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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, 7739 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.        纯化的蛋白

2.        Sequencing grade Modified Trypsin (V5113, Promega)

3.        Amicon Ultra centrifuge filters Ultracel 10k MWCO   (Millipore)

4.        盐酸胍6M

5.        Tris HCl pH8.0

6.        1 M DTT

7.        三乙胺 (TEA, Sigma)

8.        高效液相色谱溶液A (通常是10%已腈溶于水)

 

设备

 

1.        真空离心蒸发浓缩器

2.        加热块

 

步骤

 

1.        Amicon filters上浓缩纯化的蛋白至20 ul

2.        0 ul 的蛋白溶液(~100 ug)加到终浓度为6 M 盐酸胍,50 mM Tris-HCl pH8.0, 2-4 mM DTT.  95°C 加热20分钟.

3.        冷却反应,然后加入200 mM TEA (三乙胺).  最终的盐酸胍的浓度应该小于1M.

4.        20 ul 50 mM 乙酸中溶解20 ug胰蛋白酶.

1)     按照1:50比例将胰蛋白酶加入到目的蛋白中. 37°C培养1小时或更久.

2)     在真空离心蒸发浓缩器上将样品处理到干燥,然后用溶液A重悬上高效液相色谱仪.

 

参考文献

 

1.        Internal lab protocol from Thermo Fisher.

 

 

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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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