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 - 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. ### Procedure - **HeLa Protein Preparation** 1. 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](http://dx.doi.org/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 (Thomas.Koecher@imp.ac.at), Karl Mechtler (Karl.Mechtler@imp.ac.at) *Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/2274#/reagents) (2012) doi:10.1038/protex.2012.001. Originally published online 12 April 2012*.