Rapid Characterization of Chemically-Modified Proteins by Electrospray Mass Spectrometry

Electrospray mass spectrometry (ESI-MS) has been used to examine monoclonal antibodies (MAbs), antibody fragments (Fab and Fc), modified fragments, and a range of other chemically-modified proteins as part of a study aimed at establishing ESI-MS as a method for the characterization of radioimmunoconjugates. This has been approached from two angles. Firstly, ESI-MS of complexes formed between chelators and other small molecules conjugated to hen egg white lysozyme (HEL) (14 kDa) demonstrate the considerable advantages of this powerful new technique compared with existing methods for the characterization of chemically-conjugated proteins. Molecular weights can be determined rapidly to within 0.01-0.05% and with good sensitivity (10-50 pmol total), thus providing specific structural information and opening the way for ESI-MS to be applied widely for the structural characterization of radioimmunoconjugates. Secondly, the conditions for ESI-MS of intact antibodies and antibody fragments have been examined in detail, and we have shown that the addition of up to 10 biotin molecules to the 50 kDa Fab fragment can be easily detected in ESI mass spectra, thus demonstrating the potential for the characterization of modified MAb fragments and metabolites. Finally, the strengths and limitations of ESI-MS of intact antibodies are discussed, and these results indicate that it may only be possible to detect average shifts in the mass of intact antibodies following modification.