Research ArticlePROTEOMICS

In vivo mapping of tissue- and subcellular-specific proteomes in Caenorhabditis elegans

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Science Advances  10 May 2017:
Vol. 3, no. 5, e1602426
DOI: 10.1126/sciadv.1602426
  • Fig. 1 Overview of an approach to identify tissue- and subcellular-specific protein expression in C. elegans.

    Schematic of spatially restricted enzymatic tagging in C. elegans. Animal strains that express the APX enzyme in either the cytoplasm or the nucleus in a tissue-specific manner (such as in the intestine as illustrated here) are generated. Animals are treated with biotin-phenol that diffuses into cells. The APX enzyme, in the presence of H2O2 and biotin-phenol, catalyzes the formation of a phenoxyl radical that covalently labels the neighboring proteins with biotin (red “B” with black bars) (15). Thus, biotin labeling (labeled in red) of proteins occurs in whichever specific tissues and subcellular locations the enzyme is expressed. Three strains are used to measure protein localization in each tissue: GFP only, GFP-APX-NES, and GFP-APX-NLS. Spatially restricted enzymatic tagging is performed using these three strains, and then the proteins are extracted and purified using streptavidin beads. These purified proteins are then digested into peptides and labeled using reductive dimethyl labeling for quantitative comparisons between the three strains. The peptides from each sample are then combined, and peptide ratios in each sample are measured using mass spectrometry. The peptide ratios can then be used to determine whether the protein is detected over background and enriched in either the nucleus or the cytoplasm.

  • Fig. 2 Efficient, spatially restricted enzymatic tagging in C. elegans is dependent on biotin-phenol, H2O2, and bus-8 RNAi.

    (A to C) Streptavidin-purified proteins from C. elegans protein extracts were visualized with Oriole staining. (A) Animals expressing GFP-APX (APX+) in the intestinal cytoplasm or a control GFP-only strain (APX−) were grown on plates with either control (L4440) or bus-8 RNAi. Animals were either treated or untreated with biotin-phenol (BP) and H2O2. Protein markers are indicated (labeled “M”). (B) Animals expressing GFP-APX in the intestinal cytoplasm or a GFP control strain grown on plates with bus-8 RNAi were treated with either H2O2 or biotin-phenol, or both. (C) Strains of C. elegans expressing GFP-APX in either the cytoplasm (C) or the nucleus (N) of the epidermis (Epi.), pharyngeal muscle (Pha.), body wall muscle (Bod.), or intestine (Int.). A strain expressing GFP only (G) is the negative control. All strains were grown on plates with bus-8 RNAi and treated with biotin-phenol and H2O2.

  • Fig. 3 APX-mediated biotin labeling in vivo displays tissue and subcellular specificity.

    Spatially restricted enzymatic tagging was performed on strains of C. elegans expressing APX in the cytoplasm or nucleus of the intestine, epidermis, body wall muscle, or pharyngeal muscle as indicated. A strain expressing GFP without APX was used as a negative control. Animals were fixed and stained for GFP (top, green) to determine the localization of the enzyme and streptavidin (middle, red) to determine the location of protein biotinylation. Representative images are shown for each strain. Animals are aligned so that the anterior is up. Tissue expression diagrams show the location of each tissue in C. elegans (bottom).

  • Fig. 4 Identification of C. elegans proteins with tissue- and subcellular-specific localizations.

    (A) The number of proteins identified above background from the tissue or subcellular location for each of the eight locations indicated. (B) The total number of proteins identified in our experiments that were detected in different locations or detected as being specific to a location. (C) The number of proteins we identified in the indicated tissue but not in the other three tissues. For each tissue, three categories of proteins are shown: those that are specific to the cytoplasm (orange), those that are specific to the nucleus (blue), and those that are not specific to either compartment (gray). (D) Comparison of the identified tissue-specific proteins to a data set of predicted mRNA expression (22). The data presented are the average of all the mRNA expression prediction scores for each tissue-specific protein in each of the four tissues. Higher prediction scores are more likely to be expressed in that tissue. Each column represents proteins identified as specific to that tissue, compared to the predicted mRNA expression of the tissue in each row. The highest average score in each column is shaded in green, and all other scores in the column are shaded in red. (E) Gene Ontology (GO) term enrichment analysis of proteins identified in our experiments as specific to either the cytoplasm or the nucleus. (F) Pie chart of the nucleus-specific proteins we identified in our experiment and whether they have a GO term location of either the nucleus or the cytoplasm, both, or neither.

  • Fig. 5 Validation of identified protein locations using fluorescently tagged proteins.

    Strains of transgenic C. elegans expressing GFP-tagged proteins identified to be tissue- or location-specific in our study. Animals were grown to the L4 stage, and representative images displaying protein localization are shown. The protein name is listed above each construct-expressing strain. The tissue and subcellular localization determined from our proteomic data is listed below the protein name. Animals are aligned so that the anterior is up.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/5/e1602426/DC1

    Supplementary Discussion

    fig. S1. Localization of APX to the intestinal cytoplasm of C. elegans.

    fig. S2. Use of triplex reductive dimethylated samples to quantitatively compare GFP-APX-NES, GFP-APX-NLS, and GFP-only samples.

    fig. S3. Reproducibility of the number of proteins identified from each location.

    fig. S4. Identification of cytoplasm- or nucleus-localized proteins.

    fig. S5. Effect of enrichment threshold on the identification of cytoplasm- or nucleus-localized proteins.

    fig. S6. Quantitative mass spectrometry ratios of proteins selected for validation.

    table S1. Comparison of existing techniques to determine protein localization in animals.

    table S2. List of strains used in this study.

    table S3. Pearson correlation coefficients of replicate samples.

    table S4. Mass spectrometry data of spatially restricted enzymatic tagging in C. elegans.

    table S5. Number of proteins measured compared to mRNA expressed in each tissue.

    table S6. Total spectral counts for all identified proteins in each sample and NLS/NES scaling factors.

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Discussion
    • fig. S1. Localization of APX to the intestinal cytoplasm of C. elegans.
    • fig. S2. Use of triplex reductive dimethylated samples to quantitatively compare GFP-APX-NES, GFP-APX-NLS, and GFP-only samples.
    • fig. S3. Reproducibility of the number of proteins identified from each location.
    • fig. S4. Identification of cytoplasm- or nucleus-localized proteins.
    • fig. S5. Effect of enrichment threshold on the identification of cytoplasm- or nucleus-localized proteins.
    • fig. S6. Quantitative mass spectrometry ratios of proteins selected for validation.
    • table S1. Comparison of existing techniques to determine protein localization in animals.
    • table S2. List of strains used in this study.
    • table S3. Pearson correlation coefficients of replicate samples.
    • table S5. Number of proteins measured compared to mRNA expressed in each tissue.
    • table S6. Total spectral counts for all identified proteins in each sample and NLS/NES scaling factors.

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    Other Supplementary Material for this manuscript includes the following:

    • table S4 (Microsoft Excel format). Mass spectrometry data of spatially restricted enzymatic tagging in C. elegans.

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