Smits AH et al. (2014), Global absolute quantification reveals tight re...

XB-ART-49279

Nucleic Acids Res 2014 Sep 01;4215:9880-91. doi: 10.1093/nar/gku661.

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Global absolute quantification reveals tight regulation of protein expression in single Xenopus eggs.

Smits AH , Lindeboom RG , Perino M , van Heeringen SJ , Veenstra GJ , Vermeulen M .

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While recent developments in genomic sequencing technology have enabled comprehensive transcriptome analyses of single cells, single cell proteomics has thus far been restricted to targeted studies. Here, we perform global absolute protein quantification of fertilized Xenopus laevis eggs using mass spectrometry-based proteomics, quantifying over 5800 proteins in the largest single cell proteome characterized to date. Absolute protein amounts in single eggs are highly consistent, thus indicating a tight regulation of global protein abundance. Protein copy numbers in single eggs range from tens of thousands to ten trillion copies per cell. Comparison between the single-cell proteome and transcriptome reveal poor expression correlation. Finally, we identify 439 proteins that significantly change in abundance during early embryogenesis. Downregulated proteins include ribosomal proteins and upregulated proteins include basal transcription factors, among others. Many of these proteins do not show regulation at the transcript level. Altogether, our data reveal that the transcriptome is a poor indicator of the proteome and that protein levels are tightly controlled in X. laevis eggs.

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Species referenced: Xenopus laevis
Genes referenced: gnao1

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	Figure 1. Tight regulation of the single-cell proteome in X. laevis eggs. (A) Overview of the workflow used to detect technical and biological variation in single cell proteomics. (B) Unsupervised correlation-based clustering of global proteomes mixes technical and biological replicates. See also Supplementary Tables S2 and S3.
	Figure 2. Absolute, deep proteome of individual X. laevis eggs. (A) Schematic representation of the workflow used to obtain an absolute, deep proteome. Both the number of fractions and their corresponding nanoLC gradients were optimized for the low amount of protein obtained from a single X. leavis egg. (B) The absolute abundance of all proteins in X. leavis eggs spans over seven orders of magnitude. The black line corresponds to the mean abundance and the gray plusses represent the standard deviation (five replicates). The dashed line indicates the lowest detected protein of the UPS2 spike-in (unique peptides >2) above which abundance can be accurately determined. The inset represents a typical linear regression curve of the measured iBAQ intensities and the known amounts of UPS2 standard. (C) Distribution of the protein copy numbers in Xenopus eggs. Arbitrary cut-offs define five abundance regions for which significant enriched GO terms (FDR < 0.0005) are plotted above these regions. Note the shoulder of high abundant proteins on the right side of the distribution. See also Supplementary Tables S2 and S4.
	Figure 3. Comparison of single-cell proteomes and transcriptomes. (A) Scatterplot of the mean protein and mRNA levels for genes quantified in all individual proteomes and transcriptomes (4798 genes) reveals a poor correlation between protein and mRNA abundance. (B) Unsupervised correlation-based clustering of global proteomes (left panel) and transcriptomes (right panel) indicate tight regulation of global proteome and transcriptome. (C) Scatterplot of protein and mRNA CV (4798 genes) reveals a lack of correlation between protein and mRNA variation. Genes are colored that show similar mRNA and protein variation (green, high variation; blue, low variation) and that show differential mRNA and protein variation (red, low protein and high mRNA variation; orange, high protein and low mRNA variation). Genes outside these categories are in black. (D) Enriched GO terms for the gene groups as depicted in C. Y-axis represents the –log FDR. See also Supplementary Table S4.
	Figure 4. Dynamics of proteome and transcriptome in early Xenopus embryogenesis. (A) Schematic depiction of the workflow (upper panel) and the calculated protein content of X. leavis egg and embryo 10.5 (lower panel). (B) Unsupervised correlation-based clustering of egg and embryo 10.5 proteomes clearly separates egg and embryo 10.5 proteomes. (C) Heatmap of significant regulated proteins between egg and embryo 10.5, identified by an adapted t-test (FDR = 0.005 and s0 = 1). (D) Enriched GO terms for both egg and embryo 10.5 enriched proteins, as identified in C. Y-axis represents the –log FDR. (E) Protein and mRNA ratios (egg/embryo 10.5) for both egg (left panel) and embryo 10.5 (right panel) enriched proteins reveal lack of correlation between protein and mRNA dynamics. See also Supplementary Table S4.

References [+] :

Akkers, A hierarchy of H3K4me3 and H3K27me3 acquisition in spatial gene regulation in Xenopus embryos. 2009, Pubmed, Xenbase