Table 2:

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

Mpro Cys	TCEP	Peptide*	Mr (calc)	Mr (expt)	Delta	RT
Cys85	−	77VIGHSMQNCGSHVLK88	1632.74	1632.71	0.03	13.5
Cys300	−	299QCGSHSGVTFQ306	1173.44	1173.42	0.02	10.9
Cys300**	−	299pyQCGSHSGVTFQ306	1156.44	1156.40	0.04	13.6
Cys85	+	77VIGHSMQNCVLK88	1327.66	1327.64	0.02	14.7
Cys300	+	299QCSGVTFQ306	868.36	868.36	0.00	11.2
Cys300**	+	299pyQCSGVTFQ306	851.36	851.33	0.03	14

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

^**These peptides are the result of the spontaneous deamidation that occurs with peptides containing an N-terminal glutamyl residues²¹ and the retention times and molecular masses for this peptide were confirmed with the use of synthetic peptides that were run on RP-HPLC/MS. 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 without (−) and with (+) TCEP to remove glutathione moieties. Shown are the calculated native masses [M_{r (calc)}] and the experimental masses [M_{r (expt)}]. The full TIC and 205 nm UV chromatograms for these analyses can be found in supplemental material (see Figure S5C–S5F in supplemental material).