TABLE 3.

Characterization of the purified GPx4 by mass spectrometry

Cells were cultured without or with 10 μg/ml Dox, 25 μg/ml Cp, or 100 μg/ml G418 for 72 h. Peptides were normalized to an internal control peptide from GPx4 (R.YAECGLR.I, where the period indicates where the peptide is cleaved by trypsin) to account for differences in protein expression between the different treatments. AA, amino acid.

Peptidea	AA	Untreatedb	Dox	Cp	G418
R.GFVCIVTNVASQUGK.T	Sec	1.0	0.8	0.7	0.6
R.GFVCIVTNVASQCGK.T	Cys	NQc	NQc	NQc	—d
R.GFVCIVTNVASQR.G	Arg	1.0	1.7	7.9	2.2

^a The period in each peptide designates where the peptide is cleaved by trypsin. The underline indicates the position of Sec or misincorporated amino acid.

^b Signals for each GPx4 form found in the untreated sample were normalized to 1.0 and were used as a reference for quantifying these forms in samples treated with antibiotics. The different amino acid replacements and Sec-containing peptides should not be directly compared with each other because the Arg-containing peptide was of shorter length and gave a different response. However, the relative frequency (%) of each detected peptide in the untreated samples was Sec, 88.5; Arg, 11.5.

^c NQ, not quantifiable (signal:noise < 10).

^d Peptides with Cys decoded by UGA were detected, but could not be quantified as they were missing in the untreated control.