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Protein Sample Preparation for Proteomic Analysis in Leishmania donovani
杜氏利什曼原虫中蛋白质组分析用蛋白质样本的制备   

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Abstract

Leishmania is a genus of trypanosomatid protozoa and is the parasite responsible for the disease leishmaniasis. These protozoa, regulate their gene expression in an atypical way, compared to other higher eukaryotes. The regulation of gene expression is characterized by a predominance of post-transcriptional over pre-transcriptional regulatory mechanisms (Clayton, 2002). Thus proteomic analysis has proven an essential tool for understanding pathways implicated in Leishmania infectivity, host-parasite interactions, drug resistance and others. When employing a comparative proteomics analysis between different parasitic cell lines, it is essential that these lines are cultivated in exactly the same way, in the same cell density and growth phase. More importantly when cell-cycle defects are suspected, it is essential to synchronize cell-lines in the same cell-cycle phase so as to eliminate possible artifacts. This protocol describes the preparation of whole-protein samples for proteomic analysis in Leishmania donovani (L. donovani).

Keywords: Leishmania(利什曼原虫), Proteomics(蛋白质组学研究), Sample preparation(样品的制备)

Materials and Reagents

  1. Leishmania donovani (L. donovani) parasites
  2. Glucose (Sigma-Aldrich, catalog number: G8270 )
  3. Hydroxyurea (Sigma-Aldrich, catalog number: H8627 )
  4. RPMI-1640 (Life Technologies, catalog number: 21875-034 )
  5. Fetal Bovine Serum heat inactivated (HIFBS) (Life Technologies, catalog number: 10270-106 )
  6. Penicillin/Streptomycin (Life Technologies, catalog number: 15140-122 )
  7. HEPES buffer (Life Technologies, catalog number: 15630-056 )
  8. Dry ice
  9. Ethanol
  10. E-64 (Sigma-Aldrich, catalog number: E3132 )
  11. CHAPS (Sigma-Aldrich, catalog number: 26680 )
  12. ASB-14 (Sigma-Aldrich, catalog number: A1346 )
  13. DTT (Sigma-Aldrich, catalog number: 43815 )
  14. Bio-Lyte® 3/10 Ampholyte (Bio-Rad Laboratories, catalog number: 163-2094 )
  15. Urea (Sigma-Aldrich, catalog number: U5378 )
  16. Thiourea (Sigma-Aldrich, catalog number: T7875 )
  17. Bromophenol blue (Bio-Rad Laboratories, catalog number: 161-0404 )
  18. Phosphate Buffered Saline (PBS) (see Recipes)
  19. Lysis Buffer (see Recipes)
  20. Rehydration Buffer (see Recipes)
  21. Fully supplemented medium (see Recipes)

Equipment

  1. Pipette
  2. Centrifuge
  3. Spiramix

Procedure

  1. Leishmania donovani parasites in the logarithmic phase (~5 x 106 parasites/ml) were cultured in fully supplemented culture medium at 25 °C and synchronized with the addition of 2.5 mM hydroxyurea for 12 h.
  2. After this period of time the parasites are gently washed three times with 10 ml of PBS and released in 10 ml of fully supplemented RPMI medium.
  3. 5 x 108-109 parasites are harvested by centrifugation (500 x g, 15 min, RT).
  4. The parasitic pellet is washed twice with 10 ml of PBS supplemented with 0.1% (w/v) glucose.
  5. The parasitic pellet is resuspended in 50 μl Lysis buffer using a pipette.
  6. The lysed parasites are subjected to five freeze-thaw cycles in solid CO2/ethanol.
  7. The sample is centrifuged at 20,000 x g for 30 min at 4 °C.
  8. To the supernatant 400 μl of Rehydration buffer [containing 0.2% (v/v) Bio-Lyte®] are added.
  9. The sample is incubated for 30 min at RT under gentle rotation.
  10. After the end of the incubation, the sample is centrifuged at 17,000 x g for 40 min at RT.
  11. The supernatant can now be used for proteomic analysis or can be stored at -80 °C for future use.

Notes

  1. Extra caution is required at all steps of the procedure as not the protein sample to be contaminated by environmental proteins (e.g. keratin). Preferably avoid using bare hands during the procedure.
  2. After the addition of Rehydration buffer the sample should not be exposed to temperatures below 10 °C, due to urea-mediated sedimentation.
  3. Bio-Lyte® should be added just before the use of the Rehydration Buffer.

Recipes

  1. PBS
    Dissolve
    8 g of NaCl
    0.2 g of KCl
    1.44 g of Na2HPO4
    0.24 g of KH2PO4
    In 800 ml distilled H2O
    Adjust pH to 7.4
    Add H2O to 1 L
    Sterilized by autoclaving
  2. Lysis Buffer
    400 μl Tris-HCl (pH 9.5)
    20 μl 0.5 M EDTA (pH 8.0)
    100 μl 1 mM E-64
    Add distilled H2O up to 10 ml
    Aliquot and stored at -20 °C
  3. Rehydration Buffer
    To a volume of 3 ml distilled H2O dissolve
    4.8 g urea
    1.52 g thiourea
    0.4 g CHAPS
    0.1 g DTT
    0.24 g ASB-14
    5 μl 0.1% (w/v) bromophenol blue
    Add distilled H2O up to 10 ml
    Aliquot and stored at -20 °C
  4. Fully supplemented medium
    RPMI-1640
    10% (v/v) HIFBS
    10 mM HEPES
    100 U/ml Penicillin/Streptomycin

Acknowledgments

This protocol was adapted from Clayton (2002) and Smirlis et al. (2006). Partial funding from the General Secretariat of Research and Technology action “Proposals for the development of Research Institutes, Krepis” supported this work.

References

  1. Alexandratos, A., Clos, J., Samiotaki, M., Efstathiou, A., Panayotou, G., Soteriadou, K. and Smirlis, D. (2013). The loss of virulence of histone H1 overexpressing Leishmania donovani parasites is directly associated with a reduction of HSP83 rate of translation. Mol Microbiol 88(5): 1015-1031.
  2. Clayton, C. E. (2002). Life without transcriptional control? From fly to man and back again. EMBO J 21(8): 1881-1888.
  3. Smirlis, D., Bisti, S. N., Xingi, E., Konidou, G., Thiakaki, M. and Soteriadou, K. P. (2006). Leishmania histone H1 overexpression delays parasite cell-cycle progression, parasite differentiation and reduces Leishmania infectivity in vivo. Mol Microbiol 60(6): 1457-1473.

简介

利什曼原虫属是锥虫病原生动物属,是负责疾病利什曼病的寄生虫。这些原生动物,与其他高等真核生物相比,以非典型方式调节它们的基因表达。基因表达的调节的特征在于转录后优先于转录前调节机制(Clayton,2002)。因此,蛋白质组学分析已经证明是了解利什曼原虫感染,宿主寄生虫相互作用,耐药性和其他相关途径的重要工具。当在不同寄生细胞系之间使用比较蛋白质组学分析时,以相同的方式,在相同的细胞密度和生长期中培养这些细胞系是必要的。更重要的是,当怀疑细胞周期缺陷时,必须在同一细胞周期阶段同步细胞系,以消除可能的人工因素。该方案描述了在杜氏利什曼原虫( donovani )中用于蛋白质组分析的全蛋白样品的制备。

关键字:利什曼原虫, 蛋白质组学研究, 样品的制备

材料和试剂

  1. Leishmania donovani ( L. donovani )寄生虫
  2. 葡萄糖(Sigma-Aldrich,目录号:G8270)
  3. 羟基脲(Sigma-Aldrich,目录号:H8627)
  4. RPMI-1640(Life Technologies,目录号:21875-034)
  5. 胎牛血清热灭活(HIFBS)(Life Technologies,目录号:10270-106)
  6. 青霉素/链霉素(Life Technologies,目录号:15140-122)
  7. HEPES缓冲液(Life Technologies,目录号:15630-056)
  8. 干冰
  9. 乙醇
  10. E-64(Sigma-Aldrich,目录号:E3132)
  11. CHAPS(Sigma-Aldrich,目录号:26680)
  12. ASB-14(Sigma-Aldrich,目录号:A1346)
  13. DTT(Sigma-Aldrich,目录号:43815)
  14. Bio-Lyte 3/10 Ampholyte(Bio-Rad Laboratories,目录号:163-2094)
  15. 脲(Sigma-Aldrich,目录号:U5378)
  16. 硫脲(Sigma-Aldrich,目录号:T7875)
  17. 溴酚蓝(Bio-Rad Laboratories,目录号:161-0404)
  18. 磷酸盐缓冲盐水(PBS)(参见配方)
  19. 裂解缓冲液(参见配方)
  20. 补液缓冲液(参见配方)
  21. 完全补充培养基(见配方)

设备

  1. 移液器
  2. 离心机
  3. Spiramix

程序

  1. 在完全补充的培养基中在25℃下培养处于对数期(约5×10 6个寄生虫/ml)的杜氏利什曼原虫寄生虫,并与添加2.5mM 羟基脲12小时。
  2. 在这段时间后,用10ml PBS将寄生虫轻轻洗涤三次,并释放到10ml充分补充的RPMI培养基中。
  3. 通过离心(500×g,15分钟,RT)收获5×10 8个 -10 9个寄生虫。
  4. 用10ml补充有0.1%(w/v)葡萄糖的PBS洗涤寄生沉淀两次。
  5. 使用移液管将寄生沉淀重悬浮于50μl裂解缓冲液中
  6. 将裂解的寄生虫在固体CO 2 /乙醇中进行五次冻融循环。
  7. 将样品在4℃以20,000×g离心30分钟。
  8. 向上清液中加入400μlRehydration buffer [含有0.2%(v/v)Bio-Lyte ]。
  9. 将样品在室温下温和旋转孵育30分钟。
  10. 孵育结束后,将样品在室温下以17,000×g离心40分钟。
  11. 上清液现在可用于蛋白质组学分析或可以储存在-80℃以备将来使用。

笔记

  1. 在该程序的所有步骤中需要特别小心,因为不是蛋白质样品被环境蛋白质(例如角蛋白)污染。 最好避免在手术过程中使用裸手。
  2. 加入再水合缓冲液后,由于尿素介导的沉降,样品不应暴露于低于10℃的温度。
  3. 应在使用补液缓冲液之前加入Bio-Lyte ®

食谱

  1. PBS
    溶解
    8克NaCl
    0.2克KCl
    1.44g的Na 2 HPO 4
    0.24g的KH 2 PO 4 sub/
    在800ml蒸馏的H 2 O中 将pH调节至7.4
    将H <2> O添加到1 L
    高压灭菌
    灭菌
  2. 裂解缓冲液
    400μlTris-HCl(pH9.5)
    20μl0.5M EDTA(pH 8.0)
    100μl1mM E-64
    将蒸馏的H 2 O加至最多10ml的
    等分并储存在-20°C
  3. 补液缓冲液
    向体积为3ml的蒸馏H 2 O溶解物
    中 4.8克尿素
    1.52克硫脲 0.4 g CHAPS
    0.1 g DTT
    0.24g ASB-14
    5μl0.1%(w/v)溴酚蓝
    将蒸馏的H 2 O加至最多10ml的
    等分并储存在-20°C
  4. 完全补充培养基
    RPMI-1640
    10%(v/v)HIFBS
    10 mM HEPES
    100U/ml青霉素/链霉素

致谢

该方案改编自Clayton(2002)和Smirlis等人(2006)。 来自研究和技术总秘书处的部分资助"研究所发展建议Krepis"支持这项工作。

参考文献

  1. Alexandratos,A.,Clos,J.,Samiotaki,M.,Efstathiou,A.,Panayotou,G.,Soteriadou,K。和Smirlis,D。 过表达利什曼原虫寄生虫的组蛋白H1的毒力的丧失与HSP83的减少直接相关 Mol Microbiol 88(5):1015-1031。
  2. Clayton,C. E.(2002)。没有转录控制的生活? EMBO J 21(8):1881-1888。
  3. Smirlis,D.,Bisti,S.N.,Xingi,E.,Konidou,G.,Thiakaki,M。和Soteriadou,K.P。(2006)。 利什曼原虫组蛋白H1过表达延迟寄生虫细胞周期进程,寄生虫分化和减少利什曼原虫感染力 体内。/a> Mol Microbiol 60(6):1457-1473
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Alexandratos, A. and Smirlis, D. (2014). Protein Sample Preparation for Proteomic Analysis in Leishmania donovani. Bio-protocol 4(5): e1058. DOI: 10.21769/BioProtoc.1058.
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