Structural analysis of an intact monoclonal antibody by online electrochemical reduction of disulfide bonds and Fourier transform ion cyclotron resonance mass spectrometry.
Structural confirmation and quality control of recombinant monoclonal antibodies (mAbs) by top-down mass spectrometry is still challenging due to the size of the proteins, disulfide content, and post-translational modifications such as glycosylation. In this study we have applied electrochemistry (EC) to overcome disulfide bridge complexity in top-down analysis of mAbs. To this end, an electrochemical cell was coupled directly to an electrospray ionization (ESI) source and a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer (MS) equipped with a 15 T magnet. By performing online EC-assisted reduction of interchain disulfide bonds in an intact mAb, the released light chains could be selected for tandem mass spectrometry (MS/MS) analysis without interference from heavy-chain fragments. Moreover, the acquisition of full MS scans under denaturing conditions allowed profiling of all abundant mAb glycoforms. Ultrahigh-resolution FTICR-MS measurements provided fully resolved isotopic distributions of intact mAb and enabled the identification of the most abundant adducts and other interfering species. Furthermore, it was found that reduction of interchain disulfide bonds occurs in the ESI source dependent on capillary voltage and solvent composition. This phenomenon was systematically evaluated and compared with the results obtained from reduction in the electrochemical cell.
Center for Proteomics and Metabolomics, Leiden University Medical Center , Albinusdreef 2, 2300 RC Leiden, The Netherlands.
Simone
S
Nicolardi
André M
AM
Deelder
Magnus
M
Palmblad
Yuri E M
YE
van der Burgt
D000911:Antibodies, Monoclonal / Q000032:analysis*; D017357:Cyclotrons; D004220:Disulfides / Q000737:chemistry*; D004563:Electrochemistry; D005583:Fourier Analysis; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis; D009862:Online Systems; D021241:Spectrometry, Mass, Electrospray Ionization; D017550:Spectroscopy, Fourier Transform Infrared; D053719:Tandem Mass Spectrometry
D000911:Antibodies, Monoclonal; D004220:Disulfides; D007074:Immunoglobulin G