Proteomics Biochemistry

scientificprotocols authored about 3 years ago

Authors: Thomas Köcher, Peter Pichler, Remco Swart & Karl Mechtler

Abstract

A protocol for the generation of a tryptic peptide mixture from HeLa cell culture is described. The primary purpose of the sample is to evaluate, optimize and validate LC-MS/MS procedures.

Reagents

  1. Acetone, Sigma-Aldrich Caution! It is flammable! Handle it a fume hood and wear gloves and safety goggles
  2. Ammoniumbicarbonate, Sigma-Aldrich
  3. Beta-glycerophosphate, Sigma-Aldrich
  4. Chymostatin, Sigma-Aldrich
  5. DL-Dithiothreitol (DTT), Sigma-Aldrich
  6. Dimethyl sulfoxide (DMSO), Fluka
  7. Ethylenediaminetetraacetic acid (EDTA), BioChemika
  8. Glycerol, BioChemika
  9. High glucose Dulbecco’s modified Eagle’s medium (DMEM), (Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum, (Gibco, Invitrogen)
  10. Iodoacetamide, Sigma-Aldrich Caution! It is toxic and corrosive. Handle in a fume hood and wear gloves and safety goggles.
  11. KCl, Sigma-Aldrich
  12. KOH, Merck
  13. Leupeptin, Sigma-Aldrich
  14. L-glutamine, Sigma-Aldrich
  15. Lys-C, Wako Chemicals
  16. N-(2-Hydroxyethylpiperazine-N’-(2-ethanesulfonic acid) (HEPES), Sigma-Aldrich
  17. NaF, Sigma-Aldrich
  18. Na-pyrophosphat, Sigma-Aldrich
  19. Na3VO4, Sigma-Aldrich
  20. Nocodazol, Sigma-Aldrich
  21. Penicillin, Sigma-Aldrich
  22. Pepstatin, Sigma-Aldrich
  23. Phenylmethylsulfonylfluorid, Sigma-Aldrich
  24. Streptomycin, Sigma-Aldrich
  25. Trifluoroacetic acid (TFA), Pierce Caution! It is corrosive. Handle in a fume hood and wear gloves and safety goggles.
  26. Triton X-100, Fluka
  27. Trypsin Gold Mass spectrometry grade, Promega
  28. Urea, Sigma-Aldrich

REAGENT SETUP

HeLa culture medium High glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, 0.2 mM L-glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin.

Protease Inhibitor Mix (1000× stock solution) 10 mg/mL each of leupeptin, pepstatin and chymostatin, in DMSO

Lysis buffer 50 mM HEPES-KOH pH 7.5; 5 mM EDTA, 15mM KCl; 10% glycerol, 1% Triton X-100, 20 mM beta-glycerophosphate 10 mM Na-pyrophosphat, 10 mM NaF, 1 mM DTT, Na3VO4 (200mM), 0,1 mM Phenylmethylsulfonylfluorid, and 2 x Protease Inhibitor Mix.

Procedure

  • HeLa Protein Preparation
  • Grow HeLa Kyoto cells in DMEM and arrest the growth with nocodazol over night.
  • Harvest cells with a scraper and wash three times with PBS. One tray (25 cm x 25 cm; 100 mL medium) generates approximately 4 mg of protein.
  • Suspend the cell pellet in lysis buffer using twice its volume and disrupt the cell membrane by pulling the suspension through a thin needle (first 21 then 27 gauge). Centrifuge the suspension at 500 g in a cooling centrifuge (4°C) for 15 min.
    • Acetone precipitation of proteins
  • Add 5 times the volume of chilled acetone to the protein solution and incubate the sample at minus 30 °C.
  • Centrifuge the suspension at 500 g in a cooling centrifuge (4°C) for 30 min.
  • Discard the supernatant and carefully suspend the protein pellet with 80% chilled acetone and repeat the centrifugation.
  • Air-dry the pellet avoiding complete desiccation and dissolve the protein pellet in the digestion buffer (8 M urea, 0.5 M ammoniumbicarbonat) to a protein concentration of 5 µg/µL. The protein solution should have a pH of 8.0. in our laboratory we measure protein concentration using a Bradford assay.
    • Reduction and alkylation of proteins
  • Add 1 µg DTT stock solution up to a final concentration of 0.05 µg DTT per µg protein and incubate the sample for 30 min at 56 °C, shaking.
  • Next add iodoacetamide to a final concentration of 0.25 µg per µg protein and incubate the sample for 30 min at room temperature in the dark.
  • Quench the reaction by adding DTT stock solution up to a final concentration of 0.25 µg DTT per µg protein
    • Digestion with Lys-C
  • Dilute the sample to 6 M urea with 50 mM ammoniumbicarbonat buffer and add Lys-C to a final concentration of 1 µg per 50 µg protein.
  • Incubate the sample at 30 °C for 2 h. Next, digest the predigested protein sample with trypsin starting with diluting the solution to 0.8 M urea with 50mM ammoniumbicarbonat buffer.
  • Add 1 µg trypsin per 60 µg protein and incubate the sample for 2 h at 37 °C.
  • Add another aliquot of 1 µg trypsin per 60 µg protein and incubate the sample at 37 °C over night. The digest is stopped by acidifying the sample to a pH of 2 by adding TFA.
  • Peptide mixtures should and stored in aliquots at minus 80º C.

Associated Publications

Analysis of protein mixtures from whole-cell extracts by single-run nanoLC-MS/MS using ultralong gradients. Thomas Köcher, Peter Pichler, Remco Swart, and Karl Mechtler. Nature Protocols 7 (5) 882 - 890 doi:10.1038/nprot.2012.036

Author information

Thomas Köcher & Karl Mechtler, Research Institute of Molecular Pathology (IMP), Vienna, Austria

Peter Pichler, Christian Doppler Laboratory for Proteome Analysis, University of Vienna, Vienna, Austria

Remco Swart, Dionex Corporation, Amsterdam, Netherlands

Correspondence to: Thomas Köcher ([email protected]), Karl Mechtler ([email protected])

Source: Protocol Exchange (2012) doi:10.1038/protex.2012.001. Originally published online 12 April 2012.

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