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.
Method Article
Preparation of HeLa peptides for LC-MS
https://doi.org/10.1038/protex.2012.001
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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.
Acetone, Sigma-Aldrich Caution! It is flammable! Handle it a fume hood and wear gloves and safety goggles
Ammoniumbicarbonate, Sigma-Aldrich
Beta-glycerophosphate, Sigma-Aldrich
Chymostatin, Sigma-Aldrich
DL-Dithiothreitol (DTT), Sigma-Aldrich
Dimethyl sulfoxide (DMSO), Fluka
Ethylenediaminetetraacetic acid (EDTA), BioChemika
Glycerol, BioChemika
High glucose Dulbecco’s modified Eagle’s medium (DMEM), (Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum, (Gibco, Invitrogen)
Iodoacetamide, Sigma-Aldrich Caution! It is toxic and corrosive. Handle in a fume hood and wear gloves and safety goggles.
KCl, Sigma-Aldrich
KOH, Merck
Leupeptin, Sigma-Aldrich
L-glutamine, Sigma-Aldrich
Lys-C, Wako Chemicals
N-(2-Hydroxyethylpiperazine-N'-(2-ethanesulfonic acid) (HEPES), Sigma-Aldrich
NaF, Sigma-Aldrich
Na-pyrophosphat, Sigma-Aldrich
Na3VO4, Sigma-Aldrich
Nocodazol, Sigma-Aldrich
Penicillin, Sigma-Aldrich
Pepstatin, Sigma-Aldrich
Phenylmethylsulfonylfluorid, Sigma-Aldrich
Streptomycin, Sigma-Aldrich
Trifluoroacetic acid (TFA), Pierce Caution! It is corrosive. Handle in a fume hood and wear gloves and safety goggles.
Triton X-100, Fluka
Trypsin Gold Mass spectrometry grade, Promega
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.
HeLa Protein Preparation
Acetone precipitation of proteins
Reduction and alkylation of proteins
Digestion with Lys-C
The authors declare no competing financial interests
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted
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