Lee-Liu D et al. (2018), Quantitative Proteomics After Spinal Cord Injur...

XB-ART-54576

Mol Cell Proteomics 2018 Apr 01;174:592-606. doi: 10.1074/mcp.RA117.000215.

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Quantitative Proteomics After Spinal Cord Injury (SCI) in a Regenerative and a Nonregenerative Stage in the Frog Xenopus laevis.

Lee-Liu D , Sun L , Dovichi NJ , Larraín J .

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The capacity to regenerate the spinal cord after an injury is a coveted trait that only a limited group of nonmammalian organisms can achieve. In Xenopus laevis, this capacity is only present during larval or tadpole stages, but is absent during postmetamorphic frog stages. This provides an excellent model for comparative studies between a regenerative and a nonregenerative stage to identify the cellular and molecular mechanisms that explain this difference in regenerative potential. Here, we used iTRAQ chemistry to obtain a quantitative proteome of the spinal cord 1 day after a transection injury in regenerative and nonregenerative stage animals, and used sham operated animals as controls. We quantified a total of 6,384 proteins, with 172 showing significant differential expression in the regenerative stage and 240 in the nonregenerative stage, with an overlap of only 14 proteins. Functional enrichment analysis revealed that although the regenerative stage downregulated synapse/vesicle and mitochondrial proteins, the nonregenerative stage upregulated lipid metabolism proteins, and downregulated ribosomal and translation control proteins. Furthermore, STRING network analysis showed that proteins belonging to these groups are highly interconnected, providing interesting candidates for future functional studies. Data are available via ProteomeXchange with identifier PXD006993.

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Species referenced: Xenopus laevis
Genes referenced: a2m alb apoa1 dynll1 f2 fat1 fga fgb fgg gap43 hbz hrg mtdh nucb1 plaa rpl21 serpina3 tf trarg1 tsc2 vtn
GO keywords: vasculature development [+]

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	Fig. 1. Experimental workflow. One iTRAQ 8-plex experiment for each stage (regenerative, left; nonregenerative, right) was performed, including duplicate uncut samples, and triplicate sham (1dps) and transected (1dpt) samples isolated 1 day after surgery. Two iTRAQ channels were used for labeling of duplicate uncut samples. Three iTRAQ channels were used for labeling of triplicate sham (1dps) samples, and the remaining three channels for triplicate transected (1dpt) samples. Approximately 0.95 million tandem mass spectra were acquired, which corresponded to over 50,000 peptide sequences, and 7859 identified protein groups. 6384 had quantifiable levels, corresponding to 3852 proteins in the regenerative stage, and 5629 proteins in the nonregenerative stage (overlap: 3106 proteins quantified in both stages). Analysis identified 172 differentially expressed proteins in the regenerative stage, and 240 proteins in the nonregenerative stage. Functional analyses were performed using BLAST2GO and STRING for differentially expressed proteins.
	Fig. 2. Principal Component Analysis. Reporter ion intensities for transected and sham operated samples were normalized against uncut samples, and principal component analysis performed on normalized values. Regenerative (●) and nonregenerative stage (○) samples were separated along component 1, and 1 day after transection (1dpt, black) and 1 day after sham-operation (1dps, gray) samples separated along component 2. Percentages (%) indicate the percentage of variance that each principal component represents.
	Fig. 3. Proteins showing differential expression when comparing transected and sham-operated animals. Differentially expressed proteins were determined using a Welch's t test (p value < 0.05) and an additional log2 (transected/sham) ≥ 0.10 or ≤ −0.10 fold-change filter. A, Venn diagram showing the number of proteins meeting differential expression criteria for each stage, distributed into those which did so in the regenerative, nonregenerative stage, or both. B, C, Volcano plots showing log2 (transected/sham) fold-change (x-axis) and -log10 (p value) (x-axis) for all quantified proteins in the regenerative (B) or the nonregenerative stage (C). Colored dots indicate proteins meeting differential expression criteria, and labels show gene symbols for top 15 proteins with highest fold-change. Regenerative- green; nonregenerative- red.
	Fig. 4. Gene ontology enrichment for differentially expressed proteins in the regenerative and nonregenerative stage. Gene ontology (GO) enrichment analysis was performed for proteins meeting differential expression criteria (see Fig. 3 legend) using Fisher's Exact Test, reporting terms with a false discovery rate (FDR) < 0.05. In these graphs, terms were additionally filtered so that only terms exclusively found in one stage but not the other are shown. Top 10 categories with the lowest FDR are shown when more than 10 exclusive GO terms were found.
	Fig. 5. Heatmaps showing differential expression changes for proteins belonging to enriched gene ontology (GO) categories. Gene ontology terms were classified into 5 groups (A–E). Specific GO terms included in each group are listed below. n.q. protein was not quantified (gray boxes). Color scale represents log2 (transected/sham) fold-change values with significant differential expression (p value < 0.05). Fold-changes with nonsignificant p value (≥ 0.05) are shown in white.
	Fig. 6. STRING network analysis for differentially expressed proteins in the R-stage. STRING network analysis for proteins meeting differential expression criteria (see Fig. 3 legend) in the regenerative stage. A, STRING network. Fill color represents fold-change, and node size represents the number of edges (or undirected connections) each protein has. B, List of most highly connected proteins and their See both scales in bottom-right corner.
	Fig. 7. STRING network analysis for differentially expressed proteins in the NR-stage. See Fig. 6 for legend.
	Fig. 8. Gene ontology (GO) categories enriched at both the transcriptome and proteome levels. A list of GO terms enriched among differentially expressed genes, in both previously published RNA-Seq data (8) and the present iTRAQ data was generated, after which level 5 GO terms were selected and their enrichment false discovery rate (FDR) value obtained from iTRAQ data. As in Fig. 4, terms were also filtered to include only level 5 terms exclusively found in either stage. When more than 10 exclusive GO terms were found, only the top 10 with the lowest FDR value were included in the graph (see Table I and supplemental data 6 for additional information). Note: cellular component for the nonregenerative stage is not shown because there were no level 5 terms exclusively found in this stage in this analysis.

References [+] :

Becker, Neuronal regeneration from ependymo-radial glial cells: cook, little pot, cook! 2015, Pubmed