Trypsin enzyme

The sarcoplasmic proteins (50μg) were digested in a solution of 8 M urea (1:1) followed by 25 minutes of incubation with 10 mM DTT at 56°C. Alkylation was performed by adding 14 mM iodoacetamide (IAA) during 30 minutes at room temperature, avoiding light exposure. To quench free IAA, samples were reincubated for 15 minutes with 5 mM DTT, and subsequently, 1 mM calcium chloride was added to the samples. The hydrolysis occurred in the presence of trypsin enzyme (Promega, Madison, WI) (1:50 enzyme/substrate) for 18 hours at 37°C. Trypsin was inactivated with 0.4% formic acid solution. The samples were desalinated using Sep-Pak Vac C18 cartridges (Waters, Milford, MA, USA). The peptide fragments were reduced with vacuum centrifuge and kept at -20°C until analysis by mass spectrometry.

Unless indicated otherwise all chemicals were purchased from Sigma-Aldrich (Steinheim, Germany), Merck (Darmstadt, Germany), Applichem Biochemica (Darmstadt, Germany), Carl Roth (Karlsruhe, Germany) Roche Applied Biosystems (Mannheim, Germany), Amersham Biosciences (Piscataway, NJ, USA), BD Biosciences (San Diego, CA, USA), Gibco (Thermo Fisher Scientific, Germany), Invitrogen (Darmstadt, Germany) or Fluka (Buchs, Switzerland). N3a and HSP90-inhibitor 17-AAG were obtained from Cayman Chemical (Ann Arbor, MI, USA). mTOR inhibitor, Rapamycin and PI3K/mTOR inhibitor, LY292004 were purchased from Calbiochem (San Diego, CA, USA). Protease inhibitor cocktail and the BCA kit were obtained from Pierce Thermo Fisher Scientific (Schwerte, Germany) and the phosphatase inhibitor cocktails I/II A.G. Scientific, Inc, (San Diego, CA, USA). Benzonase Nuclease was purchased from Novagen, Merk (Darmstadt, Germany). The lysis buffer used was homemade RIPA Buffer: 150 mM NaCl, 1.0% w/v IGEPAL^® CA-630, 0.5% w/v sodium deoxycholate, 0.1% w/v SDS, and 50 mMTris, pH 8.0. Bradford reagent was from Bio-Rad (Hercules, CA, USA). Western blot analyses were conducted using Bio-Rad reagents unless indicated otherwise. The ICPL-labeling kit came from Serva Electrophoresis (Heidelberg, Germany) and trypsin enzyme from Promega (Mannheim, Germany).

To identify proteins separated by 2-DE, selected protein spots were manually excised from silver stained 2-DE gels. Then the gel slices washed with double distilled water were destained with 100 mM sodium thiosulfate and 30 mM potassium ferricyanide (1:1). The sample was then vortexed for 10 min, washed with distilled water for 3–5 times until completely destained and dehydrated for 10 min with 100% acetonitrile (ACN) and dried by vacuum centrifugation. After destaining, the gel pieces were reduced with 10 mM dithiothreitol in 100 mM NH₄HCO₃ for 1 hour at 56°C and again incubated with 55 mM iodoacetamide in 100 mM NH₄HCO₃ in the dark for 40 min. The gel slices were digested in 100 mM NH₄HCO₃ with 7–8 μL (0.1 μg/μL) trypsin enzyme (Promega Corporation, Madison, WI 53711–5399, USA) and incubated at 37°C for 16 hours. The tryptic peptides were extracted from the gel grains with 5% trifluoroacetic acid (TFA) in 50% acetonitrile 3 times. The solution containing eluted peptide was concentrated up to drying by vacuum centrifugation and the resultant extracts were analyzed by mass spectrometry.