Notes

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.

together in a volume proportion of 1:1. The mixture is then added immediately on top of the membrane making sure that it is covered completely.

10. The amount of mitochondrial protein will mainly be dictated by the quality and number of isolations. We consider 0.5 mg total protein as the absolute minimum. Ideally, 0.8 mg or more should be used to obtain a high number of identified acetylation sites. In general the number of identified sites correlates with the amount of peptides used in the enrichment step [15].

11. We recommend using 4 ml centrifugation devices (for example Amicon Ultra-4, Millipore) for digesting samples in the range of 0.3 to 3 mg total protein. For larger amounts the protocol can also be scaled up to devices such as Amicon Ultra-15 (Millipore).

12. To reach more complete digestion, it is possible to additionally digest the sample with LysC. For example LysC is added at an enzyme to protein ratio of 1:100 and the sample is incubated for 2 h at room temperature, before adding trypsin and incubating overnight as specified.

13. If the peptides do not dissolve well, the sample can also be placed in an ultrasonic bath for 5 min. Higher amounts of 20% acetonitrile can also help to redissolve the pellet, however bear in mind that the antibody tolerates only low amounts of acetonitrile.

14. Formic acid remaining after desalting and evaporation on the vacuum concentrator can lower the pH substantially. After adding the TBS buffer check whether the pH is in the desired range and, if necessary, correct by adding small amounts of 1 M Tris-HCl pH 7.6.

15. We recommend desalting this aliquot directly on C18 StageTips [17] and keeping the sample until measurement at -20°C.

16. 50 µl slurry corresponds to 100 µg antibody. A ratio of 100 µg antibody per 1 mg of peptides, results in a reasonable compromise between the number of sites identified and the costs for the antibody. The antibody is quite sensitive to temperature changes and cannot be re-used in our experience.

17. Take care not to lose beads while washing. Gel-loading tips are useful for taking off the remaining buffer close to the beads.

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Table1: Preparation of resolving gel and spacer gel for second dimension. Amount of  solutions is calculated for two 16.5% Tricine SDS PAGEs. 

Resolving Gel (30 ml) Spacer Gel (10 ml)

Acrylamide 40% (w/v) 12.4 ml 2.5 ml

Tricine gel buffer 10 ml 3.4 ml

Glycerol 87% (v/v) 4 ml -

Distilled water 3.6 ml 4.1 ml

APS 10% (w/v) 100 µl 34 µl

TEMED 10 µl 3.4 µl

Table2: Preparation of sample gel for second dimension. Amount of solutions is calculated for two sample gels.

Sample Gel (10 ml) Acrylamide 40% (w/v) 2.5 ml

Gel buffer BN (6X) 3.4 ml Glycerol 100% (v/v) 1 ml

SDS 10% (w/v) 100 µl

Distilled water 2.9 ml

APS 10% (w/v) 83 µl

TEMED 8.3 µl

50

25

14 8 30

A B

Figure 1: Detection of lysine acetylation in mitochondrial protein complexes of Arabidopsis analyzed by 2D BN/SDS-PAGE and Western blot.

(A) PonceauS stain of 2D BN/SDS-PAGE of mitochondrial protein complexes. The identity of OXPHOS complexes is indicated above the gels. I+III2, supercomplex composed of complex I and dimeric complex III; I, complex I; V, complex V (ATP synthase); III2, dimeric complex III. The molecular mass scale (in kDa) is indicated on the left.

(B) Lysine-acetylated proteins from Arabidopsis mitochondria detected by Western blot analysis using the anti-acetyllysine antibody.