Bondarenko, Pavel V. and Second, Tonya P. and Zabrouskov, Vlad and Makarov, Alexander A. and Zhang, Zhongqi (2009) Mass Measurement and Top-Down HPLC/MS Analysis of Intact Monoclonal Antibodies on a Hybrid Linear Quadrupole Ion Trap-Orbitrap Mass Spectrometer. Journal of the American Society for Mass Spectrometry, In Press, Corrected Proof . - .
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Mass and top-down analyses of 150-kDa monoclonal immunoglobulin gamma (IgG) antibodies were performed on an Orbitrap analyzer. Three different sample delivery methods were tested including (1) infusion of an off-line desalted IgG sample using nano-electrospray; (2) on-line desalting followed by a step elution with a high percentage of organic solvent; and (3) reversed-phase HPLC separation and on-line mass and top-down analyses of disulfide isoforms of an IgG2 antibody. The accuracy of mass measurements of intact antibody was within ±2 Da (15 ppm). The glycoforms of intact IgG antibodies separated by 162 Da were baseline resolved. In-source fragmentation of the intact antibodies produced mainly 115 residue fragments including N-terminal variable domains of heavy and light chains. The sequence coverage (the number of cleavages) was greatly increased after reduction of disulfide bonds and HPLC/MS/MS analysis of light and heavy chains using collision-induced dissociation in the ion trap of the LTQ-Orbitrap. This is an attractive alternative to peptide mapping for characterization and monitoring of post-translational modifications attributed to minimal sample preparation, high speed of the mass/top-down analysis, and relatively minor method-induced sample modifications.
|Uncontrolled Keywords:||Mass measurement and top-down analysis of an intact 150-kilodalton monoclonal IgG antibody and its chains were performed on an Orbitrap analyzer on-line with reversed-phase HPLC.|
|Subjects:||Biomedical Science > Nanomedicine|
|Deposited On:||28 Jul 2009 15:06|
|Last Modified:||28 Jul 2009 15:06|
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