) and loadings plot (bottom panel) revealing small-molecule differencesbetween microprobe sampling and cell dissection. Key to scores plot: each data point is a different cell; cells numbered by sampling order; 95%confidence ellipses in dashed lines. Key to loadings plot: each data point is a detected molecular feature (see Table S1); signals from culture media infilled circles. (C) Statistical analysis of fold change (FC) differences between microprobed/dissected cells. Signals from media in filled circles.Horizontal line marks P = 0.05. Metabolites are listed in Table S1.



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cells of the dorsal cell clone are formed between the 8-cell and32-cell embryo (Figure 4A); this clone is the main precursor ofneural tissues (e.g., brain, spinal cord, and retina) withmoderate contributions to other organs, such as the notochord,gut, heart, and liver.42−47 We quantified metabolites in the D1Rcell (n = 6 biological replicates) in the 8-cell embryo, its midlineD11R (n = 4) and lateral D12R (n = 5) daughter cells in the16-cell embryo, and their respective animal pole daughter cellsin the 32-cell embryo, which are the D111R (n = 4) and D121R(n = 4) cells. Cells in the clone become progressively smaller asthe volume of the cell is halved during each cell divison; thevolume of the average cell corresponds to a sphere with an∼550 μm diameter in the 16-cell embryo and ∼450 μmdiameter in the 32-cell embryo (Figure 4A). Each cell extractwas measured in technical duplicate−quadruplet, yielding 50single-cell measurements that quantified the production profilesfor ∼230 different molecular features, including the ∼70metabolites that were identified across the cell lineage in thisstudy (Table S1).The quantitative data revealed complex spatial and temporal

metabolic changes as the progenitor D1R cell gave rise to itsdescendants. A brief survey of metabolite abundances usinganalysis of variance based on Fisher’s least significant analysisfound significant changes in the single-cell metabolome forclose to 100 molecular features, including ∼50 identifiedmetabolites (Table S3). For example, the daughter cells of D1Rcontained increasing levels of γ-aminobutyric acid (GABA) anddecreasing amounts of aspartate, whereas creatine wascomparably abundant across all cells in the lineage (Figure4B). In agreement, aspartate has been found to rapidly decay inthe whole developing Xenopus laevis embryo.11,57 To system-

atically evaluate metabolic profiles across the cell lineage, weexamined the quantitative metadata using unsupervisedclustering by fuzzy-c means (see Methods). A total of 122small-molecule features (excluding compounds from the culturemedia) were grouped into 4 different clusters based on differentspatial and temporal abundance profiles across cells of thedorsal clone (see Figure 4C). A complete list of these molecularfeatures and their cluster assignments are listed in Table S4. Asa general trend, metabolites in Cluster 0, including creatine,were present at steady abundance across the cells. Metabolitesin Cluster 1 yielded stable signal abundance in cells in the 8-and 16-cell embryo and then became enriched in D121R withsignificant accumulation in D111R of the 32-cell embryo. Incomparison to Cluster 1, metabolites in Cluster 2 followed aseemingly anticorrelated profile with enrichment in D11R butrapid decay in the daughter cells in the 32-cell embryo.Progressively decreasing concentration across all cells of thelineage was characteristic for metabolites in Cluster 3, includingaspartate, asparagine, carnitine, and acetylcholine. This findingis in agreement with recent isotopic pulse-chase experiments foraspartate in whole Xenopus embryos.11 Stable production untilthe 16-cell stage followed by a rapid decrease in abundancecharacterized metabolites in Cluster 4. While understanding thefunctional significance of these metabolic profiles goes beyondthe scope of this study, these results demonstrate the firstexample of using single-cell MS to track metabolic cellheterogeneity in space and time in the live, developingvertebrate embryo. The data resulting from these measure-ments show how the metabolome is reorganized as embryoniccells commit to different tissue fates during early vertebrateembryonic development.

■ CONCLUSIONSMicroprobe single-cell CE-ESI-MS enables, for the first time, thein situ characterization of metabolic changes as a singleembryonic cell forms a cell clone in the developing vertebrateembryo (X. laevis). Key advantages of the technology overclassical whole-cell dissection include rapid and direct (in situ)operation with a capability for in vivo studies (see Figures 1 and2A), scalability over broad spatial and temporal dimensions(see Figure 4A), and compatibility with the morphologicallycomplex structure of the vertebrate embryo. Compared towhole-cell dissection, microprobe CE-ESI-MS enhanceddetection sensitivity and quantitative repeatability (see Figure2) and imposed significantly lower oxidative stress on cells.Repeated analysis of the same cell in the same embryo raises atechnical capability to profile metabolite production betweenmultiple cells in the same embryo. By minimizing oreliminating biological and cell-cycle variability between cellsdissected from different embryos, microsampling CE-ESI-MSprovides higher quantitative fidelity than whole-cell dissection,which in turn affords a potential to find metabolic cellheterogeneity that would otherwise be masked by biologicalvariability using whole-cell dissection. Single-cell metabolomicMS complements single-cell transcriptomics and recentadvances in single-cell proteomics,36−38,58 helping to pave theway to multiomic analysis of single cells. With futuredevelopments in speed, throughput, automation, and stream-lined data analysis, we anticipate microprobe single-cell CE-ESI-MS to enable one to measure metabolites in largerpopulations of single cells and other model organisms. In situmicroprobe single-cell CE-ESI-MS extends the bioanalyticaltoolbox of systems cell biology16 and empowers exciting new

Figure 4. Single-cell metabolism in the dorsal cell lineage of theX. laevis embryo. (A) Optical images of the progenitor right dorsal(D1R) cell, its daughter cells, the D11R and D12R cells, which thenrespectively are precursors to animal pole D111R and D121R cells.(B) Fisher’s least discriminant analysis finding significant metabolicchanges between cells in the lineage. Key: *P < 0.05; **P < 0.001; ns= no significant difference. (C) Unsupervised fuzzy-c means clusteringof single-cell metabolite profiles between cells in the clone. Seemolecular features and their cluster assignments in Table S4. Scale bars= 250 μm.



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research directions to help understand the metabolic under-pinnings of cell differentiation during normal and impaireddevelopment.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.anal-chem.7b00880.

Molecular features detected and identified in singleembryonic cells; significant metabolite differencesbetween microprobe CE-ESI-MS and whole-cell dis-section; Fisher’s least significant discriminant analysis;results from GProX Cluster Analysis; representativemolecular features detected from the embryo culturemedia (PDF)

■ AUTHOR INFORMATIONCorresponding Author*E-mail: [email protected]. Phone: 202-994-5663.ORCIDPeter Nemes: 0000-0002-4704-4997Author ContributionsP.N. and R.M.O. designed the study. S.A.M. provided theXenopus embryos and commented on the manuscript. R.M.O.and E.P.P. measured the cells. R.M.O., E.P.P., and P.N.analyzed the data. P.N. and R.M.O. interpreted the results andwrote the manuscript.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis research was supported by the National Institutes ofHealth Grants GM114854 (to P.N.) and R03CA211635 (toP.N.), the Arnold and Mabel Beckman Foundation BeckmanYoung Investigator grant (to P.N.), and the DuPont YoungProfessor award (to P.N.). The opinions and conclusionsexpressed in this publication are solely those of the authors anddo not necessarily represent the official views of the fundingsources.

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1

In situ Microprobe Single-cell Capillary Electrophoresis Mass Spectrometry: Metabolic Reorganization in Single Differentiating Cells

in the Live Vertebrate (X. laevis) Embryo

Rosemary M. Onjiko1, Erika P. Portero1, Sally A. Moody2, and Peter Nemes1*

1Department of Chemistry and 2Department of Anatomy and Regenerative Biology,

The George Washington University, Washington DC, 20052

*Correspondence to: 800 22nd Street, NW, Suite 4000, Washington, DC 20052 (USA), (Ph) 202-994-5663, [email protected]

Table of Contents

SUPPLEMENTARY TABLES ...................................................................................................... 2

Table S1 ...................................................................................................................................... 2

Table S2 ...................................................................................................................................... 7

Table S3 ...................................................................................................................................... 9

Table S4 .................................................................................................................................... 11

SUPPLEMENTARY FIGURES ................................................................................................... 13

Figure S1 ................................................................................................................................... 13

2

SUPPLEMENTARY TABLES

Table S1. Molecular features detected and identified in single embryonic cells in the 8-to-32-cell X. laevis embryo. Key: MT, migration time; Δ m/z, difference between theoretical and measured mass (m/z value).

ID Metabolite (Abbrev.) Formula MT

(min) m/z measured

m/z theoretical

Δ m/z(mDa)

Δ m/z(ppm)

1 M 6.9 360.3225

2 Spermidine** C7H19N3 8.6 146.1652 146.1652 0.0 0.0

3 Putrescine‡ C4H12N2 9.0 89.1071 89.1073 0.2 2.2

4 Methylhistamine** C6H11N3 9.5 126.1030 126.1026 –0.4 –3.2

5 9.8 252.9739

6 9.9 140.9521

7 10.0 139.9526

8 10.1 403.8637

9 Na formate cluster M Na(NaCOOH)2 10.2 158.9640 158.9641 0.1 0.6




13 Na formate cluster M Na(NaCOOH)3 10.2 226.9516 226.9515 –0.1 –0.4



16 11.1 133.0340

17 12.1 166.1333

18 12.1 182.1641

19 12.7 208.1793

20 12.8 161.1288

21 12.8 180.1500

22 13.1 214.1329

23 13.4 194.1640

24 Ethanolamine‡ EA C2H7NO 13.6 62.0596 62.0600 0.4 6.4

25 13.6 114.0654

26 Thiamine** C12H16N4OS 14.0 265.1111 265.1118 0.7 2.5

27 14.1 238.1662

28 Choline*,** Cho C5H13NO 15.0 104.1067 104.1070 0.3 2.9

29 Ala-Lys** C9H19N3O3 15.2 218.1491 218.1499 0.8 3.7

30 Arg-Ala** C9H19N5O3 15.6 246.1555 246.1561 0.6 2.3

31 Lys-Ser** C9H19N3O4 15.6 234.1437 234.1448 1.1 4.8

32 S-adenosyl-methionine**

SAM C15H22N6O5S 15.9 399.1438 399.1445 0.7 1.8

33 Ser-Arg** C9H19N5O4 16.0 262.1503 262.1510 0.7 2.7

3


(min) m/z measured

m/z theoretical

Δ m/z(mDa)

Δ m/z(ppm)

34 Val-Lys** C11H23N3O3 16.0 246.1807 246.1812 0.5 2.1

35 16.1 106.0863

36 16.1 114.0654

37 Ornithine** Orn C5H12N2O2 16.2 133.0974 133.0972 –0.2 –1.5

38 16.3 131.1179

39 16.3 125.1077

40 Sarcosine‡ Sar C3H7NO2 16.3 90.0549 90.0550 0.1 1.1

41 Lysine*,** Lys C6H14N2O2 16.4 147.1130 147.1128 –0.2 –1.3

42 17.0 116.1069

43 Arginine*,** Arg C6H14N4O2 17.0 175.1191 175.1190 –0.1 –0.8

44 Homolysine** C7H16N2O2 17.1 161.1288 161.1285 –0.3 –2.1

45 Tyr-Lys** C15H23N3O4 17.2 310.1753 310.1761 0.8 2.7

46 γ-aminobutyrate*,** GABA C4H9NO2 17.2 104.0704 104.0706 0.2 2.0

47 N6,N6,N6-rimethyl-lysine**

TML C9H20N2O2 17.3 189.1599 189.1598 –0.1 –0.8

48 17.3 130.0860

49 Histidine*,** His C6H9N3O2 17.3 156.0769 156.0768 –0.1 –0.9

50 17.5 139.1234

51 17.8 150.0761

52 17.9 98.0944

53 Methylhistidine** C7H11N3O2 17.9 170.0920 170.0924 0.4 2.4

54 18.0 118.1216

55 Guanidinopropanoate** C4H9N3O2 18.2 132.0763 132.0768 0.5 3.4

56 18.3 142.1225

57 Acetylcholine*,** AcCho C7H15NO2 18.4 146.1177 146.1176 –0.1 –1.0

58 18.4 134.1173

59 18.4 141.0661

60 18.5 142.0968

61 18.5 184.1107

62 18.5 120.1028

63 Trolamine‡ TEA C6H15NO3 18.6 150.1145 150.1125 -2.0 –13.3

64 18.6 296.0675

65 Leu-Ala** C9H18N2O3 18.6 203.1379 203.1390 1.1 5.5

66 TRIS-relatedM 18.6 122.0813

67 18.8 150.0761

68 19.0 254.0207

69 19.0 303.1053

70 19.0 128.0447

71 19.0 154.0976

72 Guanine** C5H5N5O 19.1 152.0571 152.0567 –0.4 –2.7

4


(min) m/z measured

m/z theoretical

Δ m/z(mDa)

Δ m/z(ppm)

73 19.1 158.1179

74 19.1 152.0916

75 19.2 203.1480

76 Urocanate, cis-** cURA C6H6N2O2 19.2 139.0506 139.0502 –0.4 –2.9

77 19.2 273.1301

78 19.3 136.0968

79 19.4 194.1399

80 Carnitine*,** Car C7H15NO3 19.7 162.1125 162.1125 0.0 –0.2

81 19.7 140.9521

82 19.8 168.1127

83 Urocanate, -trans** tURA C6H6N2O2 19.9 139.0505 139.0502 –0.3 –2.2

84 20.0 160.0771

85 Methylguanine** C6H7N5O 20.4 166.0719 166.0723

86 20.5 168.0657

87 20.5 150.0580

88 Pyridoxal (Vitamin B6)**

C8H9NO3 20.6 168.0654 168.0655 0.1 0.7

89 20.9 130.1588

90 21.1 210.1322

91 21.5 278.1245

92 21.6 194.1536

93 21.6 182.1285

94 21.7 166.1070

95 Acetylcarnitine*,** AcCar C9H17NO4 21.9 204.1231 204.1230 –0.1 –0.3

96 22.0 158.1537

97 22.0 182.1285

98 22.1 139.9526

99 22.4 309.1658

100 22.4 202.1802

101 22.6 112.0498

102 22.6 198.1852

103 Propionylcarnitine** C10H19NO4 22.6 218.1381 218.1387 0.6 2.7

104 Methylaspartate** C5H9NO4 22.7 148.0607 148.0604 –0.3 –1.8

105 22.7 123.0557

106 22.7 255.0975

107 Niacinamide* C6H6N2O 22.7 123.0551 123.0553 0.2 1.5

108 Glycine* Gly C2H5NO2 22.8 76.0390 76.0393 0.3 4.0

109 22.8 248.1495

110 22.9 202.1529

111 22.9 176.0912

112 22.9 134.0814

5


(min) m/z measured

m/z theoretical

Δ m/z(mDa)

Δ m/z(ppm)

113 22.9 196.1698

114 23.0 228.1693

115 Creatine*,** CR C4H9N3O2 23.0 132.0771 132.0768 –0.3 –2.6

116 23.0 261.1957

117 23.5 285.1340

118 23.6 240.1482

119 Pro-Val** C10H18N2O3 23.6 215.1383 215.1390 0.7 3.3

120 23.7 215.1390

121 Adenosine C10H13N5O4 24.1 268.1048 268.1040 –0.8 –2.9

122 24.4 249.1811

123 25.2 307.1225

124 25.4 186.1117

125 Alanine* Ala C3H7NO2 25.7 90.0548 90.0550 0.2 1.7

126 25.9 279.1577

127 26.0 218.2103

128 26.3 320.1846

129 HEPES C8H18N2O4S 26.6 239.1066 239.1060 –0.6 –2.5

130 26.9 336.1812

131 Argininosuccinate** C10H18N4O6 27.2 291.1292 291.1299 0.7 2.4

132 27.9 274.2737

133 Valine*,** Val C5H11NO2 30.2 118.0866 118.0863 –0.3 –2.9

134 Serine*,** Ser C3H7NO3 30.3 106.0498 106.0499 0.1 0.7

135 Isoleucine* Ile C6H13NO2 30.6 132.1023 132.1019 –0.4 –3.0

136 Leucine* Leu C6H13NO2 30.9 132.1023 132.1019 –0.4 –3.0

137 M 32.6 133.0340

138 Asparagine*,** Asn C4H8N2O3 32.6 133.0611 133.0608 –0.3 –2.5

139 Threonine*,** Thr C4H9NO3 32.7 120.0655 120.0655 0.0 0.2

140 Tryptophan*,** Trp C11H12N2O2 33.4 205.0972 205.0972 0.0 –0.2

141 Methionine*,** Met C5H11NO2S 33.6 150.0586 150.0583 –0.3 –1.8

142 M 33.7 255.1550

143 Glutamine*,** Gln C5H10N2O3 33.7 147.0767 147.0764 –0.3 –1.9

144 2-Aminoadate** C6H11NO4 33.9 162.0758 162.0761 0.3 1.8

145 Acetylhomoserine† C6H11NO4 34.2 162.0760 162.0761 0.1 0.5

146 Citrulline** C6H13N3O3 34.2 176.1029 176.1030 0.1 0.4

147 Homocitrulline** C7H15N3O3 34.3 190.1183 190.1186 0.3 1.7

148 Glutamate*,** Glu C5H9NO4 34.4 148.0607 148.0604 –0.3 –1.8

149 M 34.4 360.3225

150 Phenylalanine*,** Phe C9H11NO2 35.3 166.0866 166.0863 –0.3 –2.1

151 35.6 150.0761

6


(min) m/z measured

m/z theoretical

Δ m/z(mDa)

Δ m/z(ppm)

152 Acetyllysine† C8H16N2O3 35.7 189.1226 189.1234 0.8 4.1

153 Tyrosine*,** Tyr C9H11NO3 35.9 182.0814 182.0812 –0.2 –1.3

154 36.2 175.0016

155 36.3 273.0832

156 36.4 128.0447

157 Hypoxanthine** HPX C5H4N4O 36.4 137.0460 137.0458 –0.2 –1.6

158 36.5 122.0344

159 36.5 142.0604

160 36.6 142.0604

161 36.7 263.1230

162 Proline*,** Pro C5H9NO2 36.8 116.0707 116.0706 –0.1 –0.8

163 36.8 138.0543

164 Ser-Val† C8H16N2O4 37.3 205.1182 205.1183 0.1 0.4

165 37.3 187.1071

166 37.4 241.0308

167 Cysteine*,** Cys C3H7NO2S 39.1 122.0271 122.0270 –0.1 –0.6

168 39.4 247.0975

169 Aspartate*,** Asp C4H7NO4 39.8 134.0451 134.0448 –0.3 –2.4

170 Glycine betaine* C5H11NO2 40.6 118.0863 118.0863 0.0 –0.4

171 Glutathione, oxidized**

GSSG C20H32N6O12S2 41.6 307.0832 307.0833 0.1 0.2

172 Hydroxyproline** C5H9NO3 43.7 132.0665 132.0655 –1.0 –7.4

173 Glutathione*,** GSH C10H17N3O6S 46.8 308.0916 308.0911 –0.5 –1.7

174 46.8 361.0037

Note: Asterisk (*) signifies identification based on migration-time comparison related to chemical standards. Double asterisk (**) denotes identification supplemented by tandem mass spectrometry experiments on related standards or data available in Metlin (https://metlin.scripps.edu). Dagger (†) indicates that tandem mass spectrum agrees with fragmentation predicted in Mass Frontier 7.0 (Thermo Scientific). Double dagger (‡) indicates identification for endogenous metabolite based on mass-match in Metlin (5 ppm accuracy). M indicates molecular features detected from the embryo culture media.

7

Table S2. Significant metabolite differences between microprobe CE-ESI-MS and whole-cell dissection. Metabolic differences were calculated as fold change (FC) between normalized metabolite abundances (FC = microprobe/dissection) from data presented in Fig. 3C. Statistical significance is marked at P < 0.05. Yet unidentified molecular features are listed as detected accurate mass (m/z)/migration time (min) values. Subzero FC values are shown as negative inverse ratios (–1/FC).

Mol. Features FC P Log2FC –10log(P)

Methylhistamine 26,381.0 14.7 5.33E-09 82.7

252.9739/9.8 9.3 3.2 4.71E-02 13.3

140.9521/9.9 77.0 6.3 1.73E-04 37.6

139.9526/10.0 20.9 4.4 6.79E-03 21.7

498.9012/10.2 –42.1 –5.4 6.81E-04 31.7

226.9516/10.2 –2.6 –1.4 2.72E-02 15.7

326.2288/14.2 –13.2 –3.7 3.77E-06 54.2

223.1646/15.0 –4.3 –2.1 3.96E-03 24.0

225.1451/15.3 –42.3 –5.4 1.23E-08 79.1

255.1544/15.9 –29.3 –4.9 1.14E-03 29.4

281.1509/15.9 –15.8 –4.0 4.25E-04 33.7

285.1658/16.0 –23.8 –4.6 1.57E-04 38.0

Ornithine 5.8 2.5 3.59E-03 24.4

Lysine 4.7 2.2 5.20E-03 22.8

Sarcosine 7.9 3.0 2.20E-03 26.6

125.1077/16.3 –6.4 –2.7 1.18E-02 19.3

134.0814/16.3 –189.8 –7.6 7.20E-03 21.4

Arginine 6.2 2.6 2.83E-03 25.5

Tyr-Lys 4.4 2.1 2.59E-02 15.9

GABA 16.4 4.0 1.07E-03 29.7

TML 9.1 3.2 6.57E-04 31.8

Histidine 8.5 3.1 1.61E-03 27.9

106.0863/16.1 –5.5 –2.5 1.35E-03 28.7

139.1234/17.5 –23.1 –4.5 4.78E-05 43.2

150.0761/17.8 7.6 2.9 1.68E-02 17.8

142.1225/17.8 –561.8 –9.1 1.62E-07 67.9

124.0866/17.9 377.4 8.6 6.75E-04 31.7

Methylhistidine 25.4 4.7 6.00E-03 22.2

146.1173/18.4 28.9 4.9 3.34E-02 14.8

118.1216/18.4 9.1 3.2 1.66E-02 17.8

104.0705/18.6 28,408.0 14.8 1.94E-02 17.1

134.0809/18.9 8.4 3.1 1.89E-03 27.2

178.0878/18.9 14.3 3.8 2.15E-02 16.7

154.0976/19.0 132.3 7.0 1.10E-03 29.6

116.1069/18.3 293.0 8.2 2.38E-03 26.2


142.1225/18.3 25,714.0 14.7 2.41E-10 96.2

141.0661/18.4 18.2 4.2 9.05E-03 20.4

Acetylcholine 17.6 4.1 1.08E-04 39.7

134.1173/18.4 3,880.2 11.9 4.57E-10 93.4

116.1069/18.4 271.1 8.1 2.25E-03 26.5

142.0968/18.5 59.3 5.9 2.87E-04 35.4

120.1028/18.5 252.3 8.0 3.53E-06 54.5

Methylguanine 905.1 9.8 1.35E-05 48.7

296.0675/18.6 1,368.7 10.4 1.62E-04 37.9

402.1911/19.1 –9.0 –3.2 7.34E-06 51.3

203.1480/19.2 20.3 4.3 6.17E-03 22.1

357.0827/18.9 –6.3 –2.7 3.06E-03 25.1

154.0976/19.0 76.2 6.3 1.00E-03 30.0

Guanine 11.6 3.5 3.95E-03 24.0

142.0604/19.2 36.0 5.2 1.41E-02 18.5

210.0938/19.4 788.9 9.6 6.47E-06 51.9

136.0968/19.3 –106.4 –6.7 1.23E-04 39.1

142.1592/19.5 423.8 8.7 1.32E-03 28.8

182.1285/19.6 177.6 7.5 1.53E-03 28.2

Carnitine 3.7 1.9 5.15E-03 22.9

198.1238/19.9 10.6 3.4 2.57E-02 15.9

168.1127/19.8 9.7 3.3 1.44E-02 18.4

160.0771/20.0 58.3 5.9 4.23E-03 23.7

203.1514/20.2 69.0 6.1 3.90E-04 34.1

316.1546/20.3 –168.1 –7.4 1.46E-07 68.4

318.1514/20.4 –9.9 –3.3 6.30E-04 32.0

166.0713/20.4 3.2 1.7 3.64E-02 14.4

150.0580/20.5 267.4 8.1 5.50E-04 32.6

Pyridoxal 43.0 5.4 2.25E-02 16.5

140.1437/20.5 96.4 6.6 3.07E-03 25.1

152.0916/20.7 –2.9 –1.5 2.66E-02 15.8

140.1437/20.9 18.0 4.2 6.52E-03 21.9

130.1588/20.9 4.6 2.2 2.85E-03 25.5

194.1536/20.9 214.9 7.7 1.84E-03 27.3

150.1136/21.6 –25.0 –4.6 2.60E-07 65.8

8


194.1536/21.6 30.4 4.9 2.06E-03 26.9

142.1592/21.0 9.7 3.3 2.33E-02 16.3

178.1069/21.0 –10.2 –3.4 1.40E-04 38.5

186.1117/21.0 –97.8 –6.6 1.93E-07 67.1

210.1322/21.1 –2.6 –1.4 7.64E-03 21.2

184.1107/21.3 2.0 1.0 4.35E-02 13.6

172.0964/21.4 –6.1 –2.6 1.32E-02 18.8

166.1070/21.7 –3.8 –1.9 4.89E-02 13.1

182.1285/21.6 623.3 9.3 9.66E-04 30.2

176.0912/22.9 –7.6 –2.9 6.38E-04 32.0

Glycine 25.1 4.7 5.23E-05 42.8

294.9389/10.2 –3.5 –1.8 2.86E-02 15.4

162.0204/22.6 114.9 6.8 4.62E-04 33.4

255.0975/22.7 4.0 2.0 2.32E-02 16.3

202.1529/22.9 –17.2 –4.1 1.98E-05 47.0

Creatine 6.4 2.7 1.53E-02 18.2

102.0545/23.5 10.2 3.3 1.11E-02 19.5

215.1390/23.7 7.0 2.8 7.19E-03 21.4

285.1340/23.5 5.8 2.5 2.03E-02 16.9

196.1708/23.5 111.2 6.8 1.68E-04 37.7

240.1482/23.6 12.9 3.7 8.19E-03 20.9

218.2103/26.0 7.8 3.0 3.34E-02 14.8

276.1681/24.4 6.5 2.7 3.05E-02 15.2

362.9263/10.2 –7.3 –2.9 4.52E-03 23.5

296.0675/24.4 558.2 9.1 2.84E-03 25.5

218.1500/24.4 106.5 6.7 8.75E-03 20.6

Alanine 6.6 2.7 2.42E-03 26.2

307.1185/25.2 652.8 9.4 9.59E-04 30.2

205.1177/25.7 577.5 9.2 3.52E-03 24.5

249.1811/25.6 30.9 4.9 8.97E-03 20.5

246.2421/25.7 7.3 2.9 1.38E-02 18.6

Arginino-succinate

6.1 2.6 3.59E-02 14.5

104.0705/28.5 47.4 5.6 1.09E-03 29.6

202.1063/29.6 –204.9 –7.7 4.82E-09 83.2

Valine 16.2 4.0 1.74E-02 17.6

134.0809/30.1 2,000.5 11.0 2.54E-04 36.0

Isoleucine 7.6 2.9 2.20E-03 26.6

Leucine 7.1 2.8 9.20E-04 30.4

Asparagine 6.6 2.7 6.14E-04 32.1

Threonine 6.6 2.7 2.32E-04 36.4

Methionine 9.0 3.2 4.78E-04 33.2

106.0497/33.6 275.6 8.1 5.44E-05 42.6


Glutamine 6.8 2.8 8.41E-04 30.8

2-Aminoadipate 5.6 2.5 1.91E-03 27.2

430.9138/10.2 –18.5 –4.2 1.36E-03 28.7

Citrulline 4.7 2.2 1.51E-03 28.2

160.0771/34.2 284.5 8.2 2.36E-03 26.3

Homocitrulline 4.6 2.2 9.20E-04 30.4

Glutamate 4.8 2.3 1.54E-03 28.1

Phenylalanine 5.7 2.5 4.06E-03 23.9

150.0761/35.6 16.4 4.0 5.69E-03 22.4

Hypoxanthine 5.5 2.5 1.17E-02 19.3

159.0270/36.3 6.4 2.7 2.71E-02 15.7

Proline 6.3 2.7 4.78E-03 23.2

Ser-Val 4.3 2.1 1.27E-02 19.0

Cysteine 142.7 7.2 1.80E-05 47.5

Aspartic acid 8.2 3.0 1.19E-04 39.2

GSSG –12.1 –3.6 2.16E-03 26.7

Hydroxyproline 6.6 2.7 4.42E-04 33.6

361.0037/46.8 4.4 2.1 3.76E-02 14.2

GSH 6.2 2.6 2.81E-02 15.5

9

Table S3. Fisher’s least significant discriminant (LSD) analysis of quantitative metabolic differences between cells in the neural-fated dorsal cell clone (analysis of variance: P < 0.05).

Mol. Feature P Fisher's LSD

229.0848/7.1 1.4E-05 D1 - D11; D1 - D111; D1 - D12; D1 - D121

255.0625/7.1 8.1E-04 D111 - D1; D111 - D11; D111 - D12; D111 - D121

161.1285/7.2 2.2E-02 D111 - D1; D111 - D11; D111 - D12; D111 - D121

118.0859/7.2 3.2E-03 D111 - D1; D111 - D11; D111 - D12; D111 - D121

351.0994/7.2 9.2E-03 D111 - D1; D111 - D11; D111 - D12; D111 - D121

156.0419/7.3 2.7E-03 D1 - D111; D1 - D121; D11 - D111; D12 - D111

Putrescine 2.6E-02 D11 - D1; D111 - D1; D12 - D1; D121 - D1

Methylhistamine 3.0E-02 D1 - D111; D11 - D111

359.2103/10.0 3.2E-02 D111 - D1; D121 - D1

191.0212/10.0 5.6E-03 D111 - D1; D111 - D11; D111 - D12; D111 - D121

9369.2310/10.0 2.2E-02 D111 - D1; D111 - D11; D111 - D12; D111 - D121

145.0347/11.3 5.2E-03 D1 - D11; D1 - D12; D1 - D121; D111 - D121

361.2428/11.3 4.7E-02 D111 - D1; D111 - D11; D111 - D12

135.0305/11.4 4.0E-02 D1 - D111; D1 - D121; D12 - D111; D12 - D121

144.1016/12.8 3.7E-04 D11 - D1; D1 - D111; D11 - D111; D11 - D12; D11 - D121

76.0758/14.2 3.5E-04 D1 - D111; D1 - D12; D1 - D121; D11 - D111; D11 - D12; D11 - D121

88.0754/14.3 7.8E-03 D111 - D1; D111 - D11; D111 - D12

232.1392/15.0 3.7E-02 D11 - D1; D11 - D111; D11 - D12

Arg-Ala 9.6E-04 D11 - D1; D111 - D1; D11 - D12; D111 - D12

SAM 6.2E-04 D1 - D11; D1 - D111; D1 - D12; D1 - D121

Ser-Arg 3.4E-03 D11 - D1; D11 - D111; D11 - D12; D11 - D121

243.1081/16.2 7.4E-04 D11 - D1; D11 - D111; D11 - D12; D11 - D121; D121 - D111

274.1871/16.3 1.1E-02 D11 - D1; D11 - D111; D11 - D12; D11 - D121

90.0908/16.2 3.1E-02 D111 - D1; D111 - D11; D111 - D12; D111 - D121

Lysine 2.9E-03 D1 - D12; D11 - D12; D111 - D12; D121 - D12

276.1554/16.8 6.0E-06 D1 - D111; D1 - D12; D1 - D121; D11 - D111; D11 - D12; D12 - D111; D121 - D111

106.0855/16.1 5.1E-04 D111 - D1; D111 - D11; D111 - D12; D111 - D121

102.1265/17.0 7.7E-04 D111 - D1; D111 - D11; D111 - D12; D111 - D121

Arginine 4.1E-03 D1 - D12; D11 - D12; D111 - D12; D121 - D12

GABA 3.4E-06 D11 - D1; D111 - D1; D12 - D1; D121 - D1; D111 - D11

TML 2.6E-07 D1 - D11; D1 - D111; D1 - D12; D1 - D121; D11 - D111

Histidine 1.8E-03 D1 - D11; D1 - D111; D1 - D12; D121 - D111; D121 - D12

338.1814/17.5 1.3E-02 D11 - D1; D11 - D111; D11 - D12


Methylhistidine 2.8E-06 D1 - D11; D1 - D111; D1 - D12; D1 - D121; D11 - D111; D12 - D111; D121 - D111

Acetylcholine 8.4E-10 D1 - D11; D1 - D111; D1 - D12; D1 - D121; D11 - D111; D11 - D12

100.0759/18.2 8.2E-06 D1 - D111; D1 - D12; D1 - D121; D11 - D111; D11 - D12; D11 - D121; D12 - D111

142.1216/18.3 6.2E-05 D1 - D11; D1 - D111; D1 - D12; D1 - D121; D11 - D111

Trolamine 2.8E-06 D111 - D1; D111 - D11; D111 - D12; D111 - D121

130.1220/18.6 5.6E-04 D1 - D111; D1 - D12; D1 - D121; D11 - D111; D121 - D111

144.0650/18.6 3.5E-03 D111 - D1; D12 - D1; D121 - D1; D111 - D11

89.1066/18.9 1.2E-08 D1 - D11; D1 - D111; D1 - D12; D1 - D121

218.1489/18.8 1.4E-04 D1 - D11; D1 - D111; D1 - D12; D1 - D121; D121 - D111

Guanine 3.7E-02 D1 - D111; D11 - D111

Carnitine 2.2E-10 D1 - D11; D1 - D111; D1 - D12; D1 - D121; D11 - D111

Methylguanine 5.1E-03 D11 - D1; D11 - D111; D11 - D12

150.0542/20.5 2.8E-02 D11 - D111; D12 - D111

154.0859/20.6 3.6E-02 D11 - D111; D121 - D111

Acetylcarnitine 1.1E-04 D1 - D11; D1 - D111; D1 - D12; D11 - D12; D121 - D111; D121 - D12

112.0497/22.6 6.2E-03 D111 - D1; D111 - D11; D111 - D12; D111 - D121

198.1850/22.6 1.6E-04 D1 - D111; D1 - D12; D1 - D121; D11 - D111; D11 - D12

Propionylcarnitine 4.2E-04 D111 - D1; D121 - D1; D111 - D11; D121 - D11; D111 - D12; D121 - D12

Methylaspartate 9.3E-04 D1 - D11; D1 - D111; D1 - D12; D1 - D121

255.0968/22.7 4.6E-02 D1 - D11; D1 - D111; D1 - D12; D1 - D121

240.1231/22.7 2.3E-02 D121 - D1; D121 - D11; D121 - D111; D121 - D12

Pro-Val 5.4E-08 D1 - D11; D1 - D111; D1 - D12; D1 - D121; D11 - D111; D12 - D111; D121 - D111

215.1385/23.7 1.4E-08 D1 - D11; D1 - D111; D1 - D12; D1 - D121; D11 - D111

254.1379/23.5 2.2E-02 D1 - D11; D1 - D111; D1 - D12; D1 - D121

298.0521/25.4 2.8E-02 D11 - D111; D12 - D111; D12 - D121

262.1647/25.4 4.7E-03 D1 - D11; D1 - D111; D1 - D12; D1 - D121

260.1844/24.9 1.6E-04 D1 - D111; D1 - D12; D1 - D121; D11 - D111; D11 - D12; D11 - D121

307.1219/25.2 8.6E-06 D1 - D11; D1 - D111; D1 - D12; D1 - D121

205.1177/25.2 4.3E-02 D11 - D1; D11 - D111; D11 - D12; D121 - D111

142.1216/26.3 5.0E-02 D1 - D111; D11 - D111

10


Argininosuccinate 8.0E-04 D1 - D11; D1 - D111; D1 - D12; D121 - D111

Valine 5.1E-05 D1 - D11; D1 - D111; D1 - D12; D1 - D121; D11 - D111; D11 - D12; D121 - D12

Serine 1.4E-03 D1 - D11; D1 - D111; D11 - D111; D12 - D111; D121 - D111

Isoleucine 8.4E-07 D1 - D11; D1 - D111; D1 - D12; D1 - D121; D11 - D111; D11 - D12

Leucine 5.7E-04 D1 - D11; D1 - D111; D1 - D12; D1 - D121

Asparagine 2.4E-07 D1 - D11; D1 - D111; D1 - D12; D1 - D121; D11 - D111; D11 - D12; D121 - D111

Threonine 1.5E-04 D1 - D11; D1 - D111; D1 - D12; D1 - D121; D12 - D111; D121 - D111

264.1187/32.7 1.2E-03 D1 - D11; D1 - D111; D1 - D12; D1 - D121

188.0697/33.4 8.4E-04 D1 - D111; D1 - D12; D11 - D111; D11 - D12

Methionine 2.9E-10 D1 - D11; D1 - D111; D1 - D12; D1 - D121

Glutamine 5.7E-03 D1 - D11; D1 - D111; D1 - D12; D1 - D121

2-aminoadipate 6.2E-03 D1 - D111; D1 - D12; D11 - D111; D121 - D111

170.0418/34.3 6.6E-03 D11 - D1; D11 - D111; D11 - D12

333.0606/34.3 1.3E-10 D1 - D11; D1 - D111; D1 - D12; D1 - D121; D11 - D111; D12 - D111

Glutamate 1.0E-05 D1 - D11; D1 - D111; D1 - D12; D1 - D121

Phenylalanine 3.9E-03 D1 - D11; D1 - D111; D1 - D12; D121 - D12

123.0433/35.9 1.5E-02 D1 - D111; D11 - D111; D12 - D111

Tyrosine 4.3E-03 D1 - D11; D1 - D111; D1 - D12; D1 - D121

159.0269/36.3 8.0E-03 D11 - D1; D11 - D111; D11 - D121; D12 - D111

Ser-Val 6.1E-04 D1 - D111; D1 - D12; D1 - D121; D11 - D111; D11 - D12

Cysteine 3.2E-02 D11 - D1; D11 - D111

Aspartate 2.1E-12 D1 - D11; D1 - D111; D1 - D12; D1 - D121; D11 - D111; D11 - D12

Glycine betaine 6.5E-06 D1 - D11; D1 - D111; D1 - D12; D1 - D121; D11 - D111

144.1016/42.1 3.1E-04 D1 - D111; D1 - D12; D1 - D121; D11 - D111; D12 - D111; D121 - D111

Hydroxyproline 1.1E-04 D1 - D11; D1 - D111; D1 - D12; D1 - D121

160.0962/44.7 1.4E-03 D1 - D11; D1 - D111; D1 - D12; D1 - D121

277.1025/46.2 6.8E-06 D1 - D11; D1 - D111; D1 - D12; D1 - D121; D11 - D111

346.0404/46.8 5.3E-03 D1 - D11; D1 - D111; D1 - D12; D1 - D121

437.0552/46.9 7.6E-07 D1 - D11; D1 - D111; D1 - D12; D1 - D121

234.0570/46.9 1.0E-09 D1 - D11; D1 - D111; D1 - D12; D1 - D121

130.1220/43.7 7.2E-04 D1 - D11; D1 - D111; D1 - D12; D1 - D121

GSH 6.6E-06 D1 - D11; D1 - D111; D1 - D12; D1 - D121

11

Table S4. Results from GProX Cluster Analysis of average metabolite ratios between cells in the dorsal cell lineage after normalization to the progenitor D1R cell. Yet unidentified molecular features are listed as accurate mass (m/z)/migration time (min) values.

Mol. Feature Cluster D11/D1 D12/D1 D111/D1 D121/D1

122.0252/11.4 0 0.80 0.87 1.05 0.76

Lysine 0 1.01 0.83 0.91 0.94

Argnine 0 1.08 0.82 0.96 0.98

Histidine 0 0.86 0.79 0.67 0.94

Creatine 0 0.84 0.80 0.98 0.89

Phenylalanine 0 0.86 0.80 0.75 0.84

255.0625/7.1 1 0.75 0.64 1.33 0.89

333.1584/7.2 1 0.94 0.66 1.11 0.72

161.1285/7.2 1 0.39 0.93 4.98 1.98

118.0859/7.3 1 0.31 0.39 4.83 2.23

351.0994/7.3 1 0.84 0.84 1.59 1.04

255.0973/7.7 1 0.66 0.65 0.98 0.58

88.0754/7.9 1 1.87 1.83 5.59 3.24

Spermidine 1 0.34 0.65 1.54 0.84

Putrescine 1 1.99 2.01 3.21 3.08

387.2417/10.0 1 0.38 0.69 17.04 7.98

151.0083/11.3 1 0.85 0.55 1.14 0.83

361.2428/11.4 1 1.35 1.16 2.19 1.71

Arg-Ala 1 2.64 1.38 3.73 2.48

90.0908/16.2 1 0.74 0.85 5.02 1.15

106.0855/16.1 1 0.16 0.21 3.06 0.58

102.1265/17.1 1 1.25 0.48 2.77 1.48

Homolysine 1 0.62 0.65 1.89 0.67

166.0714/18.6 1 5.93 2.92 6.90 9.70

Trolamine 1 0.33 0.71 2.89 0.73

GABA 1 1.48 1.67 4.80 4.21

144.0650/18.7 1 0.86 0.95 3.33 2.37

166.0857/18.7 1 0.88 1.19 4.11 3.54

112.0497/22.6 1 3.63 0.75 6.57 1.28 Propionyl-carnitine 1 1.11 1.56 3.82 4.56

144.1016/12.8 2 2.73 0.94 0.02 0.61

Choline 2 1.77 1.14 0.38 1.26

Ala-Lys 2 4.31 2.05 3.18 4.78

Ser-Arg 2 6.21 2.72 3.53 3.93

248.1601/16.1 2 1.34 1.14 0.66 1.31

243.1081/16.2 2 9.31 3.57 1.05 4.00

Arg-Val 2 7.06 1.30 3.04 2.98

Tyr-Lys 2 2.78 1.52 2.29 2.40


160.0748/20.1 2 1.92 1.15 0.36 1.07

Methylguanine 2 11.21 2.61 0.71 4.72

150.0542/20.6 2 1.81 1.23 0.34 0.79

154.0859/20.6 2 4.47 4.01 0.10 3.18

278.1249/21.6 2 2.44 2.61 1.41 1.64

240.1231/22.8 2 2.20 2.63 0.14 4.39

226.1425/22.8 2 2.10 1.12 0.01 1.19

212.1015/26.5 2 8.29 4.98 0.00 2.57

Citrulline 2 0.68 0.67 0.62 1.57

Acetyllysine 2 5.82 0.27 2.94 2.34

175.1071/36.0 2 2.19 1.19 0.11 0.97

241.0305/37.5 2 4.58 4.40 3.31 2.20

Cysteine 2 2.97 1.63 0.54 1.51

229.0848/7.1 3 0.20 0.25 0.32 0.25

145.0347/11.4 3 0.44 0.47 0.67 0.24

SAM 3 0.68 0.63 0.62 0.62

TML 3 0.71 0.48 0.36 0.50

Acetylcholine 3 0.75 0.66 0.34 0.47

142.1216/18.4 3 0.67 0.45 0.09 0.11

89.1066/18.9 3 0.22 0.39 0.10 0.17

218.1489/18.8 3 0.45 0.52 0.00 0.53

Carnitine 3 0.68 0.70 0.40 0.44

Acetylcarnitine 3 0.76 0.39 0.42 0.73

Methylaspartate 3 0.53 0.47 0.35 0.52

255.0968/22.8 3 0.65 0.77 0.67 0.59

232.1516/23.0 3 0.53 1.08 0.86 0.93

254.1379/23.5 3 0.78 0.51 0.28 0.52

262.1647/25.5 3 0.60 0.88 0.48 0.61

307.1219/25.2 3 0.45 0.59 0.43 0.46 Arginino-succinate 3 0.41 0.37 0.19 0.70

320.1860/26.3 3 0.55 0.70 0.29 0.80

Valine 3 0.97 0.56 0.54 0.69

Isolucine 3 0.91 0.55 0.40 0.57

Leucine 3 0.67 0.48 0.55 0.54

264.1187/32.7 3 0.82 0.67 0.59 0.65

Threonine 3 0.78 0.80 0.35 0.66

Methionine 3 0.55 0.60 0.27 0.36

Glutamine 3 0.84 0.81 0.64 0.63

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333.0606/34.4 3 0.44 0.57 0.06 0.18

Glutamate 3 0.80 0.75 0.53 0.61

Tyrosine 3 0.77 0.83 0.66 0.70

Aspartic acid 3 0.66 0.54 0.38 0.40

Hydroxyproline 3 0.78 0.71 0.53 0.67

160.0962/44.7 3 0.63 0.63 0.03 0.45

277.1025/46.3 3 0.54 0.33 0.04 0.30

346.0404/46.8 3 0.71 0.68 0.56 0.54

437.0552/47.0 3 0.19 0.38 0.14 0.04

234.0570/46.9 3 0.24 0.31 0.12 0.24

130.1220/43.7 3 0.49 0.38 0.10 0.13

GSH 3 0.66 0.63 0.52 0.52

156.0419/7.3 4 0.98 1.01 0.38 0.52

Methylhistamine 4 1.37 1.11 0.31 0.80

135.0305/11.4 4 0.90 1.23 0.01 0.11

76.0758/14.3 4 1.07 0.68 0.13 0.23

112.0496/16.0 4 1.30 1.12 0.30 0.61

Ornithine 4 1.09 1.77 0.24 0.68

276.1554/16.8 4 1.10 0.69 0.00 0.39

104.1058/18.3 4 18.31 661.79 4.30 1.64

Methylhistidine 4 0.80 0.90 0.06 0.39

100.0759/18.2 4 1.12 0.49 0.01 0.33

130.1220/18.6 4 1.07 0.49 0.14 0.54

Guanine 4 1.16 0.79 0.50 0.76

128.0444/19.0 4 1.11 1.02 0.47 0.74

Pyridoxal 4 1.25 1.36 0.52 0.72

198.1850/22.7 4 0.97 0.63 0.41 0.64

123.0565/22.8 4 1.12 1.32 0.00 0.36

Pro-Val 4 0.86 0.74 0.03 0.33

222.1846/22.8 4 1.17 1.17 0.36 0.78

298.0521/25.5 4 1.84 2.19 0.39 0.54

218.1538/24.4 4 1.64 1.42 0.37 0.29

260.1844/24.9 4 1.52 0.88 0.04 0.32

320.1713/25.6 4 1.26 2.17 0.39 0.36

142.1216/26.3 4 1.50 0.93 0.11 0.65

Serine 4 0.71 0.94 0.04 0.66

Asparagine 4 0.88 0.64 0.21 0.52

188.0697/33.5 4 1.04 0.65 0.63 0.82

2-Aminoadate 4 1.29 0.71 0.14 0.74

123.0433/35.9 4 1.37 0.96 0.11 0.51

Hypoxanthine 4 0.97 1.10 0.67 0.67

Proline 4 1.70 1.48 0.37 0.77


Ser-Val 4 1.38 0.61 0.02 0.43

427.0941/39.5 4 1.21 0.94 0.83 0.51

Glycine betaine 4 0.93 0.71 0.23 0.42

144.1016/42.1 4 1.07 0.87 0.09 0.48

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SUPPLEMENTARY FIGURES

Figure S1. Representative molecular features detected from 10 nL portion of the embryo culture media. Microprobe CE-ESI-MS eliminated/minimized these signals, which are known to cause ion suppression during ESI-MS, thus efficiently enhancing detection sensitivity. Underlined numbers correspond to the molecular features listed in Table S1.

In Situ Microprobe Single-Cell Capillary Electrophoresis …, including 70 known ... cell dissection...

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Transcript of In Situ Microprobe Single-Cell Capillary Electrophoresis …, including 70 known ... cell dissection...