Proteome labeling and protein identification in specific tissues and at specific developmental stages in an animal

Abstract

Identifying the proteins synthesized at specific times in cells of interest in an animal will facilitate the study of cellular functions and dynamic processes. Here we introduce stochastic orthogonal recoding of translation with chemoselective modification (SORT-M) to address this challenge. SORT-M involves modifying cells to express an orthogonal aminoacyl-tRNA synthetase/tRNA pair to enable the incorporation of chemically modifiable analogs of amino acids at diverse sense codons in cells in rich media. We apply SORT-M to Drosophila melanogaster fed standard food to label and image proteins in specific tissues at precise developmental stages with diverse chemistries, including cyclopropene-tetrazine inverse electron demand Diels-Alder cycloaddition reactions. We also use SORT-M to identify proteins synthesized in germ cells of the fly ovary without dissection. SORT-M will facilitate the definition of proteins synthesized in specific sets of cells to study development, and learning and memory in flies, and may be extended to other animals.

Figure 1. SORT-M enables proteome tagging and labelling at diverse codons, with diverse chemistries, and in genetically targeted cells and tissues.

(a) Proteome tagging via SORT (stochastic orthogonal recoding of translation) uses an orthogonal aminoacyl-tRNA synthetase/tRNA pair. The pyrrolysyl-tRNA synthetase/tRNA pair is used in this study. This synthetase (and its previously evolved active-site variants) recognizes a range of unnatural amino acids (yellow star, and yellow hexagon), does not aminoacylate endogenous tRNAs, but efficiently aminoacylates its cognate tRNA - without regard to anticodon identity; PyltRNA is not a substrate for endogenous aminoacyl-tRNA synthetases. Orthogonal pyrrolysyl-tRNA synthetase/tRNA_XXX pairs (XXX indicates choice of anticodon, yellow) in which the anticodon has been altered compete for the decoding of sense codons (dark blue and pink) via a pathway that is orthogonal to that used by natural synthetases and tRNAs (dark blue and pink) to direct natural amino acids. SORT allows the incorporation of diverse chemical groups into the proteome, at diverse codons. Since there is no competition at the active site of the orthogonal synthetase, starvation and minimal media are not required. In addition the expression pattern of the orthogonal proteome tagging system can be genetically directed allowing tissue specific proteome labelling. Selective pressure incorporation approaches are shown in Supplementary Figure 1 for comparison to SORT. (b) The combination of encoding amino acids (1-3) across the proteome via SORT and chemoselective modification of 3 with tetrazine probes (4a-g, 5, 6 and 7) allows detection of labelled proteins via SORT-M (stochastic orthogonal recoding of translation and chemoselective modification). Amino acid structures: N^ε-((tert-butoxy)carbonyl)-l-lysine 1, N^ε-(1-propynlyoxy)carbonyl)-l-lysine 2 and N^ε-(((2-methylcycloprop-2-en-1-yl)methoxy)carbonyl)-l-lysine.

Figure 2. Site-specific incorporation of 3 into proteins at diverse codons and specific proteome labelling using SORT-M in human cells.

(a) Western blot analysis demonstrates the efficient amino acid dependant expression of an mCherry-EGFP fusion protein separated by an amber stop codon bearing a C-terminal HA-tag (mCh-TAG-EGFP-HA) in HEK293T cells. Anti-FLAG detected tagged PylRS (b) Specific labelling of mCh-TAG-EGFP-HA (immunoprecipitated from 10⁶ cells) with 4a (20μM in 50μL PBS, 1h, RT) confirms the incorporation of 3 into protein in HEK293 cells. (c) SORT-M labelling of 3 that is statistically incorporated into newly synthesised proteins across the whole proteome of mammalian cells directed by six different PylRS/PyltRNA_XXX mutants using 0.5 mM 3. Labeling with 4g (20μM in PBS, 1h, RT, as above). The amino acids in parentheses are the natural amino acids encoded by the endogenous tRNA bearing the corresponding anti-codon.

Figure 3. Site-specific incorporation of amino acid 3 into protein produced in Drosophila melanogaster.

(a) Incorporation of 3 demonstrated by a dual luciferase reporter. Dual luciferase assay on ovary extract from 10 female flies expressing Triple-Rep-L in the presence or absence of 10 mM 1 or 10mM 3. The data show a representative example from 1 of 3 biological replicates. The error bars represent the standard deviation of 3 technical replicates from a single biological replicate. (b) Site-specific incorporation of 3 (or 1) into GFP_TAG_mCherry-HA in flies expressing PylRS/PyltRNA_CUA. The full-length protein resulting from unnatural amino acid incorporation is detected by anti-HA western blot. (c) Specific labelling of encoded 3 with tetrazine probes. Flies were fed with no amino acid, amino acid 1 (500 flies) or amino acid 3 (100 flies). 5 times more flies were fed with 1 in order to generate comparable amount of reporter protein. The full-length protein containing the unnatural amino acid was immunoprecipitated from lysed ovaries with anti-GFP beads. The beads were labelled (4g, 4μM, 200μL PBS, RT, 2h) washed. Full length protein was detected by anti-HA blot and the same gel imaged on a fluorescence scanner shows specific fluorescent labelling of the protein incorporating 3 but not 1, confirming the identity of the incorporated amino acid.

Figure 4. SORT-M enables selective imaging of proteins synthesized within the germ cells of the fly ovary from stage 5 onwards.

(a) Schematic representation of fly oogenesis, anterior to the left, green indicates the germline and white the somatic tissue (follicular epithelium). (b-d) Genetically directed, cell-type specific proteome labelling within an organ. Egg chambers from stage 4 to stage 10 of oogenesis of the indicated genotypes and feeding condition are shown. Anterior to the left, DAPI (blue), Phalloidin (red), 4g (green), (b) Egg chambers receiving 3 (10mM), expressing PylRS/PyltRNA_UGC (nos-vp16-GAL4, PylRS,PylT_UGC) were washed, fixed and labelled with 4g (4μM, PBS, RT, 2h). (c) Egg chambers not receiving 3, but expressing PylRS/PyltRNA_UGC and labelled with 4g. (d) Egg chambers receiving 3, but not expressing PylRS/PyltRNA_GCU, and labelled with 4g. scalebar shown on merge images is 20μm.

Figure 5. SORT-M facilitates tissue specific labelling of the fly proteome.

The ovary proteome, but not the rest of the body proteome from 20 female flies expressing PylRS/PyltRNA_UGC (nos-vp16-GAL4, PylRS,PylT_UGC) fed with 3 (10 mM) is labelled with 4g (20μM, PBS, RT, 2h, lysate 30 mg/ml)) . Labelling requires feeding 3 and the PylRS/PyltRNA_UGC pair.

Figure 6. Tissue and developmental stage specific proteome labelling, protein identification and validation.

(a) In-gel fluorescence image of a 2D-gel of protein extracts from a dissected fly ovary that has been labelled non-specifically with an amine reactive NHS-Cy3 dye and then imaged. This gel acts as a reference for an ovary proteome. (b) In-gel fluorescence image of a 2D-gel of a protein extract from fly bodies that have had their ovaries removed. The extract has been labelled non-specifically with an amine reactive NHS-Cy5 dye. This gel acts as a reference for a fly body only proteome. (c) In-gel fluorescence image of protein extracts from ovaries dissected from flies fed with 3, then labelled with 4e and mixed with protein extracts from the remaining body parts. (d) In-gel fluorescence image of protein extracts from ovaries dissected from flies expressing PylRS/PyltRNA_UGC fed with 3, mixed with protein extracts from the body and then labelled with 4e. The gut of body samples was removed to limit proteolysis in protein extracts. All experiments used nos-vp16-GAL4, PylRS/PylT_UGC flies. Circled spots were excised for mass spectrometry. Scale bar on all 2D gels is 10 mm (e) The timing of Vasa protein expression and its pattern of immunostaining overlaps with that of SORT-M labelling, validating its identification by SORT-M. Ovaries from nos-vp16-GAL4, PylRS, PylT_UGC flies fed with or without 3, were dissected and immunostained for Vasa (identified as Spot 13 from the 2D gels) and DAPI and labelled with 4g. Scale bar is 20 μm.