Detection of phosphorylated proteins

Mitochondria were isolated from INS-1E cells as described above. Pelleted mitochondria (100-120 μg of total protein) were thawed in 37°C waterbath and resuspended in 100 μl of 100 mM Tris-HCl (pH 7.4) containing 1% (v/v) SDS. Mitochondria were solubilized by heating for 5 min at 95°C, followed by 5 min cooling on ice. Mitochondrial proteins were precipitated by chloroform/methanol to remove lipids and salts. This was done by adding 400 μl methanol, 100 μl chloroform and 300 μl ddH20. The sample was vortexed after each addition. The mixture was centrifuged at 13000 rpm (~14000 g) for 5 min at 4°C. The upper phase was discarded and white precipitated disc (proteins) at the interphase was kept. The protein pellet was washed with 300 μl of methanol followed by centrifugation at 13000 rpm (~14000 g) for 5 min at 4°C. Methanol was removed and precipitated proteins were air-dried in a vacuum centrifuge (SpeedVac) for 5 min.

For one-dimensional SDS-PAGE, the dry protein pellets were resuspended in 1x sample buffer: 62.5 mM Tris-HCl, 2% (w/v) SDS, 0.005% (w/v) bromophenol blue, 10% (v/v) glycerol, 5% (v/v) β-mercaptoethanol, and heated for 5 min at 100°C. The molecular weight marker PeppermintStick^TM (Invitrogen, Cat. No. P27167) was used in one-dimensional SDS-PAGE (Figure 1 and 2).

For 2D PAGE protein pellets were resuspended in 8 M urea, 4% CHAPS, 65 mM dithioerythritol (DTE), 2% Resolyte 4–8, containing trace amounts of bromophenol blue.

120 27.2. Alkaline phosphatase treatment of mitochondrial protein samples

INS-1E cells were plated in tissue culture plates (147.8 cm² growth area, TPP, Switzerland, Cat. No. 93150) at a density of 8-9 x 10⁶ cells/plate. Cells were used 3-4 days after plating.

For the experiment shown in Figure 1, mitochondria were obtained by 9000 g centrifugation of INS-1E cell homogenates prepared from two 147.8 cm² plates (“crude mitochondrial fraction” as described above). The mitochondrial pellet (200 μg total protein) was washed twice by resuspending it in 400 μl of 250 mM sucrose, 20 mM Tris-HCl followed by centrifugation at 10000 rpm (~8500 g) for 4 min in a pre-cooled (4°C) bench-top centrifuge.

Washed mitochondria were resuspended in 400 μl of 250 mM sucrose, 20 mM Tris-HCl and separated into two 200 μl aliquots. The tubes were centrifuged at 10000 rpm (~8500 g) for 4 min at 4°C.

One aliquot (100 μg of total protein) was used to prepare mitochondrial lysates. To this end, pelleted mitochondria were resuspended in 350 μl of lysis buffer (250 mM sucrose, 20 mM Tris-HCl, 1% (v/v) SDS) and sonicated 5 times, 3 sec. each. Then 350 μl of lysis buffer + 77.8 μl of 10x NEB Buffer 3 (New England Biolabs; 500 mM Tris-HCl, 100 mM MgCl2, 1000 mM NaCl, 10 mM dithiothreitol, pH7.9) were added. The suspension was split to two 388 μl aliquots (A and B). Aliquot A received 50 units of calf intestinal alkaline phosphatase (New England Biolabs, Cat. No. M0290S, 10 units/μl). Aliquot B received 5 μl of lysis buffer.

Mitochondria in the other aliquot (100 μg of total protein) were left intact. They were resuspended in 700 μl of 250 mM sucrose, 20 mM Tris-HCl + 77.7 μl of 10x NEB Buffer 3.

The suspension was split to two 388 μl aliquots (C and D). Aliquot C was treated with alkaline phosphatase, while aliquot D was left untreated.

Aliquots A, B, C and D were then incubated for 50 min in a 37°C waterbath. Then tubes were shifted for 10 min to 96°C to heat-inactivate alkaline phosphatase.

To dissolve mitochondria from aliquots C and D, 10% SDS solution was added (to bring SDS concentration to 1%) followed by 10 min heating at 100°C.

Proteins from aliquots A, B, C and D were precipitated in chloroform/methanol and separated on SDS polyacrylamide gel.

For the experiment shown in Figure 5 mitochondria were isolated by Percoll gradient centrifugation (as described above). The frozen mitochondrial pellet (160 μg of total protein) was thawed and immediately resuspended in 300 μl of 40 mM Tris-HCl containing a protease inhibitor cocktail (Roche Diagnostics, Cat. No. 04 693 132 001), 1% (v/v) SDS and sonicated

121 5 times, 3 sec. each. Then 400 μl of the same solution + 78 μl of 10x NEB Buffer 3 were added. The suspension was split to two 390 μl aliquots (80 μg total protein each). The first aliquot received 76 units of calf intestinal alkaline phosphatase. The second aliquot received 8 μl of 500 mM NaF (inhibitor of Ser/Thr protein phosphatases) and 8 μl of 100 mM Na3VO₄ (inhibitor of protein tyrosine phosphatases). Both aliquots were then incubated for 50 min in a 37°C waterbath. Then tubes were shifted for 10 min to 96°C to heat-inactivate alkaline phosphatase. Proteins were precipitated in chloroform/methanol and separated on 2D polyacrylamide gel.

27.3. Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and protein identification

2D PAGE and protein identification by mass spectrometry were performed at the Proteomics Core Facility of the Faculty of Medicine by the facility personnel.

For 2D PAGE, proteins were first separated on the gel in which a pH gradient has been established (immobilized pH gradient gel (IPG) Immobiline Drystrip pH 3-10 NL 7 cm, GE Healthcare Bio-Sciences, Cat. No. 17-6001-12). Electrophoresis was performed using Ettan IPGphor II System electrophoresis unit (GE Healthcare Bio-Sciences) at 15 °C with the following program:

Stage 1. 300 V 1 min Stage 2. 3500 V 30 min Stage 3. 3500 V 90 min

The IPG strip with separated proteins was attached to the 12.5% polyacrylamide gel using solution containing 0.5% (w/v) agarose, 25 mM Tris, 198 mM glycine and 0.1% w/v SDS (pH 8.3) heated to ~70°C. Proteins were separated by electrophoresis in a direction perpendicular to the first separation using SE260 Mighty Small II electrophoresis unit (Hoefer, Holliston, MA, USA, Cat. No. SE26010A75) at 100V for 3 h at room temperature.

The gels were fixed in a solution of 50% methanol and 10% acetic acid.

Protein identification was performed using peptide fragmentation sequencing by Maldi-Tof-Tof and NanoLC-ESI MS/MS.

122 27.4. Phosphoprotein staining protocol

Important: Polypropylene containers should be used for all incubations. Glass dishes should not be used. All fixation, staining and washing steps were performed with gentle agitation (on an orbital shaker 50-60 rpm) at room temperature.

Mitochondrial proteins were separated on 10% polyacrylamide gels. Gels were left for 40 min in a solution of 50% methanol and 10% acetic acid. Then fresh solution was added and gels were fixed overnight. Gels were rinsed 5-6 times with ddH₂O followed by three 15 min washes in 100 ml of water. Next, gels were stained with Pro-Q Diamond solution (Invitrogen, Cat. No. P33301, ~40 ml for 6 x 8 cm minigel) for 90 min. During the staining and subsequent wash steps gel containers were covered with aluminum foil to protect them from light.

After staining the Pro-Q Diamond solution was removed and gels were washed three times (30 min each) in 100 ml of destain solution: 50 mM sodium acetate, 20% acetonitrile. The gels were then washed four times (5 min each) in 100 ml of ddH2O.

Gels were scanned on a Typhoon 9400 scanner using 532 nm excitation filter and 580 nm emission filter (580 BP30).

27.5. Total protein staining protocol

For subsequent total protein staining, gels were washed in ddH2O and incubated overnight with Sypro Ruby (Invitrogen, Cat. No. S12001), ~40 ml for 6 x 8 cm minigel. Then gels were washed twice (20 min each) in 100 ml of solution of 10% methanol, 7% acetic acid. Next, gels were washed three times in 100 ml of ddH2O. Gels were scanned in a Typhoon 9400 instrument using 532 nm excitation filter and 610 nm emission filter (610 BP30).

Gels were kept in 10% ethanol at 4°C for later excision of protein spots and analysis by mass spectrometry.