Materials
A UP 200S Sonicator (Dr. Hielscher GmbH, Stuttgart, Germany) was used to homogenate the samples. For the first dimension, immobilized pH gradient (IPG) strips (17 cm, pH 5-8 and 7-10) were purchased from Bio-Rad (Hercules, CA, USA). The pre-fractionation was carried out with a Rotofor Cell (Bio-Rad). The focusing chamber was a Protean Isoelectric Focusing (IEF) Cell (Bio-Rad). For the second dimension a Protean plus Dodeca Cell (Bio-Rad) was used.
Reagents (tris, urea, thiourea, CHAPS, dithiothreitol, bromophenol blue, glycerin, sodium dodecyl sulfate, glycin, temed, ammonium peroxodisulfate, ammonium sulfate, ammonium bicarbonate, colloidal coomassie blue and acrylamide) were purchased from Roth (Karlsruhe, Germany). Iodacetamide was from SERVA (Heidelberg, Germany). Benzonase was purchased from Novagen (Darmstadt, Germany). Ampholytes (biolyte 3-10) were purchased from Bio-Rad. DeStreak was purchased from Amersham Bioscience (Freiburg, Germany).
Animal care
A total of n = 6 C57/Bl6 male mice (aged 6-8 months) weighing 25-33 g were housed in Makrolon® Type III cages. Drinking water and food (V1124-000, SSNIFF, The Netherlands) were given ad libitum. The temperature and relative humidity were 22 ± 2°C and 40-70%, respectively. Furthermore, a 12-h day and night cycle was used. For liver explantation, mice were anesthetized with Ketamin 10% 100 μL/100 g and Xylazin 2% 50 μL/100 g, and after surgical removal the liver was washed until free of blood.
Mouse liver sample preparation
Approximately 0.1 g of the liver sample was ground in a mortar under liquid nitrogen flow. Then, the samples were processed with 0.5 mL of a buffer containing 40 mM tris base, 7 M urea, 4% CHAPS, 100 mM DTT and 0.5% (v/v) biolyte 3-10 first (LB2). The suspensions were homogenized by sonication (3 × 20 s) and after addition of 3 μL of benzonase (endonuclease that degrades DNA and RNA) were incubated at room temperature for 20 min. The samples were then centrifuged at 12,000 g for 20 min. The pellets were washed and sonicated for 5 min with a further 0.5 mL of LB2 and centrifuged at 12,000 g for another 20 min, and the resulting two fractions of supernatant were collected (extract A) (Figure 1). Finally, the pellets were dissolved with 0.5 mL of buffer containing 40 mM tris base, 5 M urea, 2 M thiourea, 4% CHAPS, 100 mM DTT, 0.5% (v/v) biolyte 3-10 (LB3), sonicated and centrifuged at 12,000 g for 20 min. The pellet was collected, and the supernatant was marked as extract B (Figure 1).
From the same animals, a further 0.1-g portion was ground in a mortar, but treated this time with 0.5 mL of LB3. The suspensions were sonicated, incubated with benzonase and centrifuged. The pellets were then washed with another 0.5 mL of LB3, sonicated and centrifuged, and the supernatants were collected (extract C) (Figure 1).
Proteome mapping was done under a variety of conditions, e.g. extraction with lysis buffers 2 and 3. In addition, proteins were separated at two different pH ranges (5-8 and 7-10). A total of 4 experiments were carried out, and duplicate measurements were run for each experiment (Figure 2). Overall approximately 9600 spots, cut from 24 gels, could thus be investigated. The protein concentration of all extracts was determined using the Bradford assay.
Liquid-phase IEF pre-fractionation
The liquid-phase IEF pre-fractionation was performed in the Rotofor Cell system (Bio-Rad) following the instructions of the supplier. Ion exchange membranes were equilibrated overnight in the appropriate electrolyte (anion exchange membranes in NaOH 0.1 M and cation exchange membranes in H3PO4 0.1 M). After four runs, ion exchange membranes were always discarded and replaced by new membranes for the other samples. For each run, the electrode chambers were filled with fresh appropriate electrolytes (30 mL). Initially, the cell was filled with pure water and run for 5 min at 5 W constant power to remove residual ionic contaminants from the membrane core and ion exchange membranes. Approximately 32 mL of LB2 were used to fill the cell. A total of 60 mg of total proteins in approximately 2 mL of LB2 were added to the cell to reach the maximum loadable volume (40 mL). Focusing started at 12 W constant power. After approximately 4 h, the voltage increased to 3000 V and the wattage decreased to 3 W. The focused proteins were harvested in 20 ~ 1.5 mL fractions, and pH values were checked. Fractions having pH values between 3 and 7.0 were collected and denoted as "A-a" (acid) (Figure 2). Fractions having pH values > 7.0 were collected and denoted as "A-b" (basic) (Figure 2). Again the protein concentration was determined for both fractions (A-a and A-b) by the Bradford method. Approximately 30 mg of protein were recovered at the end of the liquid-phase IEF pre-fractionation from an initial 60 mg load. The losses are accounted for by the multi-step pre-fractionation procedure, but are not the result of a precipitate that could not be dissolved in our lysis buffer. Other investigations have reported similar losses during pre-fractionation; for instance, see Fountoulakis et al. [18], or P.G. Righetti et al. [19]. After each run, the membrane core was cleaned with NaOH 0.1 M overnight and sonicated for 5 min in water before the new focusing.
Two-dimensional gel electrophoresis
Isoelectric focusing (IEF) - first dimension
IEF was performed using precast linear IPG strips. The 17-cm IPG strips 7-10 and 5-8 were loaded with 1.5 mg of proteins by active rehydration (12 h, 50 V). Samples destined to be separated by IPG strips 7-10 received an excess of hydroxyethyl disulfide (HED) (DeStreak™) prior to the focusing run. Focusing began at 250 V for 20 min in rapid mode, increasing to 10,000 V for 5 h in linear mode and 10,000 V for 50,000 Vh in rapid mode (for the IPG strips 5-8). IEF for the strips 7-10 was carried out at 250 V for 60 min in rapid mode, then at 10,000 V for 3 h in linear mode and at 10,000 V for 50,000 Vh in rapid mode. Each sample was analyzed in duplicate (Figure 2).
2-DE - second dimension
After IEF, the IPG strips were either stored at -80°C or transferred to 10 mL of equilibration buffer (6 M urea, 30% w/v glycerin, 2% w/v SDS, 50 mM Tris-HCl pH 8.8) with 2% w/v DTT and 0.5% v/v bromophenol blue solution (0.25% w/v bromophenol blue, 1.5 M Tris-HCl pH 8.8, 0.4% w/v SDS) and incubated for 20 min at room temperature. Strips were removed and incubated in equilibration buffer with 4% w/v iodoacetamide and 0.5% v/v bromophenol blue solution for further 20 min at room temperature. Finally, the strips and 10 μL SDS-PAGE molecular weight standard on filter paper were placed on top of the 20 cm × 20.5 cm 12% second-dimension gel (12% v/v acrylamide/bis solution, 375 mM Tris, pH 8.8, 0.1% v/v SDS, 1/2000 TEMED, 0.05% v/v APS). Both were fixed in place with a 0.5% w/v agarose overlay. Gels were run in PROTEAN Plus Dodeca cell from Bio-Rad at 70 V for approximately 14 h, followed by 200 V until the bromophenol blue dye reached the bottom of the gel. The running buffer (25 mM Tris, 0.2 M glycin, 0.1% SDS) was cooled externally to 16°C.
Gels/proteins were fixed overnight in 30% ethanol and 2% phosphoric acid and washed 3 × 20 min with 2% phosphoric acid. The gels were equilibrated with 15% ammonium sulfate, 18% ethanol and 2% phosphoric acid for 15 min and finally stained with colloidal coomassie blue for 48 h.
Gel scanning and image analysis
After staining, gels were washed 10 min with pure water and scanned on a Molecular FX Scanner Bio-Rad at 100 μm resolution. Protein spots were imaged first automatically and then manually and analyzed using the PDQuest™ software Bio-Rad. The normalization was carried out in total density in gel mode according to the manufacturer's recommendation.
Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS)
A total of 9600 spots derived from 24 gels were excised using the spot cutter of Bio-Rad and placed into 96-well microtiter plates. Excised gel spots were washed manually with 20 μL of water for 10 min and destained twice, first with 15 μL ammonium bicarbonate 50 mM for 5 min and then with 15 μL 50% ammonium bicarbonate 50 mM - 50% acetonitrile for 5 min. Finally, the gel particles were covered by acetonitrile until gel pieces shrunk and left to dry for 10 min. All gels/proteins were digested manually in situ with 4 μL of ammonium bicarbonate 50 mM containing 20 ng trypsin (Sequencing Grade Modified Trypsin Promega). After 15 min, each gel piece was re-swelled with 10 μL of ammonium bicarbonate 50 mM and incubated for 4 h at 37°C. After 4 h, the reaction was stopped by adding 10 μL of trifluoroacetic acid 1% containing 1.5% (w/v) n-octyl-β-D-glucopyranoside (OGP) (AppliChem). For the application of the samples, 4 μL of peptide solution were loaded on an MTP Anchor Chip Target 600/384 (Bruker Daltonics) previously prepared with a saturated solution of matrix, α-cyano-4-hydroxy-cinnamic acid (α-HCCA) (Bruker Daltonics). An external calibration was performed by spotting on the 96 calibration positions of the Anchor Chip Target 1 μL of the peptide calibration standards (Bruker Daltonics) containing the following peptides: angiotensin II (1046.5420 Da), angiotensin I (1296.6853 Da), substance P (1347.7361 Da), bombesin (1619.8230 Da), ACTH clip 1-17 (2093.0868 Da), ACTH clip 18-39 (2465.1990 Da), somatostatin 28 (3147.4714 Da) and OGP 1.5% (w/v). Samples were analyzed in a MALDI-TOF-TOF spectrometer (Ultraflex, Bruker Daltonics) using an accelerating voltage of 25 kV for the Peptide Mass Fingerprint (PMF) mode. When necessary, MALDI-Post Source Decay (PSD) analysis was carried out using the LIFT special technique delivered by Bruker (the basic idea of LIFT is to lift the potential to fragment the selected peptides of interest). Peptide matching and protein searches were performed automatically with the MASCOT software. For the PMF search the parameters were the following: C-carbaimidomethyl (fixed modification), M-oxidation (variable modification), monoisotopic (mass value), 100 ppm (peptide mass tolerance), 1 (max missed cleavage), mammalia (taxonomy). Five matching peptides and at least 10% peptide coverage of the theoretical sequences was the minimal requirement for an identity assignment. For the MS/MS search (PSD) the parameters were the same except the peptide mass tolerance, which was 200 ppm. The identified proteins were organized with the ProteinScape™ database (Bruker Daltonics) and checked individually and only mouse proteins or highly homologous sequences from other mammalian species, like Homo sapiens or Rattus Norvegicus, having pI and Mw values close to the theoretical, were considered (a total of n = 39 proteins).
Optimization of the IEF for proteins in the basic pH range
Isoelectric focusing of proteins in the acidic and basic pH ranges is often associated with streaking, due to disappearance of the reducing agent, normally DTT, from the basic part of the IPG strip, followed by oxidation of the protein thiol groups, resulting in a heterogeneous mixture of inter and intra chain -S-S- bonds and causing a train of extra artifactual spots.
To decrease the streaking of basic proteins we tested: a) different IEF programs, b) the use of an electrode paper pad at the cathode as a source of DTT during focusing and c) the use of hydroxyethyl disulfide (DeStreak™).