IgG antibody labeled with a conjugation reagent is an essential component of immunoassay development. This conjugated IgG antibody was characterized using Q-TOF tandem mass spectrometry in order to determine antibody sites of conjugation. Intact and reduced IgG antibody conjugates were analyzed using ESI-MS (electrospray ionization mass spectrometry). ESI-MS analysis demonstrated an average of two and a maximum of four conjugation molecules per antibody with the majority of conjugation sites on the heavy chain. The heavy chain bands of the antibody conjugates were excised from SDS-PAGE and subjected to in-gel proteolytic digestion. The proteolytic digests were analyzed using gradient capillary C18 LC/MS/MS. MS results determined a maximum of eight possible sites of conjugation to lysine amino acid residues with one site on the variable region of the heavy chain. MS determination of the sites of conjugation provides additional characterization of antibody conjugates.

Characterization of antibody sites of conjugation using Q-TOF mass spectrometry

243rd National Meeting (2012)

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