A multi-parameter optimization in middle-down analysis of monoclonal antibodies by LC-MS/MS

In antibody-based drug research, regulatory agencies request a complete characterization of antibody proteoforms covering both the amino acid sequence and all post-translational modifications. The usual mass spectrometry-based approach to achieve this goal is bottom-up proteomics, which relies on the digestion of antibodies, but does not allow the diversity of proteoforms to be assessed. Middle-down and top-down approaches have recently emerged as attractive alternatives but are not yet mastered and thus used in routine by many analytical chemistry laboratories. The work described here aims at providing guidelines to achieve the best sequence coverage for the fragmentation of intact light and heavy chains generated from a simple reduction of intact antibodies using Orbitrap mass spectrometry. Three parameters were found crucial to this aim: the use of an electron-based activation technique, the multiplex selection of precursor ions of different charge states and the combination of replicates.