Mitochondrial proteomics

3.7.1.1 Preparation of mitochondrial protein lysates

Mitochondrial protein lysates from hippocampal tissue of adult HR and LR mice were gained according to the subcellular fractionation and protein extraction protocol described by Cox and Emili in Nature Protocols (Cox and Emili, 2006). According to this, hippocampal tissue (around 30 mg) was slowly thawed from -80 °C and homogenized on ice using a plastic pestle tightly fitting a 1.5 ml Eppendorf tube. After homogenization, 180 µl 250-STMDPS buffer (Table 2) were added to the hippocampal tissue and the tissue homogenate was dissolved in the buffer by slightly grinding for 3 min. The completely dissolved homogenate was centrifuged for 15 min at 80 g, supernatant was collected and stored on ice. The pellet (volume around 80 µl) was dissolved in 640 µl 250-STMDPS buffer and rehomogenized for 1 min using the plastic pestle. A second centrifugation step for 15 min at 80 g followed and the supernatant was collected again, the pellet was discarded. To pellet the mitochondrial protein fraction, the first and the second supernatant from one hippocampal tissue sample were collected and centrifuged in separate tubes for 15 min at 4,500 g. The mitochondrial pellets from one sample were resuspended in 100 µl 250-STMDPS buffer, centrifuged for 15 min at 4,500 g to repellet the mitochondrial fraction. After resuspension of the pellet in 100 µl HDP buffer (Table 2), the suspension was incubated on ice for 30 min. Samples were sonicated on ice usingfive bursts each of the Branson Sonifier Cell Disruptor B15 (Hielscher Ultrasonics, Teltow) to support protein lyses. After spinning the samples for 30 min at 10,000 g, the supernatant containing the soluble fraction of the mitochondrial matrix proteins was collected. In order to achieve an extraction of mitochondrial membrane proteins, the pellet was resuspended in 100 µl ME buffer (Table 2) and incubated for 30 min at 4 °C while gently rocking the samples using an Elmi Rotamix RM1 (ELMI, Riga, Latvia). A final centrifugation step was conducted for 30 min at 10,000 g. The resulting supernatant contained the fraction of mitochondrial membrane proteins. Both mitochondrial proteins fractions, the matrix protein fraction and the mitochondrial membrane proteins, were merged and protein concentration was determined via a Bradford assay.

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3.7.1.2 Bradford assay

5 µl of the mitochondrial protein sample were mixed with 795 µl ddH2O. A standard curve consisting of a BSA dilution series (2, 4, 6, 8, and 10 µg/ml) was pipetted, and both the diluted protein samples and the BSA standards were mixed with 200 µl Bradford reagent (Bio-Rad Laboratories, München). Absorption was detected within 30 min at 595 nm using a Beckman DV640 Spectrophotometer (Beckman Coulter, Krefeld). Samples as well as standards were measured in duplicated and mean values were used for further calculations.

3.7.1.3 Two-dimensional gel electrophoresis

After determination of protein concentrations, 200 µg mitochondrial protein lysates were diluted in IEF buffer (Table 2) to a total volume of 250 µl. The mitochondrial protein samples were centrifuged for 20 min at 24,000 g and subsequently loaded on a ReadyStrip IPG Strip (Bio-Rad Laboratories, Hercules, CA, USA). 200 µl of the samples were loaded on an IPG strip and incubated for 1 h before overlaying it with 3 ml mineral oil (Bio-Rad). After rehydrating the IPG strips in the IEF-tray (Bio-Rad) for 12 h, wicks (Bio-Rad), which were humidified with 20 µl ddH2O, were placed between IPG strips and electrodes to absorb excess salt. The first dimension, which separates the proteins contained in the lysate according to the isoelectric point of the proteins, was run for 8 - 9 h with a final voltage of 8,000 V.

In the second dimension, the proteins already separated along the isoelectric gradient, were additionally segregated along a size gradient. One criterion gel per sample was therefore prepared using a 12% separating gel consisting of 12 ml acrylamide/bis- acrylamide (37.5:1; Serva Electrophoresis, Heidelberg), 1.5 M Tris-HCl 7.5 ml, 10% SDS 300 µl, Temed 15 µl, 10% APS 150 µl (all Bio-Rad), 10.06 ml ddH2O and a 4% stacking gel consisting of acrylamide/bis-acrylamide (37.5:1) 1.98 ml, 0.5 M Tris-HCl 3.78 ml, 10% SDS 150 µl, Temed 15 µl, 10% APS 75 µl and 9 ml ddH2O. During polymerization of criterion gels, IPG strips were equilibrated for 15 min in equilibration buffer I and subsequently for 10 min in equilibration buffer II. After equilibration, IPG strips were overlaid with running buffer, inserted between the spacer glass plates above the stacking phase of the criterion gel, and additionally covered with pre-warmed 0.5% low-melting agarose solution (Bio-Rad). A placeholder for the protein ladder (Precision Plus Protein Standard; Bio-Rad) was formed. The gel was initially run at 80 V, voltage was increased to 120 V after 10 min for approximately 1.5 h.

For protein staining in colloidal coomassie, the gel was fixed in 100 ml 30% ethanol containing 2% phosphoric acid (Merck) overnight and afterwards washed in ddH2O three

45 times for 1 h, and incubated in 120 ml staining solution for 1 h. The two-dimensional (2D) gels were left in the colloidal coomassie staining solution (Table 2) rocking for three days on a Rotamax 120 shaker (Heidolph Instruments, Schwabach).

Table 2: List of buffers and staining solutions used for protein based protocols.

Buffer Function Composition (filter-sterilized, stored at 4 °C) 250-STMDPS Initial homogenization 1 mM DTT (Bio-Rad) 25 µg ml-1 spermine (Sigma) 25 µg ml-1 spermidine (Sigma) PMSF as per manufacturer´s specifications (Sigma)

Complete Protease Inhibitor Cocktail Tablets (Roche) according to

manufacturer´s instructions HDP Extraction of soluble mitochondrial proteins 10 mM HEPES (pH = 7.9; Sigma) 1 mM DTT 1 mM PMSF ME Extraction of mitochondrial membrane proteins 20 mM Tris-HCl (pH 7.8; Bio-Rad) 0.4 M NaCl (Merck) 15% glycerol (Merck) 1 mM DTT 1 mM PMSF 1.5% Triton-X-100 (Sigma) Rehydration

buffer Rehydration of IPG strips

7 M urea (Bio-Rad) 2 M thiourea (Sigma)

0.2% biolytes 3 - 10 (Bio-Rad) 2% CHAPS (Bio-Rad)

100 mM DTT

IEF Sample buffer during first

dimension

Rehydration buffer including 25x Complete Protease Inhibitor (Roche) 100 mM PMSF

100x Pepstatin (Roche) 2D running

buffer

Running buffer during electrophoresis

10 x TGS (Bio-Rad) ddH2O

Equilibration

buffer I First equilibration of IPG strip

180 g urea 20% SDS 50 ml (Bio-Rad) 1.5 M Tris (pH 8.8; Bio-Rad) 87% glycerol 115 ml (Merck) 2% DTT ddH2O up to 500 ml Equilibration buffer II

Second equilibration of IPG strip 180 g urea 20% SDS 50 ml 1.5 M Tris (pH = 8.8) 87% glycerol 115 ml 2.5% IAA (Bio-Rad) ddH2O up to 500 ml Colloidal

coomassie Gel staining solution

17% ammonium sulfate (Merck) 2% phosphoric acid

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Destaining was done by washing the 2D gels in ddH2O for 1 h until background signals were minimal and protein signals were precise. Finally, gels were scanned using a gel scanner (GS-800 Calibrated Densitometer; Bio-Rad).