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Affinity Purification of Yeast Protein-interacting Metabolites for ESI-MS Analysis   

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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, Affinity purification, Yeast, Metabolite extraction, Metabolomics


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.
Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Li, X. (2011). Affinity Purification of Yeast Protein-interacting Metabolites for ESI-MS Analysis. Bio-protocol Bio101: e61. DOI: 10.21769/BioProtoc.61.
Q&A

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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