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1 advances.sciencemag.org/cgi/content/full/3/5/e /dc1 Supplementary Materials for In vivo mapping of tissue- and subcellular-specific proteomes in Caenorhabditis elegans The PDF file includes: Aaron W. Reinke, Raymond Mak, Emily R. Troemel, Eric J. Bennett Published 10 May 2017, Sci. Adv. 3, e (2017) DOI: /sciadv 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. Other Supplementary Material for this manuscript includes the following: (available at advances.sciencemag.org/cgi/content/full/3/5/e /dc1) table S4 (Microsoft Excel format). Mass spectrometry data of spatially restricted enzymatic tagging in C. elegans.

2 Supplementary Materials Supplementary Discussion Evaluation of reproducibility To identify proteins that are present in the eight locations we analyzed, ratios of proteins identified from the APX-NES and APX-NLS strains for each tissue were compared to the GFP only control strain. We evaluated the reproducibility of our approach by comparing how many proteins with ratios greater than 2-fold over background were identified in at least one replicate compared to those identified in all three replicates (fig. S3). In the largest locations measured, the cytoplasm of the intestine and epidermis, over 60% of proteins that were identified 2-fold above background in one replicate were also identified as 2-fold above background in the two other replicates. In contrast, in the smallest location measured, the pharyngeal nucleus, only 3% of proteins identified 2-fold above background in one replicate were also 2-fold above background in the two other replicates. These differences are largely due to the variability in the number of proteins identified between replicates. For example, the number of proteins identified in the intestinal cytoplasm were between 2376 to 2575, while for the pharyngeal nucleus the range was much larger, with between 116 to 759 proteins identified (fig. S3). We also compared the correlation of ratios between each sample. Pearson correlation coefficients range from (table S3). Although many of these correlations are low, we demonstrate that for most of the locations, the majority of the proteins are detected in two out of the three replicates.

fig. S1. Localization of APX to the intestinal cytoplasm of C. elegans. Microscopy of animals expressing GFP-APX-NES under the spp-5 promoter. This strain of C.

3 fig. S1. Localization of APX to the intestinal cytoplasm of C. elegans. Microscopy of animals expressing GFP-APX-NES under the spp-5 promoter. This strain of C. elegans expresses the APX enzyme fused to GFP in the intestinal cytoplasm. Green is the fluorescence channel and green + DIC (differential interference contrast) is the merged fluorescence and DIC channel.

4 fig. S2. Use of triplex reductive dimethylated samples to quantitatively compare GFP-APX-NES, GFP-APX-NLS, and GFP-only samples. Animals expressing intestinal APX-NES, APX-NLS, and the GFP only control were used to perform spatially restricted enzymatic tagging. Biotinylated proteins were isolated and digested into peptides. (A) Peptides from the three samples were mixed together, split into thirds, with each portion being labeled with a different isotopic label. All the isotopically labeled samples were then mixed back together. Shown is the Log2 transformed protein abundance ratio. (B) Peptides from the three samples were each labeled with a different isotopic label. All the isotopically labeled samples were then mixed back together. The normalized NLS/NES ratios were adjusted using a total intensity approach based on peptide abundance (See methods).

fig. S3. Reproducibility of the number of proteins identified from each location. The number of proteins identified from the tissue or subcellular location for each of the eight locations indicated.

5 fig. S3. Reproducibility of the number of proteins identified from each location. The number of proteins identified from the tissue or subcellular location for each of the eight locations indicated. (A) Each location shows the number of proteins identified in each set. (B) Each location shows the number of protein identified in at least 1 of 3 replicates, at least 2 of 3 replicates, or in all three replicates.

6 fig. S4. Identification of cytoplasm- or nucleus-localized proteins. Cytoplasm or nuclear localized proteins were determined by examining the quantitative ratios after APX-mediated proximity labeling in the indicated tissue. Shown is the distribution of the Log2 NLS/NES ratio for proteins from each biological replicate that display NES/GFP ratios greater than 1 or NLS/GFP ratios less than 1, which filters out proteins identified in the control GFP-only labeled samples. Dashed lines demarcate the cutoff for being nucleus specific (greater than 1) or cytoplasm specific (less than -1) for each indicated tissue.

7 fig. S5. Effect of enrichment threshold on the identification of cytoplasm- or nucleus-localized proteins. GO term enrichment analysis of proteins identified as specific to either the cytoplasm or the nucleus was performed at 9 different ratio thresholds. (A) Nucleus specific proteins or (B) cytoplasm specific proteins that had NLS/NES or NES/NLS values, respectively, greater than the fold threshold were analyzed for enrichment in GO terms for either the nucleus or cytoplasm. (C) Proteins not classified as being either cytoplasmic or nucleus specific at each threshold value. Below each set of proteins is indicated the fold threshold, the p-values for enrichment of the nucleus and cytoplasm GO terms, and the number of proteins detected above the fold threshold. Go term enrichment analysis was performed using PANTHER version 11.1.

9 fig. S6. Quantitative mass spectrometry ratios of proteins selected for validation. The seven proteins from Figure 5 are shown. For each protein, the NES/GFP, NLS/GFP, and NLS/NES Log2 ratios from each of the three replicates are shown for each of the four tissues. table S1. Comparison of existing techniques to determine protein localization in animals. Technique Advantages Disadvantages References Fluorescent protein tagging Good spatial and temporal resolution. Can be done in live animals. Low throughput in generation of transgenic animals and in measurement. Tagging proteins can affect their localization. 5 and 6 Tissue dissection Can be performed on wildtype animals. Potential loss of sensitivity. Can only be done on tissues large enough to dissect. 7 and 8 Biochemical fractionation Unnatural amino acid labeling Spatially restricted enzymatic labeling Can be performed on wildtype animals. Can label whole proteomes in specific cell types. Can label whole proteomes and subcellular compartments in specific cell types. Potential loss of sensitivity. Can only be performed on subcellular compartments that can be purified. Requires transgenic animals. Has low sensitivity and no subcellular resolution. Requires transgenic animals, treatment with hydrogen peroxide, and the ability to get biotin-phenol into cells of interest. 10 and and and 15 table S2. List of strains used in this study. APX strains Tissue (promoter) Protein subcellular location (localization tag) Strain nomenclature intestine (spp-5) GFP none ERT383 jysi11 [pet536(spp-5p::gfp::unc-54 ; unc-119 (+)]II; unc-119(ed3) III intestine (spp-5) GFP_APX cytoplasm (NES) ERT385 jysi13 [pet532(spp-5p::gfp_apx_nes::unc-54 ; unc-119 (+)]II; unc-119(ed3) III intestine (spp-5) GFP_APX nucleus (NLS) ERT384 jysi12 [pet522(spp-5p::gfp_apx_nls::unc-54 ; unc-119 (+)]II; unc-119(ed3) III epidermis (dpy-7) GFP_APX cytoplasm (NES) ERT446 jysi25 [pet595(dpy-7p::gfp_apx_nes::unc-54 ; unc-119 (+)]II; unc-119(ed3) III epidermis (dpy-7) GFP_APX nucleus (NLS) ERT387 jysi15 [pet525(dpy-7p::gfp_apx_nls::unc-54 ; unc-119 (+)]II; unc-119(ed3) III body wall muscle (myo-3) GFP_APX cytoplasm (NES) ERT445 jysi24 [pet594(myo-3p::gfp_apx_nes::unc-54 ; unc-119 (+)]II; unc-119(ed3) III body wall muscle (myo-3) GFP_APX nucleus (NLS) ERT386 jysi14 [pet524(myo-3p::gfp_apx_nls::unc-54 ; unc-119 (+)]II; unc-119(ed3) III pharyngeal muscle (myo-2) GFP_APX cytoplasm (NES) ERT444 jysi23 [pet593(myo-2p::gfp_apx_nes::unc-54 ; unc-119 (+)]II; unc-119(ed3) III pharyngeal muscle (myo-2) GFP_APX nucleus (NLS) ERT443 jysi22 [pet592(myo-2p::gfp_apx_nls::unc-54 ; unc-119 (+)]II; unc-119(ed3) III TransgeneOme construct strains Protein Strain nomenclature K01G5.5 ERT482 jyex247[k01g5.5::egfp::3xflag, unc-119(+)]; ttti5605 II; unc-119(ed3) III F33C8.4 ERT484 jyex249[f33c8.4::egfp::3xflag, unc-119(+)]; ttti5605 II; unc-119(ed3) III Y45F10B.13 ERT485 jyex250[y45f10b.13::egfp::3xflag, unc-119(+)]; ttti5605 II; unc-119(ed3) III W05H9.1 ERT487 jyex252[w05h9.1::egfp::3xflag, unc-119(+)]; ttti5605 II; unc-119(ed3) III F42A10.5 ERT488 jyex253[f42a10.5::egfp::3xflag, unc-119(+)]; ttti5605 II; unc-119(ed3) III F59D12.2 ERT489 jyex254[f59d12.2::egfp::3xflag, unc-119(+)]; ttti5605 II; unc-119(ed3) III F29C6.1 ERT490 jyex255[f29c6.1::egfp::3xflag, unc-119(+)]; ttti5605 II; unc-119(ed3) III

10 table S3. Pearson correlation coefficients of replicate samples. Sample Set 1 vs. set 2 Set 1 vs. set 3 Set 2 vs. set 3 DPY7 NES/GFP DPY7 NLS/GFP DPY7 NLS/NES MYO2 NES/GFP MYO2 NLS/GFP MYO2 NLS/NES MYO3 NES/GFP MYO3 NLS/GFP MYO3 NLS/NES SPP5 NES/GFP SPP5 NLS/GFP SPP5 NLS/NES table S5. Number of proteins measured compared to mrna expressed in each tissue. Proteins detected [1] mrna detected [2] % of possible proteins identified Tissue Intestine Epidermis Body wall muscle Pharyngeal muscle [1] Number of different proteins detected in this study in either the cytoplasm or nucleus. [2] ~Number of different mrnas detected in each tissue from fig. S5 of Spencer et al. Genome Research

11 table S6. Total spectral counts for all identified proteins in each sample and NLS/NES scaling factors. Sample Total spectral counts SET1_DPY7 GFP 4381 SET1_DPY7 NES SET1_DPY7 NLS SET2_DPY7 GFP 4592 SET2_DPY7 NES SET2_DPY7 NLS SET3_DPY7 GFP 6019 SET3_DPY7 NES SET3_DPY7 NLS SET1_MYO2 GFP 7823 SET1_MYO2 NES SET1_MYO2 NLS SET2_MYO2 GFP SET2_MYO2 NES SET2_MYO2 NLS SET3_MYO2 GFP SET3_MYO2 NES SET3_MYO2 NLS SET1_MYO3 GFP 7823 SET1_MYO3 NES SET1_MYO3 NLS SET2_MYO3 GFP 8779 SET2_MYO3 NES SET2_MYO3 NLS SET3_MYO3 GFP SET3_MYO3 NES SET3_MYO3 NLS SET1_SPP5 GFP 4365 SET1_SPP5 NES SET1_SPP5 NLS SET2_SPP5 GFP 3751

12 SET2_SPP5 NES SET2_SPP5 NLS SET3_SPP5 GFP 7309 SET3_SPP5 NES SET3_SPP5 NLS Replicate NLS/NES scaling factor SET1_DPY SET2_DPY SET3_DPY SET1_MYO SET2_MYO SET3_MYO SET1_MYO SET2_MYO SET3_MYO SET1_SPP SET2_SPP SET3_SPP5 0.46

Supplementary Fig. 1. Infection of three C. elegans strains used for spatially restricted enzymatic tagging. Animals infected with N.

Supplementary Fig. 1. Infection of three C. elegans strains used for spatially restricted enzymatic tagging. Animals infected with N. parisii stained with a FISH probe (red) specific for Nematocida rrna.