Table 1.

The acetylation sites in aconitase mapped by mass spectrometric analysis.

	Acetylation site	Peptide sequence	Peptide molecular ion m/z, charge
1	K31	AK[a]VAM*SHFEPSEYIR	608.3, +2
2	K50	YDLLEK[a]NINIVR	766.4, +2
3	K86	GK[a]TYLR	390.2, +2
4	K138	VAVPSTIHC*DHLIEAQVGGEK[a]DLR	672.3, +4
5	K144	AK[a] DINQEVYNFLATAGAK	998.0, +2
6	K233	VIGVK[a]LTGSLSGWTSPK	886.5, +2
7	K309	YLSK[a]TGR	433.7, +2
8	K401	SAAVAK[a]QALAHGLK	469.6, +3
9	K411	C*K[a]SQFTITPGSEQIR	897.4, +2
10	K517	FNPETDFLTGK[a]DGK	805.9, +2
11	K523	FK[a] LEAPDADELPR	771.9, +2
12	K549	SDFDPGQDTYQHPPK[a]DSSGQR	802.0, +3
13	K591	GK[a]C*TTDHISAAGPWLK	595.3, +3
14	K628	GHLDNISNNLLIGAINIENGK[a]ANSVR	930.2, +3
15	K689	AIITK[a]SFAR	524.8, +2
16	K700	IHETNLK[a]K	512.8, +2
17	K701	K[a]QGLLPLTFADPSDYNK	975.0, +2
18	K723	IHPVDK[a] LTIQGLK	502.0, +2
19	K730	LTIQGLK[a]DFAPGKPLK	590.0, +2
20	K736	DFAPGK[a]PLK	507.8, +2
21	K739	DFAPGKPLK[a]C*VIK	505.6, +3
	unmodified	VDVSPTSQR	494.8, +2
	unmodified	VAGILTVK	400.8, +2
	unmodified	NTIVTSYNR	534.3, +2
	unmodified	SQFTITPGSEQIR	732.4, +2

Isolated mitochondria treated with up to 200μM acetic anhydride were reduced, alkylated, and digested with trypsin. The digests were analyzed using combinations of unbiased data dependent methods with database searches designed to find acetylated peptides and targeted analysis based on previously identified acetylation sites. For each site, the resulting collision induced dissociation (CID) spectra were interpreted manually to verify correct assignment of the peptide and acetylation position. The acetylated peptides were incorporated into the quantitative selected reaction monitoring methods use to quantify acetylation in subsequent experiments, with the additional unmodified peptide used for normalization. K[a] designates an acetylated lysine residue.

^*M designates an oxidized methionine residue.

^*C designates a carboxyamidomethylated cysteine residue