Reversed-phase liquid chromatography in-line with negative ionization electrospray mass spectrometry for the characterization of the disulfide-linkages of an immunoglobulin gamma antibody

摘要

In this report, we present a new approach for the determination of the disulfide bond connectivity in proteins using negative ionization mass spectrometry of nonreduced enzymatic digests. The mass spectrometric analysis in negative ion mode was optimized to allow in-line analysis coupled directly to the HPLC system used for the separation of the peptides resulting from enzymatic digestion. We determined the disulfide structure of a human immunoglobulin gamma 2 (IgG2) antibody containing 18 unique cysteine residues linked via 11 unique disulfide bonds. The efficiency of the gas-phase dissociation of disulfide-linked peptides using negative electrospray ionization was evaluated for an ion trap mass spectrometer and an orthogonal acceleration time-of-flight mass spectrometer. Both mass spectrometry techniques provided efficient in-source fragmentation for the identification of the disulfide-linked peptides of the antibody. Both instruments were limited in the number of disulfide bonds that could be dissociated. Seven of the 11 unique disulfide linkages have been determined, including the linkage of the light chain to the heavy chain. Only the disulfide connectivity of the hinge peptide H6H7H8H9 (C(6)C(7)VEC(8)PPC(9)PAPPVAGPSVFLFPPKPK) could not be determined (numbering the cysteine residues sequentially from the N-terminus and labeling the heavy chain cysteies "H" and the light chain cysteines "L"). However, we identified the dimer of peptide C(6)C(7)VEC(8)PPC(9)PAPPVAGPSVFLFPPKPK linked via four disulfide bonds based on the unique molecular weight of this dipeptide. The established linkages were H1 to H2, H10 to H11, H12 to H13, L1 to L2, L3 to L4, and L5 to H3H4. The intrachain linkages of the light chain (L1 to L2, L3 to L4), and heavy chain (H10 to H11, H12 to H13) domains were identical to the linkages found in IgG1 antibodies.