TABLE 2.

MALDI-TOF analysis of glutathionylated, Cys-containing peptides from the bovine and rat complex I 75 kDa subunit Experimental masses were obtained from isolated and denatured complex I (2 mg/ml) and treated with DTT (10 mm) and SDS (1%) for 10 min at 37°C; the DTT was removed; and the samples were treated with GSSG (20 mm) for 30 min at 37°C. For native complex I, isolated bovine complex I was incubated with GSSG (20 mm) for 30 min at 37°C; bovine or rat mitochondria (1 mg of protein/ml) were incubated with diamide (0.5 mm) for 5 min at 37°C. After incubation all samples were alkylated with NEM (25–50 mm) and separated by electrophoresis (one-dimensional SDS-PAGE for isolated CI samples and two-dimensional BN/SDS-PAGE for mitochondria) before in-gel digestion and MALDI-TOF.

Cys no.a	Sequenceb	Enzymec	FeSd	Mass (MH+) + GSSG (305.068)
				Calculatede	Observed
					Denatured complex I	Native complex I	Bovine mitochondria (b)	Rat mitochondria (r)
30	TATAASNLIEVFVDGQSVMVEPGTTVLQACEK (b)	T	No	3613.7074	3613.7356
	TGTAASNLIEVFVDGQSVMVEPGTTVLQACEK (r)			3559.6917
41	FCYHER	T	Yes	1159.4295	1159.4165
52	LSVAGNCR	T	Yes	1124.4822
55	MCLVEIEK	T	Yes	1269.5529
69	VVAACAMPVMK	T	Yes	1424.6405
105,108	DCPIC or DCPIC	D	Yes	980.3164
	DCPIC			1160.3368
114	DQGGEC	D	Yes	913.2668
153,156	CIQCTR or CIQCTR	T	Yes	1403.5430
	CIQCTR			1583.5630
159	CIR	T	Yes	696.2809
203	DICPVGALTSKPYAFTARPW	D	Yes	2498.1842	2498.1648	2498.1743
344	VDSDTLCTEEVFPTAGAGTDLR (b)	T	No	2602.1135	2602.1125
	VDSDTLCTEEIFPNEGAGTDLR (r)			2687.1451
531	MLFLLGADGGCITR (b)	T	No	1771.8175	1771.8016	1771.8175	1771.8191
	LLFLLGADGGCITR (r)			1753.8453				1753.8760
541	DCFIVYQGHHG	D	No	1580.6261	1580.6172	1580.6201
687	DSISRASQTMAKCVKAVTEGAHAVE (b)	D	No	2894.3440	2894.3035	2894.3816
	DSISRASQTMAKCVKAVTEGAQAVE (r)			2885.3441
704	AVTEGAHAVEEPSIC (b)	T	No	1817.7680	1817.7848	1817.7869	1817.7720
	AVTEGAQAVEEPSIC (r)			1808.7681

^aCysteine residue numbers are shown, which are the same for bovine and rat sequences

^bUnless specified as bovine (b) or rat (r), peptide sequences are the same for both species

^cT indicates trypsin, and D indicates Asp-N

^dData indicate whether the cysteine residue is part of an iron-sulfur cluster

^eCalculated masses of peptides were glutathionylated on the Cys residues (+305.068 Da)