Table 1:

RP/HPLC/MALDI-TOF MS Identification of peptides from chymotrypsin digestion of monoglutathionylated M^pro preparations without (−) and with (+) TCEP

Mpro Cys	TCEP	Peptide*	Mr (calc)	Mr (expt)	Delta	RT
Cys156**	−	151NIDYDCGSHVSF159	1379.50	1379.47	0.03	19.0
Cys300	−	295DVVRQCGSHSGVTF305	1514.66	1514.62	0.04	14.9
Cys300***	−	295DVVRQCGSHSGVTFQ306	1642.71	1642.68	0.03	13.6
Cys156**	+	151NIDYDCVSF159	1074.42	1074.41	0.01	20.6
Cys300	+	295DVVRQCSGVTF305	1209.58	1209.56	0.02	16.9
Cys300***	+	295DVVRQCSGVTFQ306	1337.63	1337.61	0.02	15.4

^*GSH indicates modification of the cysteine by glutathione based on a monoisotopic mass increase of 305.08.

^**These peptides containing cysteine 156 occur due to lack of cleavage at the 154:155 predicted chymotryptic cleavage site.

^***These peptides containing Cys300 occur due to incomplete cleavage at the 305:306 predicted chymotryptic cleavage site. The retention times (RT) and molecular masses for the Cys300 peptides were confirmed with the use of synthetic peptides that were run on RP-HPLC/MALDI-TOF as native, alkylated or glutathionylated peptides. Peptide samples were analyzed before and after treatment with 50 mM TCEP to remove glutathione moieties. Shown are the calculated native masses [M_{r (calc)}] and the experimental masses [M_{r (expt)}] that were obtained from the analysis. The full TIC and 205 nm UV chromatograms for these analyses can be found in supplemental material (see Figure S2C–S2F in supplemental material).