Characterizing recombinant protein and its fragmentation by top-down mass spectrometry

This study establishes a top-down mass spectrometry method for evaluating the structural integrity and fragmentation profiles of recombinant proteins. Three representative samples were analyzed: recombinant collagen 1 (RC1, 15.037 kDa; no disulfide bonds), recombinant collagen 2 (RC2, 49.859 kDa; no disulfide bonds), and interferon α2b (19.497 kDa; two disulfide bonds). Both targeted MS²-based, and data-dependent acquisition (DDA)-based top-down workflows were established to accommodate different analytical needs. The developed top-down strategies enabled direct sequencing of proteins with highly repetitive sequences, which pose challenges for bottom-up methods. The findings of this study provide insights into the identification of proteins with molecular weights (MWs) below 50 kDa. First, for proteins with MWs below 12 kDa, DDA-based top-down analysis utilizing HCD fragmentation provides high-throughput identification with 50 % (eg. of 27 fragments for protein RC1). Second, for proteins with MWs greater than 12 kDa, target-MS²-based top-down approaches improve sequence coverage, achieving up to 49.5 % for RC2 by combining three dissociation strategies. Third, for proteins containing disulfide bonds, disulfide reduce pretreatment prior to MS analysis is necessary to achieve improved residue cleavage coverage. Overall, this work demonstrates that top-down mass spectrometry provides complementary data to bottom-up methods, delivering crucial insights for comprehensive characterization of therapeutic recombinant proteins.