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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

Systems Biology > Proteomics > Whole organism
Author: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford , USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
4/20/2011, 7263 views, 0 Q&A, How to cite
DOI: http://dx.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, LC-MS, Sample preparation, In-solution digestion, Yeast

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.


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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