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PLoS One
2014 Dec 15;912:e115173. doi: 10.1371/journal.pone.0115173.
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Subcellular metabolite and lipid analysis of Xenopus laevis eggs by LAESI mass spectrometry.
Shrestha B
,
Sripadi P
,
Reschke BR
,
Henderson HD
,
Powell MJ
,
Moody SA
,
Vertes A
.
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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 eggcytoplasm. 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.
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25506922
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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.
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 â¼6 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 â¼2 laser pulses, other metabolites and lipids characteristic of the cytoplasm were detected (bottom spectrum).
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.
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 â¼1.4 mm.
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.
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).
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