Sun L et al. (2014), Quantitative proteomics of Xenopus laevis embry...

XB-ART-48686

Sci Rep 2014 Feb 26;4:4365. doi: 10.1038/srep04365.

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Quantitative proteomics of Xenopus laevis embryos: expression kinetics of nearly 4000 proteins during early development.

Sun L , Bertke MM , Champion MM , Zhu G , Huber PW , Dovichi NJ .

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While there is a rich literature on transcription dynamics during the development of many organisms, protein data is limited. We used iTRAQ isotopic labeling and mass spectrometry to generate the largest developmental proteomic dataset for any animal. Expression dynamics of nearly 4,000 proteins of Xenopus laevis was generated from fertilized egg to neurula embryo. Expression clusters into groups. The cluster profiles accurately reflect the major events that mark changes in gene expression patterns during early Xenopus development. We observed decline in the expression of ten DNA replication factors after the midblastula transition (MBT), including a marked decline of the licensing factor XCdc6. Ectopic expression of XCdc6 leads to apoptosis; temporal changes in this protein are critical for proper development. Measurement of expression in single embryos provided no evidence for significant protein heterogeneity between embryos at the same stage of development.

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Species referenced: Xenopus laevis
Genes referenced: ccdc6 cdc6 diaph1 h4c4 khsrp mcm3 mcm5 mcm6.2 mcm7 myo10 orc1 orc5 prss1 psmd6 recql4 spock2 ticrr

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	Figure 1. Experimental design.Micrographs of Xenopus laevis embryos at developmental stages used for iTRAQ measurements (A). Design and workflow of three independent iTRAQ experiments (B). Three experiments were performed. In experiment 1 (E1), two embryos at stage 1, two embryos at stage 5, two embryos at stage 8, and two embryos at stage 11 were separately lysed and digested with trypsin. The first embryo at stage 1 was labeled with the iTRAQ reagent channel 113, the second embryo at stage 1 was labeled with the iTRAQ reagent channel 114, the two embryos at stage 5 were labeled with the iTRAQ reagents channels 115 and 116, the two embryos at stage 8 were labeled with the iTRAQ reagent channels 117 and 118, and the two embryos at stage 11 were labeled with the iTRAQ reagent channels 119 and 121. These labeled peptides were pooled, subjected to strong cation exchange chromatography fractionation, and each fraction was analyzed using reversed-phase liquid chromatography and detection with a Q-Exactive mass spectrometer. Tandem mass spectra were analyzed both to identify the peptide and to quantitate the abundances of each peptide from each of the eight embryos. A similar procedure was performed in experiment 2 (E2), except that the biological duplicates consisted of single embryos taken from stages 1, 5, 13 and 22. Finally, experiment 3 (E3) employed four pools of four embryos, where each pool was taken embryos at stages 1, 8, 13 and 22.
	Figure 2. Cluster analysis of quantified proteins.Proteins with significant change of abundance are grouped into one of six clusters according to the changes in expression as a function of developmental stage. Log2(protein abundance ratio) from experiment 3 (E3) was used for cluster analysis. The number of clusters was fixed at 6. The upper regulation threshold for the log2 data was 0.26 and lower threshold was −0.32 corresponding to the original ratios of 1.2 and 0.8.
	Figure 3. Expression levels of DNA replication factors and histones.The expression levels of several DNA replication factors and histones change at the mid-blastula transition. (A) Xcdc6 exhibits a marked decline after the mid-blastula transition relative to other replication factors. (B) Levels of Cdc6 protein measured by western blot for control embryos and embryos injected with Cdc6 mRNA (1 ng). (C) Overexpression of Cdc6 triggers increased levels of apoptosis. Apoptosis was detected by staining late blastula (stage 9) embryos with PSS-380 for the fluorescent detection of phosphatidylserine. Left panel is water-injected control embryo; center panel is Cdc6 mRNA (1 ng) injected embryo; right panel is embryo incubated with 0.1 μM staurosporine to induce apoptosis. (D) Levels of maternal histone H1 (H1M/B4) decline while levels of adult H1 and core histones increase. Data from experiments E1 and E3 were used to generate (A) and (D), and the error bars for stage 8 were based on the results from experiments E1 and E3.
	Figure S1. Summary of protein identification data from three iTRAQ experiments and Gene Ontology classifications. (A) Number of proteins identified in each experiment. Classification of identified proteins is given according to (B) biological process, (C) molecular function and (D) cellular component. The biological process, molecular function and cellular component information were filtered with pValue (≤0.01) and protein counts (≥10), and 10 categories are presented.
	Figure S2. Signal of iTRAQ reporter ions. (A) Tandem spectra of two parent ions with different charges. The reporter ions of iTRAQ 8-plex reagents are also shown. (B) Fundamental signal distributions of iTRAQ reporter ions from experiment I (E1).
	Figure S3. iTRAQ biological replicate data. (A) Histogram distribution of log2 protein expression ratios from stage 5/stage 1(E1 and E2); (B) Correlations of protein expression ratios from stage 8/stage 1 (E1 and E3), stage 13/stage 1 (E2 and E3) and stage 22/stage 1 (E2 and E3).
	Figure S4. Distributions of protein ratios normalized to stage 1 (for E1 and E2, normalized to one embryo of stage 1, iTRAQ channel 113). For E1 and E2, each stage has two embryos as biological replicate, and the protein ratios are shown separately.
	Figure S5. Biological process information of proteins in clusters 1-6 presented in Figure 2, which are significantly up- or down regulated during early development from E3. Only biological processes with pValue ≤0.05 are listed
	Figure S6. Cluster analysis results of quantified proteins from E1. Proteins with significant change of abundance are grouped into one of six clusters according to the changes in expression as a function of developmental stage. Log2 (protein ratio) was used for cluster analysis. Data were segregated into six clusters and fixed regulation thresholds (upper limit as 0.26 and lower limit as -0.32, corresponding to the original ratios as 1.2 and 0.8) were used.
	Figure S7. Biological process information of proteins in clusters 1-6 presented in Figure S6, which are significantly up- or down regulated during early development from E1. Only biological processes with pValue ≤0.05 were listed.
	Figure S8. Cluster analysis results of quantified proteins from E2. Proteins with significant change of abundance are grouped into one of six clusters according to the changes in expression as a function of developmental stage. Log2 (protein ratio) was used for cluster analysis. Data were segregated into six clusters and fixed regulation thresholds (upper limit as 0.26 and lower limit as -0.32, corresponding to the original ratios as 1.2 and 0.8) were used.
	Figure S9. Biological process information of proteins in clusters 1-6 presented in Figure S8, which are significantly up- or down regulated during early development from E2. Only biological processes with pValue ≤0.05 are listed.
	Figure S10. Protein expression ratios obtained for VgRBP71 during early Xenopus development. iTRAQ data (A). Western blot data (B).
	Figure S11. Histograms of protein ratios from E1 and E2 between biological replicates of six different embryonic stages. The log2 (protein ratio) was used.
	Figure S12. Two-dimensional histogram of the variation in protein expression between embryos at the same stage of development. Variation is measured as the standard deviation of the absolute value of log2(Expression Ratio) for each experiment. The vast majority of proteins show small standard deviations in their expression ratios in each experiment, and the variation in expression ratios is uncorrelated between experiments.

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