Analysis of fragmented and unfragmented human and porcine immunoglobulin G (IgG) N-Glycoforms by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry and Ultra Performance Liquid Chromatography Mass Spectrometry
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A developed workflow for detailed structural characterization of antibodies from different sources, using methods which involve MS is discussed in this thesis. The aim of this thesis is to emphasize the usefulness of detailed characterization of glycoforms of human and pig immunoglobulin (IgG), by comparing a conventional method of (1) whole antibody digestion with trypsin with (2) a more detailed and extensive workflow method of fragmenting the antibody by papain, FabricatorTM and FabulousTM. In (2) antigen-binding fragments (Fab) and crystallisable fragments (Fc) then obtained from enzymatic fragmentation are digested in-gel with trypsin and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and ultra performance liquid chromatography (UPLC-MS). Results from Fab-Fc fragmentation are compared to those obtained using a more conventional approach where whole antibodies are trypsinised into peptides and glycopeptides (1), to indicate which method gives a more detailed and accurate determination of structure.