RPMI-1640, FCS containing LPS concentrations of either < 1 EU/mL (< 0.1 to 0.2 ng/mL) or < 30 EU/mL (< 3 to 6 ng/mL), Dulbecco's phosphate buffer saline (PBS), penicillin and streptomycin were obtained from PAA Laboratories, Colbe, Germany. Urea, thiourea, dithiothreitol (DTT), trypsin, triflouroacetic acid (TFA), acitonitrile (ACN) and ammonium bicarbonate (AMBIC) were from Sigma-Aldrich, Steinheim, Germany. CHAPS buffer was from AppliChem, Darmstadt, Germany, and ampholeytes, protein assay reagents, Immobilized pH gradient strips (IPG strips) were provided by Bio-Rad, Munich, Germany. Protease and phosphatase inhibitor cocktail were from Roche, Mannheim, Germany. Bromophenol blue and Tris base were from Carl Roth, Karlsruhe, Germany, and sodium dodecyl sulfate (SDS) was from Serva, Heidelberg, Germany. Glycerin, potassium ferricynaide and sodium thiosulfate were from Merck, Darmstadt, Germany and formic acid from BASF, Ludwigshafen, Germany. Superoxide dismutase 2 (SOD2) antibodies was a gift from Dr. Dihazi, UMG, Goettingen, Germany. β-tubulin antibody was from BioVendor, Heidelberg, Germany and antibodies to HRP labelled anti-mouse secondary antibodies were from Bio-Rad, Munich, Germany.
Human T lymphoblastic leukaemia cells (CCRF-CEM) were purchased from DSMZ (German collection of microorganisms and cell cultures, Braunschweig, Germany). Cells were grown in 75 cm2 culture flasks (Sarstedt, Numberecht, Germany) in RPMI-1640 medium containing L-glutamine, 10% FCS, 100,000 U/L penicillin and 100 μg/L streptomycin, in 95% humidity and 5% CO2 conditions at 37°C.
Heat inactivation and LPS treatment of cultured cells
FCS was heated at 56°C for 30 minutes before adding it to the RPMI-1640 medium. CCRF-CEM cells were grown in RPMI-1640 medium supplemented either with (a) FCS without heat inactivation and a normal concentration of LPS (NHE), (b) FCS with heat inactivation containing a normal concentration of LPS (HE), (c) FCS without heat inactivation having a low concentration of LPS (NHL), or (d) heated FCS with low concentration of LPS (HL). The cells were adapted in RPMI-1640 medium supplemented with four different FCS concentrations for at least five passages before starting the first harvest. The cells were grown to a density of 0.25 × 106 cells/mL under recommended conditions i.e., 37°C, 95% humidity, 20% O2, 5% CO2 and the medium was changed every second day. All experiments were repeated six times.
Cell lysis and protein estimation
Cells were washed with ice cold PBS and lysed in lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 2% ampholytes [pH 3-10], 1% DTT, 1% protease inhibitor and 1% phosphatase inhibitor cocktail). Protein concentration was measured as described by Bradford (1976) using serum albumin as a standard .
Sample preparation and two-dimensional gel electrophoresis (2-DE)
2-DE was performed as described by Gorg et al . Briefly, a 160 μg protein sample was diluted in rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.2 ampholytes [pH 3-10], 0.2% DTT and 0.25% bromophenol blue) were applied on immobilized pH gradient strip (IPG strip, 17 cm) with a non-linear pH range of 3-10 at room temperature overnight for passive rehydration. Isoelectric focusing was performed with a Bio-Rad Protean electrophoresis apparatus set to final 32000 Volts hour. The IPG strip was then equilibrated for 20 minutes in equilibration buffer (6 M urea, 30% glycerol, 2% SDS, and 50 mM Tris-HCl [pH 8.8]) containing DTT (10 g/L) and then subsequently immersed for 20 minutes in fresh equilibration buffer containing iodoacetamide (40 g/L). Following equilibration, proteins were separated by SDS-PAGE at a constant voltage of 100 Volts using a 12.5% polyacrylamide separation gel at 4°C .
Phospho-specific staining of 2-DE gels
The gels were fixed twice in solution containing 50% methanol and 10% acetic acid for 45 minutes and washed three times in double distilled water for 15 minutes each. Gels were incubated in Pro-Q Diamond phospho-stain (Invitrogen, Paisley, UK) overnight in the dark at room temperature, destained three times for 30 minutes in 20% ACN and 50 mM sodium acetate, followed by three washes in double distilled water for five minutes each. Gels were scanned using an imaging instrument (FLA -5100 Fuji photo film, Dusseldorf, Germany) at a wavelength of 532 nm.
Visualization of proteins and densitometric analysis
Proteins were visualized by silver staining, as described by Blum et al , immersed in a fixative solution (50% methanol and 12% acetic acid) for one hour and washed in 50% and 30% ethanol for 20 minutes each. Gels were sensitized in 0.02% sodium thiosulfate for 60 seconds and washed three times in water. Staining was done in silver solution (0.2% silver nitrate, 0.026% formaldehyde) for 20 minutes, followed by three washings in water. All gels were developed in a solution containing 6% sodium carbonate, 0.0185% formaldehyde and 6% sodium thiosulfate until spots appeared and the reaction was stopped by adding the stop solution (50% methanol and 12% acetic acid). Gels were scanned (CanoScan 8400F, Canon, Krefeld, Germany) dried (Gel Drier, Bio-Rad, Munich, Germany), and subjected to densitometric analysis using the Delta2D software version 4.0 (DECODON, Greifswald, Germany).
Differentially expressed spots were excised and in-gel digested according to the method described by Shevchenko and colleagues . Briefly, sliced gel spots were destained with 30 mM potassium ferricyanide and 100 mM sodium thiosulfate; followed by washing with 50% ACN and 100 mM AMBIC, which was then removed and dried in a vacuum centrifuge (UNIVAPO, uniEquip, Matinsried, Germany). The gel pieces were digested with trypsin digestion buffer (0.1 μg/μl trypsin, 1 M calcium chloride, and 1 M AMBIC) for 45 minutes on ice and then incubated overnight in digestion buffer without trypsin at 37°C. The peptides were extracted with increasing concentrations of ACN and TFA in several rounds and the extracted peptides were dried by vacuum centrifugation. Peptides were reconstituted in 0.1% FA for injection into a nano-flow HPLC.
Peptide sequence analysis using nano LC ESI Q-TOF MS/M and database search
Peptide samples (1 μl) were introduced onto two consecutive C18-reversed phase chromatography columns (C18 pepMap: 300 μm × 5 mm; 5 μm particle size, and C18 pepMap100 nanoanalytical column: 75 μm × 15 cm; 3 μm particle size; LC Packings, Germering, Germany) using a nano-flow CapLC autosampler (Waters, Eschborn, Germany). Peptides were eluted with an increasing gradient of ACN and analyzed on a Q-TOF Ultima Global mass spectrometer (Micromass, Manchester, UK) equipped with a nanoflow ESI Z-spray source in the positive ion mode, as previously described . The data were analyzed with the MassLynx (version 4.0) software. The peaklists were searched using the online MASCOT search engine (http://www.matrixscience.com) against the UniProt/SwissProt database release 15.15 (515203 entries, 181334896 elements). The data were searched against the database with following parameters: trypsin as enzyme for digestion; up to a maximum of one missed cleavage site allowed; monoisotopic mass value and with unrestricted protein mass; peptide tolerance ± 0.5Da and MS/MS tolerance ± 0.5Da. Proteins were identified on the basis of two or more peptides, whose ions score exceeded the threshold, p < 0.05 which reflects the 95% confidence level for the matched peptides.
SDS-PAGE and Western blotting
Samples were resolved on 12.5% SDS-PAGE and electro-transferred using a semi-dry transblot system (SD transblot, Bio-Rad, Munich, Germany) onto PVDF membrane (Millipore, Schwalbach, Germany) at 17 Volts in a transfer buffer (192 mM glycine, 10% methanol, and 25 mM Tris [pH 8.3]) for 30 minutes. The membrane was blocked with 5% skimmed milk powder prepared in TBS-T buffer (50 mM Tris-HCl [pH 7.5], 200 mM NaCl, and 0.05% Tween 20) for one hour at room temperature and washed three times with TBS-T buffer. Membrane was incubated with Anti-SOD2, or anti-β tubulin antibody prepared in 5% skimmed milk powder for overnight at 4°C. After three washes in TBS-T for five minutes each, the membrane was incubated in HRP labelled anti-mouse secondary antibody for one hour at room temperature. Followed by subsequent washes, the signal on the blot was detected using an enhanced chemiluminescent (ECL) reagent (GE Healthcare, Munich, Germany) and developed on Amersham Hyperfilm (GE Healthcare, Munich, Germany). Signal intensities from each immunoblot were quantified using Lab Image software version 2.71 (Kapelan, Leipzig, Germany).
Densitometric analysis of protein spots from silver and phospho-stained gel were performed using Delta2D software. Protein spots, which showed ≥ 1.5 fold change in phosphorylation signal and consistently statistically significant (p < 0.05) using the Student's t-test in at least six independent 2-DE experiments, were selected for in-gel digestion and identified using ESI Q-TOF MS/MS analysis. Error bars in results represent mean ± SD. Immunoblot intensities were quantified using LabImage software (Kapelan, Leipzig, Germany). Immunoblotting was repeated at least three times and results were expressed as mean ± SD with significance measured using the Student's t-test (p < 0.05).