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[Bio101] Affinity Purification of Yeast Protein-interacting Metabolites for ESI-MS Analysis
[Bio101] 亲和纯化 酵母蛋白-相互作用代谢产物用于电喷雾质谱分析   

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Abstract

The method described here can be used to discover in vivo protein-metabolite interactions. Metabolite-protein complexes are purified from yeast cell lysates by an affinity tag that recognizes the protein of interest. The protein-bound metabolites are extracted for identification by mass spectrometry, while the protein is concurrently analyzed by gel electrophoresis. A parallel experiment using cell lysate without target protein should be used as a negative control. The metabolite extract should be analyzed within 1-2 days to avoid undesired chemical reaction.

Keywords: Protein purification(蛋白纯化), Affinity purification(亲和纯化), Yeast(酵母), Metabolite extraction(代谢产物的提取), Metabolomics(代谢组学)


Materials and Reagents

  1. Cells
  2. 1x phosphate buffered saline (PBS)
  3. Methanol (mass spec grade)
  4. Water (mass spec grade)
  5. 2x laemmli buffer (for SDS-page)
  6. NH4Ac
  7. EGTA
  8. DTT
  9. PMSF
  10. Roche protease inhibitor tablets (Roche Diagnostics)
  11. Lysis buffer (see Recipes)
  12. Wash buffer 1 (see Recipes)
  13. Wash buffer 2 (see Recipes)

Equipment

  1. Zirconia silica beads (Bio Spec Products)
  2. Rabbit IgG-conjugated dynabeads
  3. Eppendorf protein Lobind tubes
  4. FastPrep cell lyser with an adapter for 2 ml tubes
  5. Hula mixer (Life Technologies, InvitrogenTM) or similar product
  6. Magnetic stand for 1.5/2.0 ml tubes
  7. Heat block

Procedure

  1. Add equal volume of 0.5 mm Zirconia silica beads (stored at -20 °C) to cells from 150 ml culture, add in 950 μl lysis buffer, homogenate on FastPrep 24, 3 x 40 sec min at 6.5 m/sec with 2 min interval on ice.
    Note: Wash the IgG Dynabeads 2x in 1x PBS, 3x in lysis buffer. Re-suspend in lysis buffer. Use 50 μl per sample.
  2. Spin down lysate at 14,000 rpm, 10 min, and transfer supernatant (lysate) to 2.0 ml Lobind tubes. Store at 4 °C.
  3. Add 950 μl lysis to the cell pellet and lyse again as step 1.
  4. Repeat step 2 and combine the lysate. Add 50 μl IgG beads.
  5. Incubate 30 min at 4 °C with end-over-end invertion on Hula mixer.
  6. Use magnetic stand to separate beads from lysate.
  7. Wash the beads in 0.8 ml wash buffer 1, and 0.8 ml in wash buffer 2. Transfer beads with wash buffer 2 to a new tube. Each time buffer is added to the beads, invert at 4 °C until homogenate, briefly spin down the beads (10 sec), put on magnetic stand for at least 30 sec, and pipet off the buffer. 
  8. Add 50 μl methanol (MS grade) to the beads, pipette mix, 15 min at room temperature, and separate on magnetic beads, repeat and combine the methanol extracts in MS vials.
  9. Add 30 μl 2x SDS sample buffer to the beads, boil 15 min, load 15 μl on SDS-page for protein yield evaluation.

Recipes

  1. Lysis buffer
    200 mM NH4Ac (stock: 5 M)
    1 mM EGTA (stock: 500 mM EGTA)
    1 mM DTT (stock: 1 M)
    1 mM PMSF (stock: 100 mM in ethanol, 4 °C)
    Roche Protease inhibitor tablets (1x), if no EDTA, add to final 1 mM
  2. Wash buffer 1
    500 mM NH4Ac (stock: 5 M) ligation reaction
  3. Wash buffer 2
    50 mM NH4Ac (stock: 5 M)

References

  1. Li, X., Gianoulis, T. A., Yip, K. Y., Gerstein, M. and Snyder, M. (2010). Extensive in vivo metabolite-protein interactions revealed by large-scale systematic analyses. Cell 143(4): 639-650.

简介

本文所述的方法可用于发现体内蛋白质 - 代谢物相互作用。 通过识别目标蛋白的亲和标签从酵母细胞裂解物中纯化代谢物 - 蛋白复合物。 提取蛋白质结合的代谢物用于通过质谱法鉴定,同时通过凝胶电泳同时分析蛋白质。 使用没有靶蛋白的细胞裂解物的平行实验应该用作阴性对照。 代谢物提取物应在1-2天内分析以避免不期望的化学反应。

关键字:蛋白纯化, 亲和纯化, 酵母, 代谢产物的提取, 代谢组学


材料和试剂

  1. 细胞
  2. 1×磷酸盐缓冲盐水(PBS)
  3. 甲醇(质量等级)
  4. 水(质量等级)
  5. 2x laemmli缓冲液(SDS-PAGE)
  6. NH 4 Ac
  7. EGTA
  8. DTT
  9. PMSF
  10. Roche蛋白酶抑制剂片剂(Roche Diagnostics)
  11. 裂解缓冲液(见配方)
  12. 洗涤缓冲液1(见配方)
  13. 洗涤缓冲液2(见配方)

设备

  1. 氧化锆二氧化硅珠(Bio Spec Products)
  2. 兔IgG偶联的dynabeads
  3. Eppendorf蛋白
  4. FastPrep细胞裂解器与适配器2毫升管
  5. Hula混合器(Life Technologies,InvitrogenTM)或类似产品
  6. 磁力架用于1.5/2.0ml管
  7. 热块

程序

  1. 将等体积的0.5mm氧化锆二氧化硅珠(储存在-20℃)加入到来自150ml培养物的细胞中,加入950μl裂解缓冲液,在FastPrep 24上匀浆,以6.5m/sec以2分钟间隔3×40sec min 在冰上 注意:在1x PBS中洗涤IgG Dynabeads 2x,在裂解缓冲液中洗涤3次。 重悬在裂解缓冲液中。 每个样品使用50μl。
  2. 以14,000rpm,10分钟旋转裂解物,并将上清液(裂解物)转移到2.0ml Lobind管。 储存于4°C。
  3. 加入950微升裂解细胞沉淀和裂解再次作为步骤1。
  4. 重复步骤2并合并裂解液。 加入50μlIgG珠。
  5. 在4℃下孵育30分钟,在Hula混合器上进行端接反转。
  6. 使用磁力架从裂解物分离珠。
  7. 在0.8ml洗涤缓冲液1中洗涤珠子,并在洗涤缓冲液2中洗涤珠子。将洗涤缓冲液2的珠子转移到新试管中。 每次向珠子中加入缓冲液,在4℃下倒置直至匀浆,短暂地向下旋转珠子(10秒),放在磁力架上至少30秒,然后吸取缓冲液。
  8. 加入50微升甲醇(MS级)珠,吸管混合,在室温下15分钟,并在磁珠上分离,重复和结合甲醇提取物在MS小瓶中。
  9. 加入30微升2×SDS样品缓冲液的珠子,煮15分钟,加载15微升在SDS页上蛋白质产量评估。

食谱

  1. 裂解缓冲液
    200mM NH 4 Ac(原料:5μM) 1mM EGTA(原液:500mM EGTA) 1mM DTT(原液:1M) 1mM PMSF(储备液:100mM,在乙醇中,4℃) Roche蛋白酶抑制剂片剂(1x)(如果不含EDTA)加至最终1mM
  2. 洗涤缓冲液1
    500mM NH 4 Aac(原液:5μM)连接反应
  3. 洗涤缓冲液2
    50mM NH 4 Ac(原料:5μM)

参考文献

  1. Li,X.,Gianoulis,T.A.,Yip,K.Y.,Gerstein,M。和Snyder,M。(2010)。 体内广泛的代谢物 - 蛋白相互作用 大规模系统分析。 单元 143(4):639-650。
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:Li, X. (2011). Affinity Purification of Yeast Protein-interacting Metabolites for ESI-MS Analysis. Bio-protocol Bio101: e61. DOI: 10.21769/BioProtoc.61;
提问与回复

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Kyle Hoffman
Western University
Hello,
When detecting molecules, is the mass spec instrument run in positive or negative ion mode?
Thanks,
Kyle
10/5/2014 10:41:10 AM Reply
Xiyan Li
Department of Genetics, Stanford University, USA

You should use both modes to increase the chances. Remember some metabolites can only be detected in one mode.

10/6/2014 9:14:41 AM


Kyle Hoffman
Western University
Hello,
Do you foresee any problems with grinding the cells in liquid nitrogen instead of the bead lysis method?
Thanks,
Kyle
7/28/2014 3:27:30 PM Reply
Xiyan Li
Department of Genetics, Stanford University, USA

Conceptually none, other than a slight chance that some contaminants are introduced in an uncontrollable manner.

7/28/2014 3:32:45 PM


Marie-Laure Erffelinck
VIB
Hi,
How did you dissolve n-DG in water ?
Greetings,
Marie-Laure
2/11/2014 6:25:57 AM Reply
Xiyan Li
Department of Genetics, Stanford University, USA

100x stock solution in ethanol.
This thing precipitates out easily at 4C, be careful.

2/11/2014 9:21:24 AM


Marie-Laure Erffelinck
VIB
Hi,
How does the FastPrep 24 work exactly ? How can it be replaced by some more common lab equipment with comparable results ?
Greetings,
M-L
2/4/2014 9:39:30 AM Reply
Xiyan Li
Department of Genetics, Stanford University, USA

It's basically a bead beater for yeast cell lysis, which is notorious for its sturdy walls. You may find their online video.
Any device capable of breaking the cells without additional biochemical treatment may replace FastPrep.

2/4/2014 9:53:16 AM


Marie-Laure Erffelinck
VIB
Hi,
I notice that you don't use detergents in the lysis buffer. Is there a reason for this ?
Greetings,
M-L
1/21/2014 4:02:31 AM Reply
Xiyan Li
Department of Genetics, Stanford University, USA

Common lab detergents, such as Tween and Triton, are PEG polymers. They are very bad for LC-MS analysis. Some glucoside-based detergents might be useful in this case. I had success with n-dodecyl glucoside in preparing membrane proteins such as yeast PMA1.

1/21/2014 9:06:14 AM