TABLE 1.
The average masses of the P1 and C1 fragments generated by DTT reduction and IAM alkylation, followed by RP-HPLC-TOF/MS analysis
The data for the C1 fragment were from the 2-week samples incubated with H2O2. The HC contains four methionine residues, three of which are located in the truncated Fc-HC. Most of the fragments contain oxidations indicated by “O” (+16, +32, and +48 Da), as indicated by the differences in mass compared with the theoretical mass. The two glycosylation chains are composed of biantennary glycans with a variable number of galactose residues (G). The most common isoforms are G0, G1, and G2. N-terminal sequencing indicated that the N terminus of the HC was completely cyclized to pyroglutamic acid (data not shown). Conversion of N-terminal heavy chain (HC) glutamine (Q) to pyroglutamic acid (pE) is shown. Some fragments contained adducts that were proposed to conform to carboxylic acid (–COOH, +45 Da) and α-ketoacyl groups (CH3COCO, +71 Da), which were consistent with the peptide mapping results. Therefore, the additional masses can be explained by the number of oxidation products plus one of these adducts (for example, 118 Da = 3 oxidations + 71-Da adduct, and 93 Da = 3 oxidations + 45-Da adduct). Some fragments (Thr230–Gly451) were identified in both peak 2 and peak 3 because of incomplete separation (Fig. 2).
| Peak | Residues (start to end) | Theoretical mass | Observed mass | Mass difference | Assignment |
|---|---|---|---|---|---|
| Da | Da | Da | |||
| IgG1 | |||||
| LC | |||||
| 1 | Glu1–Cys215 | 23,679.3 | 23,678.5 | −0.8 | |
| HC | |||||
| 2 | pE1–Gly451 | 51,373.9 (G0) | 51,373.9 (G0) | 0.0 | |
| P1 | |||||
| Fc | |||||
| 1 | Thr228–Gly451 | 26,997.6 (G0) | 27,029.3 (G0) | 31.7 | 2 O |
| Lys227–Gly451 | 27,125.5 (G0) | 27,156.6 (G0) | 31.1 | 2 O | |
| Asp226–Gly451 | 27,240.6 (G0) | 27,270.6 (G0) | 30.0 | 2 O | |
| Asp226–Gly451 | 27,240.6 (G0) | 27,315.9 (G0) | 75.3 | 45 Da adduct +2 O | |
| Asp226–Gly451 | 27,402.7 (G1) | 27,433.4 (G1) | 30.7 | 2 O | |
| 2 | Thr230–Gly451 | 26,759.4 (G0) | 26,774.5 (G0) | 15.1 | 1 O |
| Thr230–Gly451 | 26,921.6 (G1) | 26,937.1 (G1) | 15.5 | 1 O | |
| Thr228–Gly451 | 26,997.6 (G0) | 26,996.2 (G0) | −1.4 | ||
| Thr228–Gly451 | 27,159.8 (G1) | 27,158.8 (G1) | −1.1 | ||
| 3 | Thr230–Gly451 | 26,759.4 (G0) | 26,758.4 (G0) | −1.0 | |
| Thr230–Gly451 | 26,921.6 (G1) | 26,920.0 (G1) | −1.6 | ||
| Thr230–Gly451 | 26,921.6 (G1) | 26,993.7 (G1) | 72.1 | 71-Da adduct | |
| Thr230–Gly451 | 27,083.8 (G2) | 27,082.1 (G2) | −1.7 | ||
| Thr230–Gly451 | 26,759.4 (G0) | 26,774.9 (G0) | 15.5 | 1 O | |
| Thr230–Gly451 | 26,921.6 (G1) | 26,937.1 (G1) | 15.5 | 1 O | |
| LC | |||||
| 4 | Glu1–Cys215 | 23,679.3 | 23,710.0 | 30.7 | 2 O |
| 5 | Glu1–Cys215 | 23,679.3 | 23,678.4 | −0.9 | |
| HC | |||||
| pE1–Gly451 | 51,373.9 (G0) | 51,420.5 (G0) | 46.6 | 3 O | |
| pE1–Gly451 | 51,373.9 (G0) | 51,436.9 (G0) | 63.0 | 4 O | |
| pE1–Gly451 | 51,373.9 (G0) | 51,453.6 (G0) | 79.7 | 5 O | |
| C1 | |||||
| Fc | |||||
| 1 | Thr228–Gly451 | 26,997.6 (G0) | 27,045.2 (G0) | 47.6 | 3 O |
| Thr228–Gly451 | 27,159.8 (G1) | 27,206.2 (G0) | 46.4 | 3 O | |
| Lys227–Gly451 | 27,125.5 (G0) | 27,172.5 (G0) | 47.0 | 3 O | |
| Asp226–Gly451 | 27,240.6 (G0) | 27,288.1 (G0) | 47.5 | 3 O | |
| Asp226–Gly451 | 27,240.6 (G0) | 27,334.4 (G0) | 93.8 | 45-Da adduct + 3 O | |
| Asp226–Gly451 | 27,402.7 (G1) | 27,449.2 (G1) | 46.5 | 3 O | |
| 2 | Thr230–Gly451 | 26,759.4 (G0) | 26,806.6 (G0) | 47.2 | 3 O |
| Thr230–Gly451 | 26,921.6 (G1) | 26,968.4 (G1) | 46.8 | 3 O | |
| Thr228–Gly451 | 26,997.6 (G0) | 27,014.9 (G0) | 17.3 | 1 O | |
| Thr228–Gly451 | 27,159.8 (G1) | 27,177.3 (G1) | 17.2 | 1 O | |
| 3 | Thr230–Gly451 | 26,759.4 (G0) | 26,776.8 (G0) | 17.4 | 1 O |
| Thr230–Gly451 | 26,921.6 (G1) | 26,939.1 (G1) | 17.5 | 1 O | |
| Thr230–Gly451 | 26,759.4 (G0) | 26,877.9 (G0) | 118.6 | 71 Da adduct + 3 O | |
| Thr230–Gly451 | 26,921.6 (G1) | 27,040.0 (G1) | 118.4 | 71 Da adduct + 3 O | |
| LC | |||||
| 4 | Glu1–Cys215 | 23,679.3 | 23,709.8 | 30.5 | 2 O |
| 5 | Glu1–Cys215 | 23,679.3 | 23,677.4 | −1.8 | |
| HC | |||||
| 6 | pE1–Gly451 | 51,373.9 (G0) | 51,470.9 (G0) | 96.0 | 6 O |
| 7 | pE1–Gly451 | 51,373.9 (G0) | 51,436.9 (G0) | 63.0 | 4 O |