Skip to main content
. 2009 Oct 22;284(51):35390–35402. doi: 10.1074/jbc.M109.064147

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