A differential cysteine labeling method followed by high-resolution MS was employed to distinguish free cysteine residues from those involved in disulfide bonding. Briefly, eE2 was incubated with a molar excess of NEM under denaturing, nonreducing conditions to label all free cysteine residues. After disulfide bond reduction with DTT, the newly generated free cysteines were alkylated Regorafenib clinical with IAM. The modified protein was digested with trypsin and deglycosylated with PNGase F, and the resulting peptides were resolved by MS. All cysteine-containing peptides were identified, and only one peptide (C656NLEDRDR) was modified by NEM (Fig. (Fig.4A).4A). The expected unmodified molecular mass of this peptide is 1,020.45 Da, 1,077.45 Da if modified by IAM, or 1,145.45 Da if modified by NEM. In Fig. Fig.

4A,4A, the spectrum shows a 573.75 m/z peak, corresponding to this peptide modified by NEM and carrying a +2 charge, while no peptide appears at a position corresponding to a modification by IAM (expected ~538 m/z with a 2+ charge). All other cysteine-containing peptides were shown to have an addition of 57 Da, indicating that they were modified only after reduction with DTT. For example, the expected molecular mass of the unmodified SAC459RSIEAF peptide is 983.46 Da, 1,040.46 Da if modified by IAM, or 1,108.46 Da if modified by NEM. This peptide resolves at 521.32 m/z, which corresponds to the molecular mass when modified by IAM and carrying a +2 charge. It does not resolve as modified by NEM (expected ~554 m/z with a 2+ charge) (Fig. (Fig.4B).4B).

From the results of nonreducing SDS-PAGE, cysteine-labeling experiment and SEC, we presumed that C656 may potentially be involved in the formation of an intermolecular disulfide bond. FIG. 4. Differential labeling of free and disulfide-linked cysteines. Free and disulfide-bonded cysteines were labeled with NEM and IAM, respectively. LC-MS/MS-extracted ion chromatograms for peptides containing C656 (A) or C459 (B) are shown. The extraction … To test this hypothesis, C656 was mutated to a serine (eE2-C656S) to conserve some of the biochemical properties at this position. An HEK293T cell line that stably expresses eE2-C656S-Fc was generated, and the protein was purified Dacomitinib as before. Results from nonreducing SDS-PAGE indicated that the proportion of monomer in eE2-C656S is substantially increased relative to wild-type eE2 (compare Fig. Fig.3A3A and and4C).4C). In order to determine the native behavior, eE2-C656S was analyzed by SEC under conditions identical to those for wild-type eE2. The results demonstrated that the mutant is predominantly monomeric at pH 7.5, with a lesser amount of dimer (Fig. (Fig.4D4D).