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Cell Type-specific Metabolic Labeling of Proteins with Azidonorleucine in Drosophila
果蝇中含叠氮正亮氨酸蛋白质的细胞类型特异性代谢标记   

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Abstract

Advanced mass spectrometry technology has pushed proteomic analyses to the forefront of biological and biomedical research. Limitations of proteomic approaches now often remain with sample preparations rather than with the sensitivity of protein detection. However, deciphering proteomes and their context-dependent dynamics in subgroups of tissue-embedded cells still poses a challenge, which we meet with a detailed version of our recently established protocol for cell-selective and temporally controllable metabolic labeling of proteins in Drosophila. This method is based on targeted expression of a mutated variant of methionyl-tRNA-synthetase, MetRSL262G, which allows for charging methionine tRNAs with the non-canonical amino acid azidonorleucine (ANL) and, thus, for detectable ANL incorporation into nascent polypeptide chains.

Keywords: Metabolic labeling(代谢标记), Click chemistry(点击化学), Drosophila melanogaster(黑腹果蝇), Proteomic profiling(蛋白质组学分析), Protein synthesis(蛋白质合成)

Background

The protein composition of any given cell is intimately linked to its state of differentiation and functionality. Changes in a cell’s proteome may reflect its response to cell-intrinsic cues or to signals originating from elsewhere inside the respective organism or its environment. In turn they are indicative of the significance of those signaling cues. Deciphering proteomes and their dynamics in a cell type-specific fashion has thus become a main focus in current research, reaching at a better understanding of molecular events underlying physiological or pathophysiological processes. Any proteomic approach in this direction, however, is challenged by the heterogeneity of cell types that are interconnected within a tissue or organ of interest. In the brain, for instance, different types of neurons and glial cells form the networks required to control animal or human behavior. Moreover, it is well established that information processing within these networks leading to long-term memory is strictly dependent on de novo protein synthesis and degradation. While this has been exemplified for a number of neuronal proteins (e.g., immediate early gene proteins), it is obvious that proteins that are up- or down-regulated in just a limited number of cells (or even are regulated oppositely in different groups of cells) may easily escape conventional modes of detection, where cellular proteomes are averaged across entire brain areas.
   A number of labeling methods for cellular proteomes have been published in the last two decades, e.g., using isotope-coded affinity tags (Gygi et al., 1999) or isobaric tags for relative and absolute quantification (Ross et al., 2004), quantitative proteomic analysis using samples from cells grown in 14N or 15N media (Washburn et al., 2002; MacCoss et al., 2003), and stable isotope labeling by amino acids in cell culture (Ong et al., 2002; Andersen et al., 2005). Moreover puromycin (Schmidt et al., 2009) and non-canonical amino acids, e.g., azidohomoalanine (AHA) or homopropargylglycine, in combination with click chemistry have been used to decipher cellular proteomes (Link et al., 2003; Link and Tirrell, 2003; Beatty et al., 2006; Dieterich et al., 2006; Link et al., 2006; Dieterich et al., 2010). All of these strategies, however, fail to uncover cell-type specific proteomes within tissue or organ samples. Most recently, novel strategies to resolve this issue have been reported for C. elegans and Drosophila (Elliott et al., 2014; Erdmann et al., 2015; Yuet et al., 2015). They have in common the use of either a mutated aminoacyl-tRNA synthetase or an orthogonal aminoacyl-tRNA synthetase/tRNA for tagging of newly synthesized proteins with food-supplied non-canonical amino acids. Specifically, we could show that upon cell type-specific expression of a mutant Methionyl-tRNA synthetase (MetRSL262G) as achieved by employing the well-established Gal4/UAS-system, the non-canonical amino acid ANL can be incorporated into proteins of selectable cell types in living Drosophila larvae and adult flies. An accompanying study by Niehues et al. (2015) used this method to show the causal involvement of mutated glycyl-tRNA synthetase in a model for the neurodegenerative Charcot Marie Tooth disease.
   ANL-containing proteins can either be analyzed in protein extracts by using biochemistry and mass spectrometry or can be visualized in situ by fluorescence microscopy (Erdmann et al., 2015; Niehues et al., 2015). For more information see ‘Click Chemistry (CuAAC) and detection of tagged de novo synthesized proteins’. The following protocol details the metabolic labeling of proteins in larvae and adult flies with ANL.

Materials and Reagents

  1. Fly vials (e.g., VWR, catalog number: 734-2254 )
  2. Fly vial plugs (e.g., Carl Roth, catalog number: PK13.1 )
  3. Gal4 activator strains of choice (e.g., C57-Gal4 for muscle-specific expression [from Ulrich Thomas, Magdeburg, Germany], elavC155-Gal4 for pan-neuronal expression [from Bloomington stock center, Bloomington, Indiana, USA], repo-Gal4 for glial expression [from Christian Klämbt, Münster, Germany])
  4. UAS-dMetRSL262G effector strains [available at request from Daniela C. Dieterich & Ulrich Thomas]. As described in Erdmann et al. (2015) various lines expressing dMetRSL262G either tagged with 3xmyc or EGFP are available
    Note: We traditionally use ONM. The standard corn meal medium has also been successfully used in Niehues et al. (2015) for ANL labeling, thus, we anticipate that other media can be used as well without any limitations.
  5. Otto-normal-medium (ONM, see Recipes)
    1. Agar-Agar (Carl Roth, catalog number: 5210 )
    2. Semolina (local food store)
    3. Mashed raisins (local food store)
    4. Baker’s yeast (local food store)
    5. Sugar beet sirup (local food store)
    6. Honey (local food store)
    7. Tap water
    8. 20% (w/v) Nipagin (see Recipes)
      1. Methyl-4-hydroxybenzoat (Merck, catalog number: 106757 )
      2. Propyl-4-hydroxybenzoat (Merck, catalog number: 107427 )
      3. 100 % ethanol (Th. Geyer, catalog number: 2246 )
  6. 200 mM ANL stock solution (for the synthesis of ANL see [Link et al., 2007; Ngo et al., 2009; Erdmann et al., 2015]) (see Recipes)

Equipment

  1. Beaker (kitchen/household grade)
  2. Immersion blender (kitchen/household grade)
  3. Paintbrush (art supplies)
  4. Fly incubator (e.g., SANYO, model: MIR-553 )
  5. Hotplate (kitchen/household grade)
  6. Pot (kitchen/household grade)
  7. Tablespoon (kitchen/household grade)

Procedure

  1. Preparation of ANL-containing fly food medium (Figure 1)
    1. Thaw baker’s yeast.
    2. Add semolina and Agar-Agar to 0.33 L water. Stir from time to time until swelling is completed.
    3. Heat mashed raisins, yeast, sugar beet syrup and honey in 0.66 L water. Boil the mixture for 5 min while stirring constantly.
    4. Add the semolina-Agar-Agar-mixture and boil once again. Don’t forget to stir constantly, as the mixture might braise at the bottom of the pot.
    5. Cool down the ONM to 50 °C. Stir every 15-20 min for 1 min.
    6. Add the Nipagin and stir for at least 1 min until the Nipagin is homogenously distributed in the food.
    7. Add 2 ml of ANL stock solution to 100 ml ONM in a beaker for a final concentration of 4 mM ANL. Mix for 1 min using an immersion blender.
    8. Aliquot ANL-containing ONM (2-4 ml) into fly vials and let cool down completely at room temperature.
    9. Plug the vials.
    10. ANL-containing ONM can be stored for approximately two weeks at 4 °C. Discard vials once the ONM detaches from the vial wall.


      Figure 1. Preparation of ANL-containing fly food medium. A. Ingredients for ONM are shown: R (mashed raisins); S (semolina); BS (sugar beet syrup); H (honey). B. Semolina and Agar-Agar are added to water and allowed to swell, SAA. C. Raisins and Baker’s yeast (Y), sugar beet syrup (BS), and honey (H), are added into water and boiled for 5 min. D. Afterwards, the SAA mixture is added and boiled once more. After cooling down to 50 °C with stirring from time to time, Nigapin and ANL are added and mixed thoroughly. E and F. Media is then aliquoted and allowed to cool down completely before storage at 4 °C.

  2. Cell type-specific expression of dMetRSL262G-variants in Drosophila larvae and flies
    1. Collect virgin female flies of the respective activator strain.
      Note: The number of flies and hence the number of offspring depends on your experimental design including the type of analysis (e.g., MS/MS) and the effectiveness and degree of cell selectivity of the Gal4-activator in use.
    2. Cross virgin female flies of the activator strain to male flies of a UAS-dMetRSL262G-effector strain (Erdmann et al., 2015).
      Note: Crosses can as well be set up reciprocally, that is, you may use virgin female flies of a UAS-dMetRSL262G-effector strain and cross them to males of your activator strain.

  3. Cell type-specific labeling of proteins with ANL–examples
    1. Long-term labeling of proteins in larval body wall muscles:
      1. Raise appropriate crosses on ANL-containing ONM such that ANL is present during all developmental stages of the progeny.
      2. Mid- to late 3rd larval stage is reached after approximately 5 days when crosses are raised at 25 °C and after approximately 10-12 days when kept at 18 °C.
      3. Perform dissection of larval body walls according to ‘Click Chemistry (CuAAC) and detection of tagged de novo synthesized proteins’. See also Bellen and Budnik (2000).
        Note: Biochemical approaches on whole larval extracts have proven difficult to perform time and again, perhaps due to lytic activities. We therefore recommend separating body walls (mainly comprising muscles, epithelia, cuticle, trachea and sensory neurons) from all other tissues.
    2. Long-term labeling of proteins in fly heads (e.g., if dMetRSL262G is expressed in the CNS, compound eyes and/or antenna):
      1. Raise crosses on ANL-containing ONM so that ANL is present throughout development of the progeny.
      2. Remove parental flies before eclosure of the offspring.
      3. Prepare fly heads from adult progeny according to ‘Click Chemistry (CuAAC) and detection of tagged de novo synthesized proteins’.
    3. Short-term labeling of proteins in larval body wall muscles or brains:
      1. Raise crosses on ANL-free ONM for 1-2 days at 25 °C.
      2. Transfer parental flies onto fresh ANL-free ONM for 4-6 h. Remove parental flies.
      3. Keep vials for 72 ± 2 h at 25 °C.
      4. Wash early 3rd instar larvae out of the food with warm tap water and rinse them into a mesh basket.
      5. Transfer 3rd instar larvae onto ANL-containing ONM using a paintbrush.
      6. Keep larvae at 25 °C for 24 h.
      7. Prepare the larval body walls or larval brains according to ‘Click Chemistry (CuAAC) and detection of tagged de novo synthesized proteins’.
    4. Short-term labeling of proteins in fly heads:
      1. Raise appropriate crosses on ANL-free ONM either at 25 °C or 18 °C depending on your experimental design.
      2. Discard parental flies before progeny ecloses.
      3. Transfer progeny flies on ANL-containing ONM for a period of time according to your experimental design. For certain analyses it may also be considered to place flies back onto ANL-free food.
      4. Prepare fly heads according to ‘Click Chemistry (CuAAC) and detection of tagged de novo synthesized proteins’.

Data analysis

ANL incorporation into fly or larval proteins can be analyzed after performing copper-catalyzed azide-alkyne cycloaddition (CuAAC, ‘click chemistry’) as described in Erdmann et al. (2015) and the accompanying bio-protocol. General fly and larvae viabilities upon ANL incorporation can be analyzed by assessing e.g., locomotor behavior and/or hatching rates as described in Erdmann et al. (2015).

Notes

  1. Reproducibility and variability: Uptake of ANL and thus subsequent labeling efficiency may vary when heterogeneous animal numbers are raised in the vials, as the food will be differently mashed through depending on the number of larvae. Also of note, larvae will show in general stronger ANL incorporation compared to adult flies.
  2. ANL-containing ONM is used best within two weeks after preparation to yield reproducible results.

Recipes

  1. 20% (w/v) Nipagin
    150 g methyl-4-hydroxybenzoat
    50 g propyl-4-hydroxybenzoat
    Dissolve in 100% (abs) ethanol
  2. Otto-normal-medium (ONM)
    8.3 g Agar-Agar
    50 g semolina
    40 g mashed raisins
    2 cubes of yeast ( 42 g each)
    1 tablespoon sugar beet syrup
    1 tablespoon honey
    6.6 ml 20% (w/v) Nipagin
  3. 200 mM ANL stock solution
    34.4 mg per 1 ml ddH2O

Acknowledgments

This work was supported by the German Research Foundation (DFG) with a SFB 779 grant to D.C.D., SFB 854 grants to U.T. and D.C.D. and a Leibniz Society PAKT grant (LGS SynaptoGenetics) to D.C.D. and U.T. This protocol was adapted from Erdmann et al., 2015.

References

  1. Andersen, J. S., Lam, Y. W., Leung, A. K., Ong, S. E., Lyon, C. E., Lamond, A. I. and Mann, M. (2005). Nucleolar proteome dynamics. Nature 433(7021): 77-83.
  2. Beatty, K. E., Liu, J. C., Xie, F., Dieterich, D. C., Schuman, E. M., Wang, Q. and Tirrell, D. A. (2006). Fluorescence visualization of newly synthesized proteins in mammalian cells. Angew Chem Int Ed Engl 45(44): 7364-7367.
  3. Bellen, H. J. and Budnik, V. (2000). Drosophila, a laboratory manual. In: Ashburner, M., Hawley, S. and Sullivan. B (Eds). Cold Spring Harbor Laboratory. chap. 11.
  4. Dieterich, D. C., Hodas, J. J., Gouzer, G., Shadrin, I. Y., Ngo, J. T., Triller, A., Tirrell, D. A. and Schuman, E. M. (2010). In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons. Nat Neurosci 13(7): 897-905.
  5. Dieterich, D. C., Link, A. J., Graumann, J., Tirrell, D. A. and Schuman, E. M. (2006). Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT). Proc Natl Acad Sci U S A 103(25): 9482-9487.
  6. Elliott, T. S., Townsley, F. M., Bianco, A., Ernst, R. J., Sachdeva, A., Elsasser, S. J., Davis, L., Lang, K., Pisa, R., Greiss, S., Lilley, K. S. and Chin, J. W. (2014). Proteome labeling and protein identification in specific tissues and at specific developmental stages in an animal. Nat Biotechnol 32(5): 465-472.
  7. Erdmann, I., Marter, K., Kobler, O., Niehues, S., Abele, J., Muller, A., Bussmann, J., Storkebaum, E., Ziv, T., Thomas, U. and Dieterich, D. C. (2015). Cell-selective labelling of proteomes in Drosophila melanogaster. Nat Commun 6: 7521.
  8. Gygi, S. P., Rist, B., Gerber, S. A., Turecek, F., Gelb, M. H. and Aebersold, R. (1999). Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol 17(10): 994-999.
  9. Link, A. J., Mock, M. L. and Tirrell, D. A. (2003). Non-canonical amino acids in protein engineering. Curr Opin Biotechnol 14(6): 603-609.
  10. Link, A. J. and Tirrell, D. A. (2003). Cell surface labeling of Escherichia coli via copper(I)-catalyzed [3+2] cycloaddition. J Am Chem Soc 125(37): 11164-11165.
  11. Link, A. J., Vink, M. K., Agard, N. J., Prescher, J. A., Bertozzi, C. R. and Tirrell, D. A. (2006). Discovery of aminoacyl-tRNA synthetase activity through cell-surface display of noncanonical amino acids. Proc Natl Acad Sci U S A 103(27): 10180-10185.
  12. Link, A. J., Vink, M. K. and Tirrell, D. A. (2007). Synthesis of the functionalizable methionine surrogate azidohomoalanine using Boc-homoserine as precursor. Nat Protoc 2(8): 1884-1887.
  13. MacCoss, M. J., Wu, C. C., Liu, H., Sadygov, R. and Yates, J. R., 3rd (2003). A correlation algorithm for the automated quantitative analysis of shotgun proteomics data. Anal Chem 75(24): 6912-6921.
  14. Ngo, J. T., Champion, J. A., Mahdavi, A., Tanrikulu, I. C., Beatty, K. E., Connor, R. E., Yoo, T. H., Dieterich, D. C., Schuman, E. M. and Tirrell, D. A. (2009). Cell-selective metabolic labeling of proteins. Nat Chem Biol 5(10): 715-717.
  15. Niehues, S., Bussmann, J., Steffes, G., Erdmann, I., Kohrer, C., Sun, L., Wagner, M., Schafer, K., Wang, G., Koerdt, S. N., Stum, M., Jaiswal, S., RajBhandary, U. L., Thomas, U., Aberle, H., Burgess, R. W., Yang, X. L., Dieterich, D. and Storkebaum, E. (2015). Impaired protein translation in Drosophila models for Charcot-Marie-Tooth neuropathy caused by mutant tRNA synthetases. Nat Commun 6: 7520.
  16. Ong, S. E., Blagoev, B., Kratchmarova, I., Kristensen, D. B., Steen, H., Pandey, A. and Mann, M. (2002). Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1(5): 376-386.
  17. Ross, P. L., Huang, Y. N., Marchese, J. N., Williamson, B., Parker, K., Hattan, S., Khainovski, N., Pillai, S., Dey, S., Daniels, S., Purkayastha, S., Juhasz, P., Martin, S., Bartlet-Jones, M., He, F., Jacobson, A. and Pappin, D. J. (2004). Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics 3(12): 1154-1169.
  18. Schmidt, E. K., Clavarino, G., Ceppi, M. and Pierre, P. (2009). SUnSET, a nonradioactive method to monitor protein synthesis. Nat Methods 6(4): 275-277.
  19. Washburn, M. P., Ulaszek, R., Deciu, C., Schieltz, D. M. and Yates, J. R., 3rd (2002). Analysis of quantitative proteomic data generated via multidimensional protein identification technology. Anal Chem 74(7): 1650-1657.
  20. Yuet, K. P., Doma, M. K., Ngo, J. T., Sweredoski, M. J., Graham, R. L., Moradian, A., Hess, S., Schuman, E. M., Sternberg, P. W. and Tirrell, D. A. (2015). Cell-specific proteomic analysis in Caenorhabditis elegans. Proc Natl Acad Sci U S A 112(9): 2705-2710.

简介

先进的质谱技术将蛋白质组学分析推向了生物与生物医学研究的前沿。 蛋白质组学方法的局限性现在通常与样品制备相关,而不是蛋白质检测的灵敏度。 然而,破译组织嵌入细胞亚群中的蛋白质组学及其上下文相关动力学仍然是一个挑战,我们通过我们最近建立的细胞选择性和时间上可控的代谢标记的详细版本满足了果蝇中蛋白质的代谢标记。 该方法基于甲硫氨酰-tRNA合成酶的突变变体MetRSL262G 的靶向表达,其允许用非标准氨基酸叠氮基亮氨酸(ANL)装载甲硫氨酸tRNA,因此,用于 可检测的ANL并入新生多肽链。
【背景】任何给定细胞的蛋白质组成与其分化和功能状态密切相关。细胞蛋白质组的变化可能反映其对细胞内在线索的反应或源自相应生物体或其环境中其他地方的信号。反过来,它们表明这些信号提示的意义。因此,以细胞类型特异性方式对蛋白质组及其动力学进行解密已成为当前研究的重点,更好地了解生理或病理生理过程的分子事件。然而,在这个方向上的任何蛋白质组学方法都受到在感兴趣的组织或器官内相互连接的细胞类型的异质性的挑战。在大脑中,例如,不同类型的神经元和胶质细胞形成控制动物或人类行为所需的网络。此外,已经确定,这些网络中导致长期记忆的信息处理严格依赖于新生蛋白质的合成和降解。虽然已经为许多神经元蛋白(例如,即时早期基因蛋白)例示了这一点,但显而易见的是,仅在有限数量的细胞中(或甚至是)的上调或下调的蛋白质在不同的细胞群中相反地调节)可以容易地逃避常规的检测模式,其中细胞蛋白质组在整个脑区域被平均化。
 在过去二十年中,使用同位素编码的亲和标签(Gygi等人,1999),已经公布了许多用于细胞蛋白质组学的标记方法,例如,或者用于相对和绝对定量的同量异序标签(Ross等人,2004),使用在 14中生长的细胞的样品的定量蛋白质组学分析, N媒体(Washburn等人,2002; MacCoss等人,2003),以及细胞培养中氨基酸的稳定同位素标记(Ong等人, ,2002; Andersen等人,2005)。此外,嘌呤霉素(Schmidt等人,2009)和非标准氨基酸,例如,叠氮基丙氨酸(AHA)或高炔丙基甘氨酸与点击化学结合已被用于解密细胞蛋白质组(Link等人,2003; Link和Tirrell,2003; Beatty等人,2006; Dieterich等人,2006) ; Link 等人,2006; Dieterich等人,2010)。然而,所有这些策略未能发现组织或器官样本内的细胞型特异性蛋白质组。最近报道了解决这个问题的新策略。电针杆和果蝇(Elliott等人,2014; Erdmann等人,2015; Yuet等人,2015) 。它们通常使用突变的氨酰-tRNA合成酶或正交氨酰-tRNA合成酶/ tRNA来用食物供应的非正常氨基酸标记新合成的蛋白质。具体来说,我们可以显示,通过使用公认的Gal4 / UAS系统实现的突变型甲硫氨酰-tRNA合成酶(MetRS L262G )的细胞类型特异性表达,非规范氨基酸ANL可以并入生活在果蝇幼虫和成年苍蝇中的可选细胞类型的蛋白质中。 Niehues等人(2015)的随机研究使用该方法显示突变的甘氨酰-tRNA合成酶在神经变性Charcot Marie Tooth疾病模型中的因果关系。
 可以通过使用生物化学和质谱法在蛋白质提取物中分析含ANL的蛋白质,或者可以通过荧光显微镜(Erdmann等人,2015)进行原位可视化 Niehues等人,,2015)。有关更多信息,请参阅“Click Chemistry(CuAAC)”和检测标记的“no novo”合成蛋白质。以下方案详述了具有ANL的幼虫和成年苍蝇中蛋白质的代谢标记。

关键字:代谢标记, 点击化学, 黑腹果蝇, 蛋白质组学分析, 蛋白质合成

材料和试剂

  1. 飞小瓶(例如,,VWR,目录号:734-2254)
  2. 飞瓶插头(例如,,Carl Roth,目录号:PK13.1)
  3. 选择的Gal4活化剂菌株(例如,来自Ulrich Thomas,Magdeburg,Germany的肌肉特异性表达)(例如,C57-Gal4 ,elav -Gal4 用于泛神经元表达[来自布卢明顿股票中心,布卢明顿,印第安纳州,美国],用于神经胶质表达的repo-Gal4 来自ChristianKlämbt ,明斯特,德国])
  4. UAS-dMetRS 效应菌株[可根据Daniela C.Daterich&乌尔里希·托马斯]。如Erdmann等人(2015)所述,表达具有3×myc或EGFP标记的dMetRS L262G 的各种线可用
    注意:我们传统上使用ONM。标准的玉米粉介质也已经在Niehues等人成功使用(2015)ANL标签,因此,我们预计其他媒体也可以没有任何限制。
  5. 奥托普通培养基(ONM,见食谱)
    1. 琼脂(Carl Roth,目录号:5210)
    2. Semolina(当地食品店)
    3. 捣碎葡萄干(当地食品店)
    4. 贝克酵母(当地食品店)
    5. 甜菜sirup(当地食品店)
    6. 蜂蜜(当地食品店)
    7. 自来水
    8. 20%(w / v)尼泊金(见食谱)
      1. 甲基-4-羟基苯甲酸(Merck,目录号:106757)
      2. 丙基-4-羟基苯甲酸酯(Merck,目录号:107427)
      3. 100%乙醇(Th。Geyer,目录号:2246)
  6. 200mM ANL储备溶液(用于ANL的合成参见[Link; et al。,2007; Ngo等人,2009; Erdmann等人, ,2015])(见配方)

设备

  1. 烧杯(厨房/家庭年级)
  2. 浸入式搅拌机(厨房/家庭年级)
  3. 画笔(艺术用品)
  4. 飞行孵化器(例如,,SANYO,型号:MIR-553)
  5. 电炉(厨房/家庭年级)
  6. 锅(厨房/家庭年级)
  7. 汤匙(厨房/家庭年级)

程序

  1. 含ANL的飞行食物介质的制备(图1)
    1. 解冻面包师的酵母。
    2. 将粗粉和琼脂加入0.33L水中。不时搅拌直至肿胀完成。
    3. 热量炖葡萄干,酵母,甜菜糖浆和蜂蜜在0.66升水中。在搅拌下不断搅拌混合物5分钟。
    4. 加入粗粉 - 琼脂 - 混合物再次煮沸。不要忘记不断搅拌,因为混合物可能会在锅底炖。
    5. 将ONM冷却至50°C。每15-20分钟搅拌1分钟
    6. 加入尼泊金,搅拌至少1分钟,直到尼泊金均匀分布在食物中
    7. 在烧杯中加入2毫升ANL储备溶液至100毫升ONM,最终浓度为4 mM ANL。使用浸入式搅拌机混合1分钟
    8. 将等份含ANL的ONM(2-4ml)置于飞小瓶中,并在室温下完全冷却
    9. 插上小瓶。
    10. 含ANL的ONM可以在4℃下储存约两周。一旦ONM从小瓶壁上拆下,就丢弃小瓶。


      图1.含ANL的飞行食品介质的制备。 :一种。显示ONM的成分:R(捣碎葡萄干); S(粗面粉); BS(甜菜糖浆); H(蜂蜜)。 B.将浸泡剂和琼脂 - 琼脂加入水中并使其膨胀。将葡萄干和贝克酵母(Y),甜菜糖浆(BS)和蜂蜜(H))加入水中煮沸5分钟。 D.然后再加入SAA混合物并再次煮沸。不时搅拌冷却至50℃后,加入尼古丁和ANL并彻底混合。 E和F.然后将介质等分,并在4℃下储存之前完全冷却。

  2. 在果蝇幼虫和苍蝇中的dMetRS L262G 变体的细胞类型特异性表达
    1. 收集相应活化剂菌株的原始雌性蝇。
      注意:苍蝇的数量以及后代的数量取决于您的实验设计,包括分析类型(例如MS / MS)以及Gal4激活剂在使用中的细胞选择性的有效性和程度。
    2. UAS-dMetRS L262G 效应菌株(Erdmann等人,2015)的雄性蝇的活化剂菌株的原生女性苍蝇。
      注意:十字架也可以相互设置,也就是说,您可以使用UAS-dMetRS的处女女苍蝇 L262G - 将其与您的激活菌株的雄性相交叉。

  3. 用ANL实例的蛋白质的细胞类型特异性标记
    1. 幼虫体壁肌肉长期标记蛋白质:
      1. 在含ANL的ONM上提高适当的杂交,使ANL在子代的所有发育阶段都存在。
      2. 在25℃升高大约5天后,保持在18°C的大约10-12天后,可以达到中期至晚期3小时阶段。
      3. 根据“点击化学(CuAAC)”和检测标记的新生合成蛋白质,进行幼体体壁的解剖。另见Bellen和Budnik(2000)。
        注意:整个幼虫提取物的生物化学方法证明很难再次执行,可能是由于溶解活动。因此,我们建议从所有其他组织分离体壁(主要包括肌肉,上皮,角质层,气管和感觉神经元)。
    2. 在CNS,复眼和/或天线中表达在飞头中的蛋白质的长期标记(例如,如果dMetRS L262G ):
      1. 在含ANL的ONM上提高杂交,使ANL在后代的发育中存在。
      2. 在子孙后代,清除父母的苍蝇。
      3. 根据“Click Chemistry(CuAAC)”和检测标记的“no novo”合成蛋白质,从成人后代准备飞头。
    3. 蛋白质在幼体体壁肌肉或大脑中的短期标记:
      1. 在25°C下将ANL-ON ONM上的十字架提升1-2天。
      2. 将父母转入新鲜ANL-ON ONM 4-6小时。清除父母的苍蝇。
      3. 在25°C保持小瓶72±2小时。
      4. 用温水自来水冲洗早餐3小时,将其冲洗干净。
      5. 使用油漆刷将3日龄幼体转移到含有ANL的ONM上。
      6. 将幼虫在25°C保存24 h
      7. 根据“Click Chemistry(CuAAC)”和检测标记的“no novo”合成蛋白质,准备幼虫体壁或幼虫大脑。
    4. 飞头蛋白质短期标记:
      1. 根据您的实验设计,在25°C或18°C时,在ANL-ON ONM上提高适当的交叉。
      2. 在子代生长之前放弃亲本苍蝇。
      3. 根据您的实验设计,在含ANL的ONM上转移后代苍蝇一段时间。对于某些分析,也可以考虑将苍蝇放回无ANL食物。
      4. 根据“Click Chemistry(CuAAC)”和检测标记的“no novo”合成蛋白质制备飞头。

数据分析

如Erdmann等人(2015)所述,在执行铜催化的叠氮炔环加成(CuAAC,“点击化学”)后,可以分析飞行或幼虫蛋白质中的ANL以及随附的生物方案。可以通过评估如Erdmann等人(2015)中描述的运动行为和/或孵化率来分析ANL并入后的一般飞行和幼虫存活率。

笔记

  1. 重复性和变异性:当异种动物数量在小瓶中升高时,ANL的摄取和随后的标记效率可能会变化,因为根据幼虫的数量,食物将被不同的捣碎。另外值得注意的是,与成年苍蝇相比,幼虫通常会显示更强的ANL结合
  2. 含ANL的ONM在制备后的两周内最好使用,以产生可重复的结果。

食谱

  1. 20%(w / v)尼泊金
    150克甲基-4-羟基苯甲酸酯 50克丙基-4-羟基苯甲酸酯 溶解在100%(abs)乙醇中
  2. 奥托普通媒介(ONM)
    8.3克Agar-Agar
    50克粗粉+
    40克捣碎葡萄干
    2个酵母块(每个42克)
    1汤匙甜菜糖浆
    1汤匙蜂蜜
    6.6 ml 20%(w / v)尼泊金
  3. 200毫升ANL储备溶液
    34.4mg / 1ml ddH 2 O

致谢

这项工作得到了德国研究基金会(DFG)的支持,并向美国国务院发放了779美元的SFB,向联合国提供了854美元的SFB。和D.C.D.和莱布尼茨协会PAKT授权(LGS SynaptoGenetics)到D.C.D.和U.T.该协议由Erdmann等人于2015年进行了改编。

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Erdmann, I., Marter, K., Kobler, O., Niehues, S., Bussmann, J., Müller, A., Abele, J., Storkebaum, E., Thomas, U. and Dieterich, D. C. (2017). Cell Type-specific Metabolic Labeling of Proteins with Azidonorleucine in Drosophila. Bio-protocol 7(14): e2397. DOI: 10.21769/BioProtoc.2397.
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