Subcellular metabolite and lipid analysis of Xenopus laevis eggs by LAESI mass spectrometry.

Xenopus laevis eggs are used as a biological model system for studying fertilization and early embryonic development in vertebrates. Most methods used for their molecular analysis require elaborate sample preparation including separate protocols for the water soluble and lipid components. In this study, laser ablation electrospray ionization (LAESI), an ambient ionization technique, was used for direct mass spectrometric analysis of X. laevis eggs and early stage embryos up to five cleavage cycles. Single unfertilized and fertilized eggs, their animal and vegetal poles, and embryos through the 32-cell stage were analyzed. Fifty two small metabolite ions, including glutathione, GABA and amino acids, as well as numerous lipids including 14 fatty acids, 13 lysophosphatidylcholines, 36 phosphatidylcholines and 29 triacylglycerols were putatively identified. Additionally, some proteins, for example thymosin β4 (Xen), were also detected. On the subcellular level, the lipid profiles were found to differ between the animal and vegetal poles of the eggs. Radial profiling revealed profound compositional differences between the jelly coat vitelline/plasma membrane and egg cytoplasm. Changes in the metabolic profile of the egg following fertilization, e.g., the decline of polyamine content with the development of the embryo were observed using LAESI-MS. This approach enables the exploration of metabolic and lipid changes during the early stages of embryogenesis.

Introduction

The early stages of embryonic development following fertilization in animals are characterized by synchronous cell divisions, the onset of transcription of genes that will pattern the embryo, and local signaling events that transition a ball of equipotent cells into regions that express different tissue fates [1], [2]. These events are common across different species of animals, but can occur for different lengths of times and at different stages of morphogenesis.

The African clawed frog, Xenopus laevis, has been used extensively to study the cellular and molecular events of early embryogenesis. This species is used because they are easily bred in the laboratory, are disease resistant, have a reproductive response that is independent of season, and produce a large number of eggs per spawning. In particular, the large size of the oocytes, ova (unfertilized eggs) and embryos make them easy to manipulate and analyze by standard biochemical and molecular biology approaches [3], [4], [5], [6], [7].

X. laevis oocytes and eggs have been an important source of material for elucidating the molecular regulation of the cell cycle. For example, traditional protein chemistry approaches led to the identification of maturation promoting factor and the cyclin proteins [8], [9]. More recently, the oocyte has been used to identify the proteins involved in nuclear transport and DNA replication [6]. To reveal the biochemical makeup, elucidate low abundance proteins, and study the kinetics of developmental and cellular events, X. laevis oocytes have been analyzed by high performance liquid chromatography (HPLC) [10], [11] [12], time-of-flight (TOF) secondary ion mass spectrometry (SIMS) [13], gas chromatography (GC) mass spectrometry (MS) [14], 2D gel electrophoresis followed by electrospray ionization (ESI) MS [15], and liquid chromatography-tandem MS (LC-MS/MS) [2]. To study the types and amounts of lipids extracted from Xenopus eggs, HPLC methods were developed for the detection of multiple species [10], [12]. Similarly, with an HPLC method for characterizing the carbohydrates in the jelly coat of Xenopus eggs, specific oligosaccharides were identified and assigned to the three specific layers of the coat [11], [16]. In another study, LC-MS/MS was used to monitor the amounts of alanine in the early embryo, resulting in a new hypothesis for the increase in cell cycle time at the mid-blastula transition [2]. Using a GC-MS method for analyzing metabolites from volume-limited samples, e.g., X. laevis eggs, good reproducibility was shown for volumes as small as 10 nL. However, the method required silylation of some metabolites to increase their volatility for analysis using GC [14]. On a subcellular scale, 3D molecular images of freeze dried Xenopus oocytes were obtained under optimized sample preparation conditions using TOF-SIMS [13]. While all of these methods for X. laevis oocytes and embryo analysis have been shown to capture important aspects of its biochemistry, most of them require elaborate sample preparation prior to analysis that might alter subcellular localization of important developmental molecules.

An advantage of direct analysis methods is that they can report on the unperturbed composition of biological specimens, ideally in their living, natural state. Recently, the analysis of single oocytes and embryos of mice and bovine species by desorption electrospray ionization (DESI) mass spectrometry provided new insight into lipid metabolism changes during early embryonic development [17], [18].

In another direct analysis technique, laser ablation electrospray ionization (LAESI) MS, the energy from a focused mid-infrared laser pulse is absorbed by the water molecules in a cell or tissue leading to the ablation sampling of adjacent cellular content [19]. Each consecutive laser pulse ablates a successive layer of the sample enabling analysis at a defined subcellular depth or of an entire cell [20], [21]. Combination of LAESI with microdissection of plant cells reveals subcellular metabolite gradients between the cytoplasm and the nucleus [22]. Diverse applications of LAESI-MS include the analysis of virally infected immune cells as well as comparative analysis of plant cell phenotypes [23], [24]. Because the developing egg is a spatially and temporally dynamic system that is sensitive to environmental and thus preparatory changes, we used the direct LAESI-MS analysis of individual X. laevis eggs and embryos to explore subcellular asymmetries and potential changes during significant developmental transitions. Using LAESI-MS to probe these specimens required minimal sample preparation, was performed at atmospheric pressure, and each ablation event occurred instantaneously (in less than a second) on the developmental time scale. The analyses were performed on living eggs and embryos that were minimally perturbed from their natural state.

Some of the metabolic profiles of the unfertilized egg obtained from the LAESI-MS analyses were compared to results reported previously in the literature and showed good agreement, thus validating the approach. In addition we identified subtle differences between the unfertilized egg, in which the first meiotic division is completed, and the fertilized egg and early cleavage stage embryos. Importantly, we found differences between the animal and vegetal poles of the egg, which are predisposed to different developmental fates. Since these early developmental events are shared across animals, a direct analysis of the cellular constituents should provide fundamental information about the common biochemical processes that regulate them. This simple approach using a single, live embryo in its natural state should be ideal for analyses of other embryos that are difficult to collect and are small in size.

Materials and Methods

Ova and Embryos

X. laevis eggs and sperm were obtained from mature animals kept in 30 gallon aquaria at 21°C in natural daylight and fed frog brittle 2 times per week. Oocyte maturation was induced by injecting frogs with 1000 U human chorionic gonadotropin (Sigma) 12 hours prior to removal of eggs as described elsewhere [25]. Mature eggs were gently squeezed from female frogs into Petri dishes; some were collected prior to fertilization and others were fertilized in vitro and collected at various stages after sperm entry. Sperm were obtained from minced testes of sacrificed males that were anesthetized by submersion in an ice bath with tricaine methanesulfonate. Eggs were fertilized in vitro by adding the released sperm to the eggs and adding 0.1× MBS (1× = 88 mM NaCl, 1 mM KCl, 1 mM MgSO4, 0.7 mM CaCl2, 5 mM HEPES, pH 7.8 and 2.5 mM NaHCO3) [26]. The jelly coat was removed from some samples by washing the eggs with 2% cysteine solution (pH 8.0, adjusted by 10 M NaOH) for 4 minutes followed by washing in 0.1× Steinberg's solution (1× = 60 mM NaCl, 0.67 mM KCl, 0.83 mM MgSO4, 0.34 mM Ca(NO3)2, 4 mM Tris-HCl, 0.66 mM Tris Base and pH 7.4). For some experiments the vitelline membrane was manually removed from each egg using sharpened forceps. Unfertilized eggs of X. laevis were also purchased from Xenopus Express, Inc. (Brooksville, FL) for some confirmatory studies. All animals use in these experiments followed the U.S. Public Health Service Policy of Humane Care and Use of Laboratory Animals and were approved by the George Washington University (GWU Animal Study Protocol: #A-3205-01; A237).

Chemicals

All chemicals for egg preparation were reagent grade and obtained from Sigma. For the electrospray solution, highly purified spectral-grade methanol and water solvents were obtained from Acros Organics (Geel, Belgium), and glacial acetic acid was purchased from Fluka (Munich, Germany). All of the chemicals were used without further purification.

LAESI-MS

The LAESI ion source used was similar to those described earlier [19]. Briefly, mid-infrared laser pulses at 2940 nm wavelength and 20 Hz repetition rate were produced by a Nd:YAG laser driven optical parametric oscillator system (Opolette 100, Opotek, Carlsbad, CA). Laser pulses with an average 320 µJ/pulse energy were focused to about 200–300 µm diameter spot with either a 150 mm focal length CaF2 lens or a 75 mm ZnSe lens. The eggs/embryos were placed on a pre-cleaned microscope glass slide held 15 mm below the electrospray axis that was in line with the mass spectrometer orifice. In the home-built electrospray system, a syringe pump supplied 50% methanol solution containing 0.1% acetic acid through a tapered tip metal emitter (length 5 cm, tube OD 320 µm and tip ID 50 µm) at a flow rate of 300–400 nL/min. The electrospray emitter was held at 2800–3300 V. Positive ions were collected by either an orthogonal acceleration TOF mass spectrometer (Q-TOF Premier, Waters Co., Milford, MA) with a mass range of m/z 50–2000 and a typical resolution of 8,000 (FWHM) or with a high performance TOF mass spectrometer (Synapt G2S, Waters Co., Milford, MA) with a mass range of m/z 50–2000 and a typical resolution of 30,000 (FWHM) [27].

Data Analysis

The electrospray background ion signal was subtracted from the collected LAESI mass spectra in the MassLynx 4.1 software (Waters Co., Milford, MA). Putative peak assignments for metabolites and lipids were based on accurate mass measurements, isotope distribution patterns, database searches, data mining of the related literature, and in some cases tandem MS analysis. Multiple databases were used for the metabolite and lipid searches, including METLIN [28], MetaCyc [29], Lipid Maps [30], HMDB [31] and KEGG [32], [33]. They were last accessed on August 12, 2014. The NIST Isotope Calculator program (ISOFORM, Version 1.02) was used to calculate monoisotopic masses.

Results and Discussions

Ovum Analysis

A single X. laevis egg is ∼1.4 mm in diameter and has an approximate volume of 400 nL. A typical LAESI mass spectrum from a single unfertilized X. laevis egg without the removal of its jelly coat is shown in Fig. 1. Most of the ions below m/z 450 correspond to small metabolites, whereas many ions between m/z 450 and 900 are assigned to lipids. For a typical unfertilized X. laevis ovum, putative assignments of the ions generally found in the m/z <550 region reveals 52 small metabolites (see Table 1). Many of these metabolites, e.g., amino acids, organic acids, and redox buffering agents, fulfill an essential role in cell development and serve as building blocks for cellular biosynthesis. Using liquid extraction and LC-MS/MS, a previous study followed 48 metabolites in X. laevis embryos [2]. In Table 1, sixteen metabolites found by both the LC-MS/MS and LAESI-MS methods are marked by asterisks. The advantage of LAESI-MS over conventional techniques is that it rapidly (within seconds) identifies metabolites in a single egg with minimum perturbation before analysis. It also provides complementary data for improved coverage of the small metabolites.

Figure 1

A typical positive ion LAESI mass spectrum of a single unfertilized X. laevis egg is dominated by numerous metabolite and lipid peaks.

Protonated thymosin β4 (Xen) peptide in the 6+ and 5+ charge states (m/z 835 and 1002, respectively) is also present in the spectrum. Deconvolution of the peptide peaks, shown in the inset, yields a mass of 5004 Da corresponding to the molecular mass of thymosin β4 (Xen).

Table 1

Putative identification of metabolite ions from LAESI-MS of an unfertilized X. laevis egg.

Metabolite	Chemical formula	Monoisotopic mass	Measured mass	Δm (mDa)
carbamate	CH2NO2	82.998 (Na+)	82.997	−1
putrescine	C4H12N2	89.108 (H+)	89.106	−2
alanine*	C3H7NO2	90.056 (H+)	90.054	−2
choline*	C5H13NO	104.108 (H+)	104.106	−1
propionic acid	C3H6O2	113.001 (K+)	113.012	11
creatinine	C4N3H7O	114.067 (H+)	114.064	−3
propanediol	C3H8O2	115.016 (H+)	115.022	6
proline*	C5H9NO2	116.071 (H+)	116.071	0
		138.053 (Na+)	138.053	0
niacinamide	C6H6N2O	123.056 (H+)	123.049	−7
hydroxyethylphosphonate	C2H7O4P	127.016 (H+)	127.023	7
butyric acid	C4H8O2	127.016 (K+)	127.023	7
octenol	C8H16O	129.128 (H+)	129.135	7
creatine	C4H9N3O2	132.077 (H+)	132.075	−2
imidazole acetaldehyde (or)	C5H6N2O	133.038 (Na+)	133.033	−5
N-acetylimidazole	C5H6N2O	133.038 (Na+)	133.033	−5
malic acid*	C4H6O5	135.029 (H+)	135.030	1
adenine	C5H5N5	136.062 (H+)	136.053	−9
threonic acid or erythronic acid	C4H8O5	137.045 (H+)	137.044	−1
hexenal	C6H10O	137.037 (K+)	137.044	7
valeric acid or pentanoic acid	C5H10O2	141.032 (K+)	141.038	6
gamma aminobutyric acid amide	C4H10N2O	141.043 (K+)	141.038	−5
gamma-hydroxybutyric acid	C4H8O3	143.011 (K+)	143.018	7
spermidine	C7H19N3	146.166 (H+)	146.164	−2
guaiacol	C7H8O2	147.042 (Na+)	147.048	6
glutamate*	C5H9NO4	148.061 (H+)	148.058	−3
methionine*	C5H11NO2S	150.059 (H+)	150.047	12
guanine* or hydroxyadenine	C5H5N5O	152.057 (H+)	152.054	−3
		174.039 (Na+)	174.038	−1
		190.013 (K+)	190.012	−1
dihydroorotic acid	C5H6N2O4	159.041 (H+)	159.040	−0
homospermidine	C8H21N3	160.181 (H+)	160.180	−1
urocanic acid	C6H6N2O2	161.033 (Na+)	161.029	−4
carnitine*	C7H15NO3	162.113	162.102	11
aspartic acid*	C4H7NO4	172.001 (K+)	172.008	7
glycerol 3-phosphate*	C3H9O6P	173.022 (H+)	173.028	6
arginine	C6H14N4O2	175.120 (H+)	175.117	−3
cys gly	C5H10N2O3S1	179.049 (H+)	179.046	−3
phosphocholine	C5H14NO4P	184.074 (H+)	184.070	−4
		206.056 (Na+)	206.050	−6
histidine*	C6H9N3O2	194.033 (H+)	194.033	0
phosphocreatine	C4H10N3O5P	212.044 (H+)	212.042	−2
N-formylmethionine	C6H11NO3S	216.010 (H+)	216.010	0
citrate*	C6H8O7	215.0168 (Na+)	215.010	−7
dopaquinone	C9H9NO4	234.017 (K+)	234.022	5
homocarnosine or balenine	C10H16N4O3	241.130 (H+)	241.131	1
pantothenate*	C9H17NO5	242.100 (Na+)	242.088	−12
		258.074 (K+)	258.070	−4
methylthio propylmalic acid	C8H14O5S	245.046 (Na+)	245.050	4
		261.020 (K+)	261.027	7
acetyldihydrolipoamide	C10H19NO2S2	250.094 (H+)	250.091	−3
glutathione*	C10H17N3O6S	308.092 (H+)	308.090	−2
		330.074 (Na+)	330.068	−6
		346.048 (K+)	346.047	−1
hydroxydesipramine	C18H22N2O	321.137 (K+)	321.127	−10
disaccharide or trehalose*	C12H22O11	365.106 (Na+)	365.107	1
		381.080 (K+)	381.080	0
cholesterol	C27H46O	369.352 (H+, -H2O)	369.354	2
adenosine monophosphate (AMP)	C10H14N5O7P	386.027 (K+)	386.017	−10
adenosine diphosphate (ADP)*	C10H15N5O10P2	428.037 (H+)	428.032	−5
		450.019 (Na+)	450.006	−13
		465.993 (K+)	465.981	−12
adenosine triphosphate (ATP)*	C10H16N5O13P3	508.004 (H+)	508.002	−2
		529.986 (Na+)	529.975	−11

*Metabolites also detected in Ref. [2].

In the mass spectra we noticed the presence of multiply charged peaks at m/z 835, 1,002, and 1,251 with charge states of 6+, 5+ (see Fig. 1) and 4+, respectively. Deconvolution of these peaks revealed that they corresponded to thymosin β4 (Xen) (see the inset in Fig. 1). This 44-residue peptide in Xenopus has been highly conserved throughout evolution as it only differs from the human thymosin β4 in three amino acid residues [34]. A typical defolliculated oocyte contains 0.5 and 10 picomoles of thymosin β4 (Xen) in stage I and stage VI of the oogenesis, respectively [34]. Our results show that unfertilized eggs, the next step after stage VI oocytes, continue to contain thymosin β4 (Xen) in significant quantities.

Radial Profiling of Ovum

The X. laevis egg is surrounded by a jelly coat that must be penetrated by the sperm during fertilization. The LAESI mass spectra of the jelly coat were dominated by sodiated disaccharide, trisaccharide, and tetrasaccharide molecules at m/z 365.108, 527.192 and 689.286, respectively. The Xenopus egg jelly is known to contain a 21,073 Da protein, allurin, that serves as a chemoattractant for the sperm [35]. The LAESI mass spectra of the jelly coat did not show the ions for allurin, but exhibited strong signal for multiply charged ions corresponding to a protein with a molecular weight of 11,728±1 Da (see Fig. 2) [36].

Figure 2

Positive ion LAESI mass spectrum of X. laevis egg jelly coat primarily shows sodiated oligosaccharide ions with a set of multiply charged ions at higher m/z corresponding to a species with molecular weight of 11728±1 Da.

Radial profiling of an unfertilized X. laevis egg with a jelly coat was conducted by ablating the same area with multiple laser pulses, producing a mass spectrum for each shot (see Fig. 3). The first approximately six laser shots produced mass spectra similar to that of the jelly coat (see the top panel in Fig. 3). This observation was consistent with the ∼30 µm ablation depth per laser shot and the ∼200 µm thickness of typical jelly coats. Once the jelly coat-related signal diminished, multiply charged thymosin β4 (Xen) peptide ions were detected (see the middle panel in Fig. 3). From its radial localization, it seems that this peptide is associated with the vitelline and/or plasma membranes. After approximately two additional laser shots, metabolites and lipid species characteristic to the egg cytoplasm composition were detected in the mass spectra (see bottom panel in Fig. 3).

Figure 3

Radial profiling of jellied unfertilized X. laevis egg by consecutive laser shots generated mass spectra at each shot corresponding to increasing depths.

The first ∼6 laser shots produced mass spectra similar to that of the jelly coat (top spectrum). This was followed by a mass spectrum that showed presence of multiply charged thymosin β4 (Xen) peptide (middle spectrum). After an additional ∼2 laser pulses, other metabolites and lipids characteristic of the cytoplasm were detected (bottom spectrum).

Lipid Composition of Unfertilized Ovum

The cytoplasm of the unfertilized egg contains a high percentage of yolk platelets composed of proteins and lipids for nourishment of the embryonic cells [37]. The major peaks observed between m/z 400 and 900 in the LAESI mass spectrum are attributed to lipids. Earlier studies on phospholipid extracts from X. laevis eggs showed that the major lipid components were phosphatidylcholines (PC) at ∼20 nmol/egg, followed by phosphatidylethanolamines (PE) at ∼10 nmol/egg, phosphatidylinositols (PI) at ∼4 nmol/egg, lysophosphatidylcholines (LPC) at ∼2 nmol/egg, and phosphatidylserines (PS) at ∼1 nmol/egg [12]. In addition to phospholipids, triacylglycerols (TAG) were the major neutral lipid components found in the Xenopus eggs along with fatty acids (FA), cholesterol and diacylglycerols (DAG) [38].

The LAESI mass spectra gave additional insight into the nature of the lipids present in the ovum. A wide variety of lipid classes were identified, including 14 FAs, 13 LPCs, 36 PCs and 29 TAGs. Most of the abundant lipid peaks in these spectra corresponded to PCs, where one of the acyl chains was palmitic acid (16:0), whereas the other acyl chain length and saturation varied between (14:0) and (22:6) (see Table 2). The acyl chain length assignments were based on the tandem mass spectra of the lithiated lipids, [M+Li]+, generated by reactive LAESI. In this technique Li+ ions from the electrospray solution react with the lipid molecules from the ablated sample [39]. For example, tandem MS of a protonated lipid ion at m/z 760.582 produced a single fragment ion corresponding to its phosphocholine head group at m/z 184.073 resulting in the overall assignment of PC(34:1) showing only the combined length and number of double bonds for the acyl chains (see Fig. 4a). Further information on the individual acyl chains was obtained by producing and fragmenting the corresponding lithiated species, which showed fragments for the loss of oleic acid (18:1) and palmitic acid (16:0) at m/z 510.3631 and 484.3452, respectively (see Fig. 4b). Another major class of lipids in the spectra is LPC that is structurally similar to PC without the acyl group at sn-2. Phospholipase A2 is known to degrade PC into LPC and facilitate fertilization. Similar to PC, the major LPC is 16:0, and others include acyl chains between 14:0 and 22:6 (see Table 2). Because LPCs contain a free –OH group on the glycerol moiety, they undergo a loss of H2O during protonation and most ions are detected as [MH-H2O]+. These fragments are often more abundant than the protonated peaks. A few weak peaks corresponding to PE and PS can also be detected in the spectra. The major lipids found in the LAESI experiments and their fatty acid compositions are in good agreement with the literature reports based on extraction, HPLC separation and GC-based fatty acid analysis [12]. A putative list of the assigned lipids observed in the LAESI mass spectra of de-jellied unfertilized X. laevis egg is shown in Table 2. LAESI mass spectra with annotations for selected FA, LPC and PC species are shown in Fig. 5.

Table 2

Putative peak assignments for fatty acid and lipid ions from LAESI-MS of an unfertilized X. laevis egg.

Lipid	Chemical formula	Monoisotopic mass	Measured mass	Δm (mDa)
Propionic acid (3:0)	C3H6O2	113.001 (K+)	112.992	−9
Butenoic acid or Crotonic acid (4:1)	C4H6O2	125.001 (K+)	124.998	−3
Butyric acid (4:0)	C4H8O2	127.016 (K+)	127.010	−6
Pentanoic acid	C5H10O2	141.032 (K+)	141.039	7
Caprylic acid (8:0)	C8H16O2	183.079 (K+)	183.072	−7
FA(14:0)	C14H28O2	267.173 (K+)	267.167	−6
FA(15:0)	C15H30O2	281.188 (K+)	281.184	−4
FA(16:1)	C16H30O2	277.214(Na+)	277.200	−14
		293.188 (K+)	293.179	−9
FA(16:0)	C16H32O2	279.230 (Na+)	279.217	−13
		295.204 (K+)	295.195	−9
FA(18:5)	C18H26O2	297.183 (Na+)	297.194	11
FA(18:4)	C18H28O2	299.199 (Na+)	299.186	−13
		315.173 (K+)	315.157	−16
FA(18:3)	C18H30O2	317.188 (K+)	317.183	−5
FA(18:2)	C18H32O2	303.230 (Na+)	303.223	−7
		319.204 (K+)	319.195	−9
FA(18:1)	C18H34O2	321.220 (K+)	321.214	−6
FA(18:0)	C18H36O2	323.235 (K+)	323.226	−9
FA(20:5)	C20H30O2	325.214 (Na+)	325.204	−10
		341.188 (K+)	341.181	−7
FA(20:4)	C20H32O2	327.230 (Na+)	327.222	−8
		343.204 (K+)	343.200	−4
FA(22:6)	C22H32O2	351.230 (Na+)	351.239	9
		367.204 (K+)	367.201	−3
FA(22:5)	C22H34O2	369.220 (K+)	369.210	−10
LPC(14:0)	C22H46NO7P	468.309 (H+)	468.313	4
	C22H44NO6P	450.302 (H+, -H2O)	450.299	−4
LPC(15:0)	C23H48NO7P	482.325 (H+)	482.333	8
	C23H46NO6P	464.314 (H+, -H2O)	464.329	15
LPC(16:1)	C24H48NO7P	494.325 (H+)	494.329	4
		266.644 (H+K)2+	266.639	−5
	C24H46NO6P	476.314 (H+, -H2O)	476.312	−2
LPC(16:0)	C24H50NO7P	496.340 (H+)	496.342	2
		518.322 (Na+)	518.329	7
		267.652 (H+K)2+	267.64	−5
		478.330 (H+, -H2O)	478.330	0
		258.647 (H+K)2+	258.641	−6
LPC(18:4)	C26H46NO7P	516.309 (H+)	516.315	6
LPC(18:3)	C26H48NO7P	518.329 (H+)	518.325	−4
	C26H46NO6P	500.314 (H+, -H2O)	500.301	−13
LPC(18:2)	C26H50NO7P	520.340 (H+)	520.339	−1
	C26H48NO6P	502.330 (H+, -H2O)	502.333	3
LPC(O-18:1)	C26H54NO6P	508.377 (H+)	508.380	3
		273.670 (H+K)2+	273.663	−7
LPC(18:1)	C26H52NO7P	522.356 (H+)	522.353	−3
		280.660 (H+K)2+	280.656	−4
	C26H46NO6P	504.345 (H+, -H2O)	504.347	2
LPC(18:0)	C26H54NO7P	524.372 (H+)	524.381	9
	C26H52NO6P	506.361 (H+, -H2O)	506.360	−1
LPC(20:5)	C28H48NO7P	542.325 (H+)	542.327	2
		290.644 (H+K)2+	290.648	4
		524.314 (H+, -H2O)	524.320	6
LPC(20:4)	C28H50NO7P	544.340 (H+)	544.349	9
	C28H48NO6P	526.330 (H+, -H2O)	526.324	−6
LPC(22:6)	C30H50NO7P	568.340 (H+)	568.348	8
		303.652 (H+K)2+	303.652	0
		550.349 (H+, -H2O)	550.349	0
PA(O-37:1)	C40H79O7P	703.564 (H+)	703.575	11
PC(16:0/14:1)*	C38H74NO8P	704.523 (H+)	704.524	1
		371.743 (H+K)2+	371.747	4
PC(16:0/14:0)*	C38H74NO8P	706.530 (H+)	706.541	11
PC(31:2) or PE(34:2)	C39H74NO8P	716.523 (H+)	716.538	15
PC(O-32:1) or PE(O-35:1)	C40H80NO7P	718.566 (H+)	718.567	1
PC (16:0/15:0)	C39H78NO8P	720.554 (H+)	720.557	3
PE(O-36:6)	C41H72NO7P	722.513 (H+)	722.512	−1
PC(32:2) or PE(35:2)	C40H76NO8P	730.538 (H+)	730.544	6
PC(16:0/16:1)*	C40H78NO8P	732.554 (H+)	732.553	−1
		385.759 (H+K)2+	385.752	−7
PC(33:5) or PE(36:5)	C39H74NO9P	738.507 (H+)	738.522	15
PC(O-34:2))	C42H82NO7P	744.591 (H+)	744.587	−4
PC(O-34:1)	C42H84NO7P	746.606 (H+)	746.593	−13
PS(O-34:1)	C40H78NO9P	748.541 (H+)	748.542	1
PC(34:4) or PE(37:4)	C42H76NO8P	754.531 (H+)	754.536	5
PC(16:0/18:3)*	C42H78NO8P	756.554 (H+)	756.557	3
PC(16:0/18:2)*	C42H80NO8P	758.570 (H+)	758.570	0
PC(16:0/18:1)*	C42H82NO8P	760.586 (H+)	760.582	−4
		399.775 (H+K)2+	399.778	3
PC(16:0/18:0)*	C42H84NO8P	762.601 (H+)	762.592	−9
PS(34:0) or	C40H78NO10P	764.544 (H+)	764.552	8
PC(O-36:6)	C44H78NO7P	764.559 (H+)	764.552	−7
PC(35:5)	C43H76NO8P	766.538 (H+)	766.555	17
PC(35:4)	C43H78NO8P	768.554 (H+)	768.561	7
PC(16:0/20:6)*	C44H76NO8P	778.539 (H+)	778.547	8
PC(16:0/20:5)*	C44H78NO8P	780.553 (H+)	780.551	−2
		409.759 (H+K)2+	409.760	1
PC(16:0/20:4)*	C44H80NO8P	782.570 (H+)	782.572	2
PC(16:0/20:3)*	C44H82NO8P	784.586 (H+)	784.588	2
PC(16:0/20:2)*	C44H84NO8P	786.601 (H+)	786.596	−5
PS(36:1) or	C42H80NO10P	790.560 (H+)	790.568	8
PC(O-38:7)	C46H80NO7P	790.575 (H+)	790.568	−7
PS(36:0)	C42H82NO10P	792.576 (H+)	792.584	8
PC(37:5) or PE(40:5)	C45H80NO8P	794.570 (H+)	794.5828	13
PS(O-38:5)	C44H78NO9P	796.549 (H+)	796.551	2
PC(38:8)	C46H76NO8P	802.5387 (H+)	802.547	8
PC(38:7)	C46H78NO8P	804.554 (H+)	804.558	4
PC(16:0/22:6)*	C46H80NO8P	806.570 (H+)	806.569	−1
		422.767 (H+K)2+	422.762	−5
PC(16:0/22:5)*	C46H80NO8P	808.586 (H+)	808.587	1
PS(38:4)	C44H78NO10P	812.544 (H+)	812.551	7
PS(38:1)	C44H84NO10P	818.591 (H+)	818.595	4
PC(39:6) or PE(42:6)	C47H82NO8P	820.586 (H+)	820.584	−2
PS(O-40:6)	C46H80NO9P	822.565 (H+)	822.574	9
PS(O-40:5)	C46H82NO9P	824.580 (H+)	824.578	−2
PC(40:9)	C48H78NO8P	828.554 (H+)	828.550	−4
PC(40:7)	C48H82NO8P	832.586 (H+)	832.585	−1
PS(40:5)	C46H80NO10P	838.560 (H+)	838.566	6
PS(40:4)	C46H82NO10P	840.575 (H+)	840.584	9
PS(38:1)	C44H84NO10P	840.573 (Na+)	840.584	11
PG(42:9)	C48H77O10P	845.533(H+)	845.529	−4
PG(40:6)	C46H79O10P	845.531 (Na+)	845.529	−2
PC(42:10)	C50H80NO8P	854.570 (H+)	854.561	−9
TAG(54:8)	C57H94O6	875.713 (H+)	875.710	−3
TAG(52:5)	C55H96O6	875.711 (Na+)	875.710	−1
TAG(54:7)	C57H96O6	877.729 (H+)	877.725	−4
TAG(52:4)	C55H98O6	877.726 (Na+)	877.725	−1
TAG(54:6)	C57H98O6	879.744 (H+)	879.740	−4
		901.726(Na+)	901.727	1
TAG(52:3)	C55H100O6	879.742 (Na+)	879.740	−2
TAG(54:5)	C57H100O6	881.760 (H+)	881.755	−5
		903.749 (Na+)	903.741	−8
TAG(52:2)	C55H102O6	881.757 (Na+)	881.755	−2
TAG(54:4)	C57H102O6	883.775 (H+)	883.771	−4
		905.757 (Na+)	905.756	−1
TAG(52:1)	C55H104O6	883.773 (Na+)	883.771	−2
PC(44:4)	C52H96NO8P	894.695 (H+)	894.708	13
PC(44:3)	C52H98NO8P	896.711 (H+)	896.721	10
		918.693 (Na+)	918.702	9
PC(42:0)	C50H100NO8P	896.708 (Na+)	896.721	13
PC(44:2)	C52H100NO8P	898.726 (H+)	898.733	7
PC(44:1)	C52H102NO8P	900.742 (H+)	900.741	−1
TAG(56:9)	C59H96O6	901.729 (H+)	901.727	−2
TAG(56:8)	C59H98O6	903.744 (H+)	903.741	−3
		925.726 (Na+)	925.739	13
TAG(56:7)	C59H100O6	905.760 (H+)	905.754	−6
		927.742 (Na+)	927.741	−1
TAG(56:6)	C59H102O6	907.776 (H+)	907.771	−5
		929.757 (Na+)	929.754	−4
TAG(54:3)	C57H104O6	907.773 (Na+)	907.772	−2
TAG(56:5)	C59H104O6	909.791 (H+)	909.788	−3
TAG(54:2)	C57H106O6	909.789 (Na+)	909.788	−1
PC(46:6)	C54H96NO8P	918.695 (H+)	918.702	7
TAG(58:11)	C61H96O6	925.727 (H+)	925.739	12
TAG(58:10)	C61H98O6	927.744 (H+)	927.741	−3
		949.726 (Na+)	949.737	11
TAG(58:9)	C61H100O6	929.760 (H+)	929.754	−6
		951.749 (Na+)	951.749	0
PC(46:5)	C50H97NO13	942.686 (Na+)	942.697	11
PC(46:3)	C54H102NO8P	946.724 (Na+)	946.726	2
TAG(60:13)	C63H96O6	949.729 (H+)	949.737	8
		971.711 (Na+)	971.714	3
TAG(60:12)	C63H98O6	951.744 (H+)	951.749	5
TAG(60:11)	C63H100O6	953.760 (H+)	953.757	−3
		975.742 (Na+)	975.754	12
TAG(58:8)	C61H102O6	953.757 (Na+)	953.757	0
TAG(60:10)	C63H102O6	955.776 (H+)	955.765	−11
TAG(58:7)	C61H104O6	955.773 (Na+)	955.765	−8
TAG(59:13)	C62H94O6	957.695 (Na+)	957.688	−7
TAG(62:16)	C65H94O6	971.713 (H+)	971.714	1
TAG(62:14)	C65H98O6	975.744 (H+)	975.754	10

*Lipids assignment based on tandem mass spectrometry.

Figure 4

(a) Tandem LAESI mass spectrum of a protonated PC lipid produced a single head group fragment, (b) whereas the tandem MS of its lithiated counterpart produced structure specific fragments enabling the identification of its acyl side chains.

Figure 5

Positive LAESI mass spectra of unfertilized X. laevis egg with annotations for selected (a) fatty acids (FA), (b) lysophosphatidylcholines (LPC), and (c) phosphatidylcholines (PC).

A complete list of the detected lipid ions is presented in Table 2.

Animal Pole vs. Vegetal Pole

The unfertilized X. laevis egg is polarized, with a highly pigmented animal pole and a weakly pigmented vegetal pole separated by an unpigmented equatorial zone. During oogenesis, mRNAs and proteins are synthesized and differentially stored in the cytoplasm of these regions for use by the embryo after fertilization. In particular, mRNAs that direct ectodermal development are enriched in the animal pole whereas mRNAs that direct endoderm and gamete development are enriched in the vegetal pole [40], [41]. This molecular asymmetry is altered after fertilization by a cytoplasmic reorganization that occurs in response to sperm entry. This reorganization is critical for establishing the dorsal axis of the embryo. Because the mRNAs in the animal versus vegetal regions of the egg have very different developmental functions that are essential to establishing the vertebrate body plan, the corresponding metabolic and lipid profiles were investigated by LAESI-MS.

As seen in Fig. 6, numerous lipids were found in higher abundance at the vegetal pole compared to the animal pole. This finding was consistent with magnetic resonance imaging studies of the animal and vegetal poles in X. laevis oocytes that had shown two to three times higher abundance for TAGs in the vegetal region [42]. Among the many lipids detected at the vegetal pole, we found an LPC at m/z 478, which was consistent with the presence of yolk platelets there. A previous report suggested that LPC lipids played a role in assisting fertilization [12]. In addition, there were many small metabolites found at both poles and their putative identifications can be found in Table 1. Although differences in the small metabolite abundances at the two poles were clearly discernable, they exhibited significant variance between eggs. As an example, the ion abundance ratios between vegetal and animal poles are shown in Table 3 for certain metabolites and lipids. To establish reliable abundance ratios, further analysis is needed.

Figure 6

Comparison of positive ion LAESI mass spectra of animal and vegetal poles for unfertilized X. laevis ovum.

The vegetal pole shows increased abundance of lipids relative to the animal pole. Average diameter of the egg in the overlaid image is ∼1.4 mm.

Table 3

Metabolite and lipid ion abundance ratios for vegetal and animal poles of X. laevis egg.

Metabolite or Lipid	Ion	m/z	Abundance ratio vegetal/animal	Abundance ratio animal/vegetal
choline	[+]	104.1	3.7	0.3
adenine	[H+]	136.1	1.0	1.0
guanine or hydroxyadenine	[Na+]	174.0	4.9	0.2
arginine	[H+]	175.1	1.8	0.6
phosphocholine	[H+]	184.1	5.2	0.2
monosaccharide	[Na+]	203.1	1.2	0.9
acetyldihydrolipoamide	[H+]	250.1	2.8	0.4
glutathione	[Na+]	330.1	0.6	1.7
glutathione	[K+]	346.0	0.7	1.5
disaccharide or trehalose	[Na+]	365.1	1.9	0.5
disaccharide or trehalose	[K+]	381.1	2.8	0.4
unknown m/z 374.033	-	374.0	7.5	0.1
cholesterol	[H+-H2O]	369.4	4.7	0.2
adenosine diphosphate (ADP)	[H+]	428.0	0.5	1.9
adenosine diphosphate (ADP)	[Na+]	450.0	1.7	0.6
unknown m/z 467.101	-	467.1	0.1	17.0
LPC(16:1)	[H+-H2O]	476.3	4.1	0.2
LPC(16:0)	[H+-H2O]	478.3	4.8	0.2
adenosine triphosphate (ATP)	[H+]	508.0	1.4	0.7
adenosine triphosphate (ATP)	[Na+]	530.0	0.6	1.8
unknown m/z 522.590	-	522.6	1.3	0.7
trisaccharides	[Na+]	527.2	2.4	0.4
unknown m/z 541.126	-	541.1	1.2	0.8
Cer(d 40:1)	[Na+]	660.6	0.1	11.6
unknown m/z 689.201	-	689.2	2.0	0.5
PC(16:0/16:1)	[H+]	732.6	5.6	0.2
PC(34:4) or PE(37:4)	[H+]	754.5	9.2	0.1
PC(16:0/18:3)	[H+]	756.6	3.9	0.3
PC(16:0/18:2)	[H+]	758.6	3.7	0.3
PC(16:0/18:1)	[H+]	760.6	4.3	0.2
PC(16:0/18:0)	[H+]	762.6	3.3	0.3
PC(16:0/20:5)	[H+]	780.6	5.9	0.2
PC(16:0/20:4)	[H+]	782.6	6.6	0.2
PC(16:0/20:3)	[H+]	784.6	3.7	0.3
PS(O-38:5)	[H+]	796.5	3.0	0.3
PC(16:0/22:6)	[H+]	806.6	5.8	0.2
PC(16:0/22:5)	[H+]	808.6	5.5	0.2
TAG(54:6)+[H+] or	[H+] or	879.7	18.0	0.1
TAG(52:3)+[Na+]	[Na+]	879.7	18.0	0.1
TAG(52:3)	[K+]	895.7	9.9	0.1
TAG(52:2)	[K+]	897.7	9.4	0.1
TAG(56:9)	[H+]	901.7	9.2	0.1
TAG(56:8)	[H+]	903.7	16.8	0.1
TAG(54:6)	[K+]	917.7	9.8	0.1
TAG(54:5)	[K+]	919.7	7.3	0.1
TAG(54:4)	[K+]	921.7	6.7	0.1
TAG(60:13)	[H+]	949.7	7.3	0.1

Fertilized Ovum and Early Embryos

A number of critical developmental events occur after the X. laevis egg is fertilized, including: sperm nucleus entry into the cytoplasm and an influx of calcium, the completion of meiosis II, the initiation of synchronous cell cycles, and the initiation of low levels of gene transcription at the 32-cell stage [37]. In the unfertilized egg, the cell cycle is arrested in metaphase of the second meiotic division, but all of the components for cell division, cytokinesis, gene transcription and protein synthesis have previously been produced in the oocyte.

We directly analyzed unperturbed eggs by LAESI-MS at 30 minute intervals after fertilization, and cleavage stage embryos at 8-cells and 32-cells. Most of the major metabolites and lipids found during these early stages after fertilization were similar to those in unfertilized Xenopus eggs (see Fig. 7). These results are consistent with the prevailing concept that the synthesis of the major cellular components occurs during oogenesis. However, some subtle differences between the some key metabolites were observed in the spectra. Most notably, spermine was consistently detected in the unfertilized eggs, but was absent in the 32-cell stage embryos. It is well documented that the concentration of spermine decreases during oogenesis leading to the reduction of total polyamines required for maturation [43], [44].

Figure 7

Positive ion LAESI mass spectra of an unfertilized Xenopus ovum and a 32-cell embryo show a very similar metabolite profiles with some key differences, e.g., the absence of spermine in the embryo spectrum.

The inset shows a Xenopus embryo at the 32-cell stage. The scale bar in the inset is 200 µm.

In both jellied and dejellied systems, the intensities for the following ions from stage 8 embryos were consistently lower than from unfertilized eggs: m/z 113.884, 114.892, 150.009, 175.118 (arginine), 199.991, 231.934, 239.919 and 277.199. Conversely, the following ions showed higher abundances in stage 8 embryos: 113.104, 131.111 (acetylputrescine), 157.127, 261.075, 239.101, 277.054, 283.066, 299.038, 315.015, 239.236, 241.098, 284.066, 295.021, 542.052 and 660.618 (Cer(d18:0/23:0)). Furthermore, there is a higher abundance of most TAGs in stage 8 embryos compared to unfertilized dejellied Xenopus eggs.

Conclusions

There has been extensive of the transcriptome that regulates the major developmental transitions in X. laevis at the subcellular level. These investigations have been limited to the detection of mRNAs by in situ hybridization and RT-PCR approaches and to the detection of proteins by immunohistochemistry or Western blotting techniques. The latter have been hampered by the lack of antibodies against proteins of interest. In addition, these approaches reliably detect mRNAs and proteins that are in high abundance. Metabolites and lipids have not been studied in detail at in single eggs because of the lack of appropriately sensitive technologies that can assess these molecules in the living egg/embryo in its natural state. Therefore, evaluation of eggs and embryos with LAESI-MS was performed to determine whether subtle changes in metabolites and lipids and their subcellular profiles are detectable.

In this report the direct atmospheric pressure analysis of X. laevis eggs and early cleavage stages embryos is presented. The results of the LAESI-MS metabolic and lipidomic profiles of the Xenopus eggs were compared to previous literature results and showed a wide coverage for lipids and small metabolites. Reactive-LAESI MS/MS, with in-plume cationization of PC lipids without sample preparation, was utilized to identify the acyl chains. An investigation of the animal and vegetal poles of the X. laevis ovum showed increased abundance of lipids in the vegetal pole relative to the animal pole. Radial profiling of a jelly coated egg by LAESI-MS utilizing consecutive laser pulses revealed dramatic compositional changes between the jelly coat the vitelline/plasma membranes and the cytoplasm.

Finally, LAESI-MS was used to demonstrate that subtle metabolic profile changes could be detected after the egg was fertilized and synchronous cell cycles were initiated. Of particular note, spermine was depleted after fertilization, which consistent with the reduction of total polyamines required for embryo maturation [43], [44]. Major differences between the metabolites of unfertilized egg and the early cleavage stage embryos were not expected because all the needed organelles and biochemical building blocks needed to carry the embryo through the first 8 hours of development had previously been synthesized and stored in the oocyte. Nonetheless, the sensitivity of LAESI-MS enabled the detection of a few subtle differences, and uncovered novel molecules that are asymmetrically distributed between the animal, ectoderm-forming, and vegetal, endoderm-forming, regions of the egg. This simple approach, partially validated here for a vertebrate embryo for which there is abundant traditional biochemical data, should be ideal for the direct analyses of other embryos that are rare and smaller in size.