A Phosphopeptide Purification Protocol for the Moss Physcomitrella paten

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Journal of Experimental Botany
May 2014



Protein phosphorylation is one of the most common post-translational modifications in eukaryotic cells and plays a critical role in a vast array of cellular processes. Efficient methods of protein extraction and phosphopeptide purification are required to ensure the detection of high quality of proteins. In our hands, phenol extraction of proteins and TiO2 chromatography enrich phosphorylated peptides more efficiently than other methods in the moss Physcomitrlla patens (P. patens).

Materials and Reagents

  1. Seven-day old protonemata of the moss P. patens
  2. Liquid nitrogen
  3. Tris-HCl (pH 7.5)-saturated phenol (AMRESCO, catalog number: K168 )
  4. Ammonium acetate (Sigma-Aldrich, catalog number: A1542 )
  5. Methanol (Sigma-Aldrich, catalog number: 34860 )
  6. DTT (Promega Corporation, catalog number: V3155 )
  7. Acetone (Sigma-Aldrich, catalog number: 650501 )
  8. Iodoacetamide (Sigma-Aldrich, catalog number: I6125 )
  9. Ammonium bicarbonate (pH 8.5) (Sigma-Aldrich, catalog number: 40867 )
  10. Trypsin (Promega Corporation, catalog number: V5111 )
  11. Sucrose (Sigma-Aldrich, catalog number: S5390
  12. Tris (Sigma-Aldrich, catalog number: T1378 )
  13.  EDTA (Sigma-Aldrich, catalog number: E6635
  14. 1,4-dithiothreitol (DTT) (Promega Corporation, catalog number: V3155) 
  15. Protease inhibitors cocktail (Sigma-Aldrich, catalog number: P9599 )  
  16. Phosphatase inhibitors cocktail 2 (Sigma-Aldrich, catalog number: P0044
  17. NH4OH (Sigma-Aldrich, catalog number: 320145 )   
  18. Urea (Sigma-Aldrich, catalog number: U6504 )   
  19. CHAPS (Sigma-Aldrich, catalog number: C9426 )  
  20. Acetonitrile (ACN) (Sigma-Aldrich, catalog number: 34851 )   
  21. Trifluoroacetic acid (TFA) (Sigma-Aldrich, catalog number: 302031 )  
  22. Glutamic acid (Sigma-Aldrich, catalog number: G0355000 )
  23. Protein extraction buffer (see Recipes)
  24. Protein resuspension buffer (see Recipes)
  25. Loading buffer (see Recipes)
  26. Washing buffer I (see Recipes)
  27. Washing buffer II (see Recipes)
  28. Elution buffer (see Recipes)


  1. Porcelain Mortar and Pestle (90 mm)
  2. Tubes and tube holder (2 ml)
  3. Titanium dioxide micro-columns (320 µm x 50 mm) (Column Technology, Freemont)
  4. Centrifuge
  5. Vortex
  6. Freeze dryer (Christ, model: Alpha 1-4 LSC )


This procedure includes proteins extraction, protein digestion and phosphopeptide purification. The following should all be carried out at cold room temperature except for special instructions.

  1. Add protease inhibitor mix (protease inhibitors and phosphatase inhibitors) to the ice-cold protein extraction buffer according to the manufacturer’s instructions.
  2. Pour liquid nitrogen into mortar until nearly full in order to pre-cool the mortar and pestle.
  3. Add the fresh or frozen (-80 °C freezer) tissue (about 1 g) into mortar, grind them to fine powder in liquid nitrogen, and then thaw on ice for 10-15 min.
  4. Add 2 ml ice-cold protein extraction buffer into mortar, homogenize by grinding on ice for 10 min, and then transfer 0.8 ml to each tube.
  5. Add an equal volume of ice-cold Tris-HCl (pH 7.5)-saturated phenol to each tube, vortex tubes for 5-10 min.
  6. Centrifuge samples at 13,000 rpm for 15 min at 4 °C.
  7. Collect phenol phase and mixed with three volumes of 100 mM ammonium acetate in methanol.
  8. Precipitate proteins from mixture of phenol phase and 100 mM ammonium acetate overnight at -20 °C.
  9. Centrifuge proteins at 13,000 rpm for 15 min at 4 °C.
  10. Rinse proteins 3 times with ice-cold acetone containing 13 mM DTT and then lyophilize by freeze dryer for 5-10 min.
  11. The protein concentration was determined according to Peterson (1977) using BSA as a standard.
  12. Dissolve 500 µg protein power in 100 µl protein resuspension buffer at room temperature.
  13. Reduce protein (500 µg protein in 100 µl resuspension buffer) with 20 mM DTT at 37 °C for 2.5 h and alkylate protein with 40 mM iodoacetamide for 40 min at room temperature in the dark.
  14. Precipitate the protein mixture with 1 ml 100% acetone overnight at -20 °C.
  15. Collect protein by centrifugation, dissolve protein in 100 mM ammonium bicarbonate (pH 8.5), and digest protein with trypsin (50:1 w/w, protein to trypsin ratio) at 37 °C for 16 h.
  16. Lyophilize the digested peptide mixture, and then dilute in a 200 µl loading buffer.
  17. Rinse the titanium dioxide micro-columns (TiO2 columns, 320 µm x 50 mm) with 100 µl loading buffer.
  18. Load the 200 µl samples onto TiO2 columns by applying air pressure created with a plastic syringe.
  19. Wash the TiO2 columns with 100 µl loading buffer, 100 µl washing buffer I, and finally with 100 µl washing buffer II.
  20. Elute the columns which bound phosphopeptides with 100 µl of elution buffer.
  21. Pool and lyophilize the eluates which is purification of phosphopeptides [Representative data is shown in Wang et al. (2014a) and Wang et al. (2014b)].


  1. Protein extraction buffer
    250 mM sucrose
    20 mM Tris-HCl (pH 7.5)
    10 mM EDTA 1 mM 1,4-dithiothreitol
    Protease inhibitors cocktail
    Phosphatase inhibitors cocktail 2
  2. Protein resuspension buffer
    8 M urea
    4% CHAPS
    65 mM DTT
    40 mM Tris-HCl (pH 7.5)
  3. Loading buffer
    65% ACN and 2% TFA solution saturated with glutamic acid
  4. Washing buffer I
    65% ACN
    0.5% TFA
  5. Washing buffer II
    65% ACN
    0.1% TFA
  6. Elution buffer
    300 mM NH4OH
    50% ACN


This work was supported by grants from Project of Ministry Science & Technology of China (2013CB967300, 2007CB948201) to Prof. He, grant from National Natural Science Foundation of China (No. 31371243, 30970250), China Postdoctoral Science Foundation and State Education Ministry Scientific Research Foundation for the Returned Overseas Chinese Scholars to Dr. Wang.


  1. Peterson, G. L. (1977). A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem 83(2): 346-356.
  2. Wang, X., Qi, M., Li, J., Ji, Z., Hu, Y., Bao, F., Mahalingam, R. and He, Y. (2014a). The phosphoproteome in regenerating protoplasts from Physcomitrella patens protonemata shows changes paralleling postembryonic development in higher plants. J Exp Bot 65(8): 2093-2106.
  3. Wang, X. Q., Yang, P. F., Liu, Z., Liu, W. Z., Hu, Y., Chen, H., Kuang, T. Y., Pei, Z. M., Shen, S. H. and He, Y. K. (2009). Exploring the mechanism of Physcomitrella patens desiccation tolerance through a proteomic strategy. Plant Physiol 149(4): 1739-1750.
  4. Wang, X., Zhou, S., Chen, L., Quatrano, R. S. and He, Y. (2014b). Phospho-proteomic analysis of developmental reprogramming in the moss Physcomitrella patens. J Proteomics 108: 284-294.


蛋白磷酸化是真核细胞中最常见的翻译后修饰之一,并在大量细胞过程中起关键作用。 需要蛋白质提取和磷酸肽纯化的有效方法以确保高质量的蛋白质的检测。 在我们的手中,蛋白质和TiO 2层析的酚提取比在小麦Physcomitrlla patens中的其它方法更有效地富集磷酸化肽。 )。


  1. 苔藓的7天龄的质子。 patens
  2. 液氮
  3. Tris-HCl(pH7.5) - 饱和酚(AMRESCO,目录号:K168)
  4. 乙酸铵(Sigma-Aldrich,目录号:A1542)
  5. 甲醇(Sigma-Aldrich,目录号:34860)
  6. DTT(Promega Corporation,目录号:V3155)
  7. 丙酮(Sigma-Aldrich,目录号:650501)
  8. 碘乙酰胺(Sigma-Aldrich,目录号:I6125)
  9. 碳酸氢铵(pH8.5)(Sigma-Aldrich,目录号:40867)
  10. 胰蛋白酶(Promega Corporation,目录号:V5111)
  11. 蔗糖(Sigma-Aldrich,目录号:S5390)
  12. Tris(Sigma-Aldrich,目录号:T1378)
  13.   EDTA(Sigma-Aldrich,目录号:E6635) 
  14. 1,4-二硫苏糖醇(DTT)(Promega Corporation,目录号:V3155)
  15. 蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P9599)
  16. 磷酸酶抑制剂混合物2(Sigma-Aldrich,目录号:P0044)
  17. NH 4 OH(Sigma-Aldrich,目录号:320145)  
  18. 尿素(Sigma-Aldrich,目录号:U6504)   
  19. CHAPS(Sigma-Aldrich,目录号:C9426) 
  20. 乙腈(ACN)(Sigma-Aldrich,目录号:34851)  
  21. 三氟乙酸(TFA)(Sigma-Aldrich,目录号:302031) 
  22. 谷氨酸(Sigma-Aldrich,目录号:G0355000)
  23. 蛋白质提取缓冲液(参见配方)
  24. 蛋白质重悬缓冲液(见配方)
  25. 加载缓冲区(参见配方)
  26. 洗涤缓冲液I(见配方)
  27. 洗涤缓冲液II(参见配方)
  28. 洗脱缓冲液(见配方)


  1. 瓷砂浆和杵(90毫米)
  2. 管和试管架(2 ml)
  3. 二氧化钛微柱(320μm×50mm)(Column Technology,Freemont)
  4. 离心机
  5. 涡流
  6. 冷冻干燥机(基督,型号:Alpha 1-4 LSC)



  1. 根据制造商的说明,将蛋白酶抑制剂混合物(蛋白酶抑制剂和磷酸酶抑制剂)加入冰冷的蛋白提取缓冲液中。
  2. 将液氮倒入砂浆中,直到几乎完全,以预先冷却砂浆和杵。
  3. 将新鲜或冷冻(-80℃冷冻)的组织(约1g)加入到研钵中,在液氮中研磨成细粉,然后在冰上解冻10-15分钟。
  4. 加入2毫升冰冷的蛋白质提取缓冲液在砂浆中,通过在冰上研磨10分钟,然后转移0.8毫升到每个管。
  5. 向每个管中加入等体积的冰冷Tris-HCl(pH 7.5)饱和酚,涡旋管5-10分钟。
  6. 在4℃下以13,000rpm离心样品15分钟
  7. 收集苯酚相并与三体积的100mM乙酸铵的甲醇溶液混合。
  8. 在-20℃下从苯酚相和100mM乙酸铵的混合物中沉淀蛋白质过夜
  9. 在4℃下以13,000rpm离心15分钟蛋白
  10. 用含13mM DTT的冰冷丙酮冲洗蛋白质3次,然后用冷冻干燥器冻干5-10分钟。
  11. 蛋白质浓度根据Peterson(1977)使用BSA作为标准来确定
  12. 在室温下在100μl蛋白质重悬浮缓冲液中溶解500μg蛋白质功率
  13. 用20mM DTT在37℃下减少蛋白质(500μg蛋白质,在100μl重悬缓冲液中)2.5小时,烷基化蛋白质与40mM碘乙酰胺在室温下在暗处培育40分钟。
  14. 用1ml 100%丙酮在-20℃下沉淀蛋白质混合物过夜
  15. 通过离心收集蛋白质,将蛋白质溶解在100mM碳酸氢铵(pH8.5)中,并用胰蛋白酶(50:1w/w,蛋白质/胰蛋白酶比)在37℃消化蛋白16小时。
  16. 冻干消化的肽混合物,然后在200μl上样缓冲液中稀释
  17. 用100μl上样缓冲液冲洗二氧化钛微柱(TiO 2柱,320μmx 50 mm)。
  18. 通过使用塑料注射器产生的空气压力将200μl样品加载到TiO 2柱上
  19. 用100μl上样缓冲液,100μl洗涤缓冲液I,最后用100μl洗涤缓冲液II洗涤TiO 2柱。
  20. 用100μl洗脱缓冲液洗脱结合磷酸肽的柱子
  21. 池和冷冻干燥磷酸肽的纯化的洗脱液[代表性数据显示在Wang等人(2014a)和Wang等人(2014b)]。


  1. 蛋白提取缓冲液
    250mM蔗糖 20mM Tris-HCl(pH7.5) 10mM EDTA 1mM 1,4-二硫苏糖醇 蛋白酶抑制剂鸡尾酒
  2. 蛋白质重悬缓冲液
    8 M尿素
    65 mM DTT
    40mM Tris-HCl(pH7.5)
  3. 加载缓冲区
  4. 洗涤缓冲液I
  5. 洗涤缓冲液II
  6. 洗脱缓冲液
    300mM NH 4 OH v/v 50%ACN


这项工作得到了部科学技术项目赠款的支持。 中国科学技术(2013CB967300,2007CB948201)教授,中国国家自然科学基金(31371243,30970250),中国博士后科学基金和国家教育部退出侨团科学研究基金会王博士 。


  1. Peterson,G.L。(1977)。 Lowry等人的蛋白质测定方法的简化,这是更普遍适用。 Anal Biochem 83(2):346-356。
  2. Wang,X.,Qi,M.,Li,J.,Ji,Z.,Hu,Y.,Bao,F.,Mahalingam,R.and He,Y.(2014a)。 从小立碗藓再生原生质体中的磷酸化蛋白表现出与胚胎发育并行的变化在高等植物中。 65(8):2093-2106。
  3. Wang,X.Q.,Yang,P.F.,Liu,Z.,Liu,W.Z.,Hu,Y.,Chen,H.,Kuang,T.Y.,Pei,Z.M.,Shen,S.H.and He,Y.K。 通过蛋白质组学策略探索小立碗藓的干扰耐受性。/a> Plant Physiol 149(4):1739-1750
  4. Wang,X.,Zhou,S.,Chen,L.,Quatrano,R.S。和He,Y.(2014b)。 青苔中发育重编程的磷酸蛋白质组学分析 小立碗藓。 J Proteomics 108:284-294。
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引用:Wang, X. and He, Y. (2015). A Phosphopeptide Purification Protocol for the Moss Physcomitrella paten. Bio-protocol 5(14): e1527. DOI: 10.21769/BioProtoc.1527.