Identification of lysine-acetylated peptides 1 Protein digestion using FASP

All steps are carried out at room temperature unless indicated else. Placing SDS or urea containing buffers on ice or at 4 °C can lead to precipitation.

1. Purified mitochondria (see Chapter 2) (corresponding to at least 0.5 to 1 mg mitochondrial protein) are pelleted at 15000 x g for 10 min at 4°C in a microcentrifuge tube (see Note 10).

For better coverage, we recommend processing at least three independent biological replicates. Pellets can be snap-frozen in liquid nitrogen and stored at -80°C until further processing.

2. Resuspend the pellet in 400 µl SDS lysis buffer, vortex until the pellet is resuspended and place for 5 min in a heat block at 95°C, vortexing two times for 20 seconds in between.

3. Sonicate sample in an ultrasonic bath for 1 min.

4. Centrifuge for 20 min in a benchtop centrifuge at top speed (15000 to 21000 x g) and room temperature.

5. Transfer the supernatant to a new tube without disturbing any sedimented material.

6. Repeat steps 4 and 5 once.

7. Determine the amount of protein using the 660 nm protein assay with compatibility reagent according to the manufacturer’s instructions.

8. Add 2 ml urea buffer to the CFD and centrifuge for 5 min to condition the membrane. Stop the centrifuge after approximately one minute and check the buffer retention. In rare cases unusually fast flow-through occurs, which indicates a leak in the membrane. Such damaged units cannot be used and must be replaced.

9. Dilute the sample with urea buffer to a total volume of 4 ml and transfer to the CFD (see Note 11) – final SDS concentration must not exceed 0.5%.

10. Centrifuge at 4000 x g for 15 min (or until at least ten-fold concentration).

11. Discard flow-through, add another 4 mL urea buffer and repeat centrifugation.

12. Discard flow-through, add 1 ml IAA solution, mix gently for 1 min with a pipette without touching the membrane and incubate for 30 min at room temperature in darkness.

13. Centrifuge the CFD at 4000 x g for 10 min, discard flow-through.

14. Add 4 ml of urea buffer to the CFD and centrifuge at 4000 x g for 15 min or until at least ten-fold concentration. Discard the flow-through and repeat this step twice.

15. Add 4 ml ABC buffer to the CFD and centrifuge at 4000 x g for 15 min or until at least ten-fold concentration. Discard the flow-through and repeat this step twice.

trypsin for 1 mg protein) and gently mix with a pipette (do not touch the membrane while pipetting), (see Note 12).

18. Tighten the lid of the tube well and incubate the CFD at 37°C overnight.

19. Centrifuge the CFD at 4000 x g for 10 min. The eluate now contains the peptides.

20. Add 1 ml ABC buffer to the CFD (rinsing the filters when adding the buffer) and repeat the centrifugation.

21. Repeat step 20.

22. Assess the peptide yield on a microvolume UV-VIS photometer at 280 nm, assuming an absorption of 1 for a peptide concentration of 1 mg ml^-1. Typical yields are approximately 50 to 60% of the starting amount.

23. Acidify the eluate to a final concentration of 1% TFA.

24. Condition a C18 SepPak cartridge by flushing it with 3 ml methanol, 3 ml buffer B, and then 3 ml buffer A.

25. Slowly load peptide sample on the cartridge.

26. Wash with 3x 1 ml of buffer A

27. Elute two times with 0.6 ml buffer B into a 1.5 ml Protein LoBind tube.

28. Evaporate the sample to complete dryness on a vacuum concentrator, if necessary overnight. After drying, peptides can be stored for short term at 20°C or for long term at -80°C.

3.2.2. Enrichment of lysine-acetylated peptides

Sample preparation can be carried out at room temperature to facilitate dissolution of peptides. All steps involving the anti-acetyl lysine antibody should be carried out on ice or at 4°C.

1. Re-dissolve dried peptides in 50 µL 20% acetonitrile, vortex vigorously and let rest for 10 min. Then add 450 µl TBS and vortex (see Note 13).

2. Check the pH of the solution by pipetting 1 µl of sample on pH indicator paper (see Note 14).

3. Assess the peptide yield on a microvolume UV-VIS photometer at 280 nm, assuming an absorption of 1 for a peptide concentration of 1 mg ml^-1.

4. Take an aliquot of 10 µg and store it for measuring the background proteome (see Note 15)

5. Transfer 50 µL antibody bead slurry to a 1.5 ml Protein LoBind tube with a cut 200 µl pipette tip (see Note 16).

6. Wash beads 3 x 5 min with 1 ml TBS on a rolling wheel. Centrifuge at 1000 x g in between washes and carefully remove the supernatant (see Note 16).

7. Add the re-dissolved peptides to the beads and incubate overnight at 4°C on a rolling wheel.

8. Wash beads 4 x 5 min with 1 ml TBS on a rolling wheel at 4°C.

9. Wash beads 2 x shortly with ultrapure water to reduce the salt concentration, centrifuging in between at 1000 x g for 1 min.

10. Elute 3 x 5 min with 50 µl 0.1% TFA, mixing the beads gently with the pipet and letting them rest for the remaining time period. Centrifuge in between at 2000 x g and try to recover as much liquid as possible without transferring beads using gel-loading tips.

11. Assess the peptide concentration of the combined eluates on a microvolume UV-VIS photometer at 280 nm as in step 3. Usually the concentration is very low (only a few µg yield in total). Higher concentrations might indicate massive background contamination.

12. Clean-up combined eluates on StageTips and run the samples on an LC-MS/MS system.

3.2.3 Guidelines for measuring the samples on LC-MS/MS

In general every high-resolution accurate mass nano-UHPLC-MS/MS setup used for shotgun proteomics can be used for measuring these samples. Typically we run them on self-pulled capillary columns with 75 µm diameter, packed with 20 to 50 cm C18 reversed-phase material (for example: 1.9 μm ReproSil-Pur C18-AQ, Dr. Maisch GmbH). The column is kept at 50°C in a column oven throughout the run. We inject max. 1 to 2 µg peptides and separate them at a flow rate of 250 nl min^-1 with a 90 min linear gradient from 2% to 30% buffer B in buffer A (buffer A: 0.1% (v/v) formic acid; buffer B: 80% (v/v) acetonitrile 0.1% (v/v) formic acid), followed by a 5 min linear gradient to 60% buffer B, and then a 5 min linear gradient to 95% buffer B. If sample complexity is high the gradient time should be prolonged accordingly (for example to a total of 4 h runtime) or fractionation should be considered. We typically detect the peptides on a Q-Exactive MS (Thermo Scientific) in positive mode with a scanned mass range of 300-1650 m/z, at a resolution of 70,000 and an AGC target value of 3e6. The ten most intense ions are selected for MS2 at a resolution of 35,000 with an isolation window of 1.6 m/z and an AGC target of 1e6. Peptides with a charge of +1 or with unassigned charge state are excluded from fragmentation for MS2, dynamic exclusion prevents repeated selection of selected masses for 20 s. We process raw data using MaxQuant software

missed cleavages at a protein and peptide false discovery rate of 1%. Carbamidomethylation of cysteine residues were set as fixed, oxidation of methionine, N-terminal acetylation and lysine acetylation as variable modifications.