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Proteolytic Fragment Isolation and Analysis (ex. N-terminal GST-tagged CLAVATA3 Protein GST-CLV3)
水解蛋白片段的分离和分析   

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Abstract

It has become clear that the post-embryonic growth and development of plants requires properly controlled short distance cell-to-cell communication not only through the historically well-known phytohormones, but also through secreted small peptide signals. This protocol demonstrates an example of how to isolate small peptides (< 10 daltons) from complex protein mixtures (e.g. cauliflower meristem protein extraction) for MS/MS analysis.

Keywords: CLAVATA3(CLAVATA3), Meristem protein isolation(分生组织蛋白质分离), Proteolytic processing(蛋白水解处理)

Materials and Reagents

  1. Glutathione Sepharose 4B (Amersham biosciences , catalog number: 17-0756-01 )
  2. Glutathione (Thermo Fisher Scientific, catalog number: 78259 )
  3. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P9599 )
  4. Triton X-100 (Pierce, catalog number: 85111 )
  5. GST-mCLV3 proteins
  6. Tris-HCl
  7. Hepes (Sigma-Aldrich, catalog number:  H3375 )
  8. EDTA (Sigma-Aldrich, catalog nummber: ED100g )
  9. Phenylmethylsulfonyl fluoride (Sigma-Aldrich, catalog number:  78830-1G )
  10. Aprotinin (Sigma-Aldrich, catalog number: A4529-1MG )
  11. Chymostatin (Sigma-Aldrich, catalog number:  C7268-1MG )
  12. Leupeptin (Sigma-Aldrich, catalog number: L2884-5MG )
  13. Eluting buffer (see Recipes)
  14. Cauliflower extraction buffer (see Recipes)

Equipment

  1. Rotor
  2. Microcon YM-10 centrifugal filter (EMD Millipore)
  3. Tabletop centrifuge
  4. Mass spectrometer (4700 proteomics analyzer)

Procedure

  1. Cauliflower (Brassica oleracea) meristem protein extracts were prepared as described before (Trotochaud et al., 1999) with or without 0.1% Triton X-100. Note that 1 ml of protease inhibitor cocktail for use with plant cell extracts was added in the extraction buffer per 300 x g of tissues. Before use, the extracts were centrifuged at 40,000 x g for 30 min at 4 °C. It is very important to use fresh cauliflower. We usually try to get cauliflower from local farms. The meristem tissues were collected using razor blade “shaving” the head of the cauliflower.

  2. Incubate ~100 μg purified GST-mCLV3 proteins (mature CLV3 protrein) with ~400 μl cauliflower protein extracts for 2 h at room temperature (RT) on a rotor.

  3. For N-terminal tagged fragment (~ 32-34 kD) isolation.
    1. Bound protein mixtures to Glutathione Sepharose 4B.
    2. Elute the N-terminal GST containing fragments from the beads with buffer containing 10 mM Glutathione, 50 mM Tris-HCl (pH 8.0).
    3. If needed, concentrate protein elution using Microcon YM-50 centrifugal filter at 14,000 x g, 4 °C for 30 min and elute in desired volume and buffer (e.g. 0.1% TFA trifluoroacetic acid/water) for MS analysis.

  4. For C-terminal untagged fragment (~ 2-4 kD) isolation.
    1. Subject protein mixtures to centrifuge through Microcon YM-10 centrifugal filter at 14,000 x g, 4 °C for 30 min.
    2. Collect the flow-through fractions for intact MS analysis.
    3. Further characterize peaks of interest by MS/MS analysis (control: Cauliflower protein extracts only; GST-CLV3 incubated with cauliflower extraction buffer).

Recipes

  1. Eluting buffer
    50 mM Tris-HCl
    10 mM reduced glutathione (pH 8.0)
  2. Cauliflower extraction buffer
    50 mM Hepes (pH 7.4)
    10 mM EDTA
    0.1% Triton X-100
    1 mM phenylmethylsulfonyl fluoride
    5 mg/ml aprotinin
    10 mg/ml chymostatin
    1 mg/ml leupeptin
    For extracts without membrane proteins, exclude Triton X-100 from the buffer.

Acknowledgments

This protocol has been adapted from previous publications including Ni and Clark (2006), Ni et al. (2011) and Trotochaud et al. (1999).

References

  1. Ni, J. and Clark, S. E. (2006). Evidence for functional conservation, sufficiency, and proteolytic processing of the CLAVATA3 CLE domain. Plant Physiol 140(2): 726-733.
  2. Ni, J., Guo, Y., Jin, H., Hartsell, J. and Clark, S. E. (2011). Characterization of a CLE processing activity. Plant Mol Biol 75(1-2): 67-75.
  3. Trotochaud, A. E., Hao, T., Wu, G., Yang, Z. and Clark, S. E. (1999). The CLAVATA1 receptor-like kinase requires CLAVATA3 for its assembly into a signaling complex that includes KAPP and a Rho-related protein. Plant Cell 11(3): 393-406.

简介

已经变得清楚的是,植物的后胚胎生长和发育需要适当控制的短距离细胞与细胞的通信,不仅通过历史上熟知的植物激素,而且通过分泌的小肽信号。 该方案展示了如何从复杂蛋白质混合物(例如花椰菜分生组织蛋白质提取)中分离小肽(<10道尔顿)用于MS/MS分析的实例。

关键字:CLAVATA3, 分生组织蛋白质分离, 蛋白水解处理

材料和试剂

  1. 谷胱甘肽琼脂糖4B(Amersham biosciences,目录号:17-0756-01)
  2. 谷胱甘肽(Thermo Fisher Scientific,目录号:78259)
  3. 蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P9599)
  4. Triton X-100(Pierce,目录号:85111)
  5. GST-mCLV3蛋白质
  6. Tris-HCl
  7. Hepes(Sigma-Aldrich,目录号:H3375)
  8. EDTA(Sigma-Aldrich,目录号:ED100g)
  9. 苯甲基磺酰氟(Sigma-Aldrich,目录号:78830-1G)
  10. 抑酶肽(Sigma-Aldrich,目录号:A4529-1MG)
  11. 胰凝乳蛋白酶抑制剂(Sigma-Aldrich,目录号:C7268-1MG)
  12. 亮肽素(Sigma-Aldrich,目录号:L2884-5MG)
  13. 洗脱缓冲液(参见配方)
  14. 花椰菜提取缓冲液(见配方)

设备

  1. 转子
  2. Microcon YM-10离心过滤器(EMD Millipore)
  3. 台式离心机
  4. 质谱仪(4700蛋白质组学分析仪)

程序

  1. 如前所述(Trotochaud等人,1999),使用或不使用0.1%Triton X-100制备花椰菜(芸苔)分生组织蛋白提取物。 注意,在每300×g组织的提取缓冲液中加入1ml用于植物细胞提取物的蛋白酶抑制剂混合物。 在使用前,将提取物在4℃以40,000×g离心30分钟。 这是非常重要的使用新鲜的花椰菜。 我们通常尝试从当地农场获得花椰菜。 使用剃刀刀片"刮"花椰菜的头部收集分生组织
  2. 在转子上室温(RT)下用约400μl花椰菜蛋白提取物孵育约100μg纯化的GST-mCLV3蛋白(成熟的CLV3 protrein)2小时。

  3. 对于N-末端标记的片段(〜32-34kD)分离。
    1. 结合蛋白混合物与谷胱甘肽琼脂糖4B
    2. 用含有10mM谷胱甘肽,50mM Tris-HCl(pH8.0)的缓冲液洗脱含有来自珠子的片段的N-末端GST。
    3. 如果需要,使用Microcon YM-50离心过滤器在14,000×g,4℃浓缩蛋白洗脱30分钟,并在所需体积和缓冲液(例如0.1%TFA三氟乙酸 酸/水),用于MS分析

  4. 对于C端未标记片段(〜2-4kD)分离。
    1. 将主题蛋白质混合物通过Microcon YM-10离心过滤器在14,000×g,4℃下离心30分钟。
    2. 收集流通级分用于完整的MS分析
    3. 通过MS/MS分析(对照:仅花椰菜蛋白提取物; GST-CLV3与花椰菜提取缓冲液孵育)进一步表征感兴趣的峰。

食谱

  1. 洗脱缓冲液
    50mM Tris-HCl
    10mM还原型谷胱甘肽(pH8.0)
  2. 花椰菜提取缓冲液
    50mM Hepes(pH 7.4)
    10 mM EDTA
    0.1%Triton X-100 1mM苯甲基磺酰氟 5mg/ml抑肽酶
    10mg/ml抑肽素
    1mg/ml亮抑酶肽
    对于没有膜蛋白的提取物,从缓冲液中排除Triton X-100

致谢

该方案已经改编自以前的出版物,包括Ni和Clark(2006),Ni et al。 (2011)和Trotochaud (1999)。

参考文献

  1. Ni,J.and Clark,S.E。(2006)。 CLAVATA3 CLE域功能保守,充足性和蛋白水解加工的证据 em> Plant Physiol 140(2):726-733。
  2. Ni,J.,Guo,Y.,Jin,H.,Hartsell,J.and Clark,S.E。 CLE处理活动的表征。植物分子生物学 75(1-2):67-75
  3. Trotochaud,A.E.,Hao,T.,Wu,G.,Yang,Z.and Clark,S.E。(1999)。 CLAVATA1受体样激酶需要CLAVATA3将其装配成信号复合物,包括KAPP和Rho相关蛋白。植物细胞 11(3):393-406
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ni, J. (2012). Proteolytic Fragment Isolation and Analysis (ex. N-terminal GST-tagged CLAVATA3 Protein GST-CLV3). Bio-protocol 2(14): e232. DOI: 10.21769/BioProtoc.232.
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