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This method describes the preparation of total yeast protein extract for mass spectrometry analysis. The protein extract is digested by trypsin in a solution with strong denaturants. The digested sample is dried and re-constituted in a mixture compatible with HPLC separation. Samples of isobaric labels should be processed in parallel experiments starting from trypsin digestion.

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[Bio101] In-Solution Digestion Of Purified Yeast Protein For LC-MS
[Bio101] 溶液酶解法纯化酵母蛋白用于液质联用分析

系统生物学 > 蛋白质组学 > 整个机体
作者: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford , USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
4/20/2011, 8188 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.62

[Abstract] This method describes the preparation of total yeast protein extract for mass spectrometry analysis. The protein extract is digested by trypsin in a solution with strong denaturants. The digested sample is dried and re-constituted in a mixture compatible with HPLC separation. Samples of isobaric labels should be processed in parallel experiments starting from trypsin digestion.
Keywords: Proteomics(蛋白质组学), LC-MS(LC-MS), Sample preparation(样品制备), In-solution digestion(在消解液), Yeast(酵母)

[Abstract] 该方法为用于质谱分析的总蛋白提取物做准备。蛋白提取物通过含有强变性剂的溶液中的胰蛋白酶进行消化处理,高压液相色谱分离的相匹配的样品干燥后重新形成复合物。具有等压特征的样品从胰蛋白酶消化开始就要设计进行平行对照试验。

Materials and Reagents

  1. Purified protein
  2. Sequencing grade modified trypsin (Promega Corporation, catalog number: V5113 )
  3. 6 M guanidine HCl
  4. Tris HCl (pH 8.0)
  5. 1 M DTT
  6. Triethylamine (TEA) (Sigma-Aldrich)
  7. HPLC solvent A (usually 10% acetonitrile in water)
  8. Acetic acid

Equipment

  1. Amicon Ultra centrifuge filters Ultracel 10 k MWCO (EMD Millipore)
  2. SpeedVac
  3. Heat block
  4. High Performance Liquid Chromatography (HPLC)
  5. Amicon filters

Procedure

  1. Concentrate purified protein on Amicon filters to 20 μl.
  2. Take 20 μl protein solution (~100 μg), add to final of 6 M guanidine HCl, 50 mM Tris-HCl (pH 8.0), 2-4 mM DTT. Heat at 95 °C for 20 min.
  3. Cool the reaction, then add 200 mM TEA. Final guanidine HCl concentration should be below 1 M.
  4. Dissolve a vial of trypsin (20 μg) in 20 μl 50 mM acetic acid.
    a.  Add trypsin to target protein solution in a ratio of 1:50. Incubate at 37 °C for 1 h or longer.
    b.  SpeedVac the reation to dryness, then re-suspend with solvent A in HPLC.

References

  1. Empirical lab protocol from Thermo Fisher.

材料和试剂

  1. 纯化蛋白
  2. 测序级改良的胰蛋白酶(Promega Corporation,目录号:V5113)
  3. 6M盐酸胍
  4. Tris HCl(pH8.0)
  5. 1 M DTT
  6. 将三乙胺(TEA)(Sigma-Aldrich)
  7. HPLC溶剂A(通常为10%乙腈的水溶液)
  8. 乙酸

设备

  1. Amicon Ultra离心过滤器Ultracel 10 k MWCO(EMD Millipore)
  2. SpeedVac
  3. 热块
  4. 高效液相色谱(HPLC)
  5. Amicon过滤器

程序

  1. 将纯化的蛋白质在Amicon过滤器上浓缩至20μl。
  2. 取20μl蛋白溶液(〜100μg),加入到最后的6M盐酸胍,50mM Tris-HCl(pH 8.0),2-4mM DTT中。 在95℃加热20分钟。
  3. 冷却反应,然后加入200mM TEA。 最终胍盐浓度应低于1 M.
  4. 将一瓶胰蛋白酶(20μg)溶于20μl50mM乙酸中 a。  向目标蛋白溶液中加入胰蛋白酶,比例为1:50。 在37℃孵育1小时或更长时间。
    b。  SpeedVac反应至干燥,然后在HPLC中用溶剂A重悬

参考文献

  1. 来自Thermo Fisher的经验实验室方案。
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How to cite this protocol: Li, X. (2011). In-Solution Digestion Of Purified Yeast Protein For LC-MS. Bio-protocol Bio101: e62. DOI: 10.21769/BioProtoc.62; Full Text



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