Purpose: Development of protein product quality control method using mass spectrometric intact/top-down and bottom-up analysis. Methods: For bottom up experiments, proteins were analyzed by nanoLC/MS. Intact and fragment samples were analyzed by standard flow LC- MS using a Thermo Scientific™ MabPac ™RP (2.1x50mm, 4 μm) column and a Thermo Scientific™ Orbitrap Fusion Lumos™ mass spectrometer. For top-down MS/MS experiments, ETD, EThcD, CID or HCD MS2 fragmentation were used at a resolution of 120K@ m/z 200. Results: We identified and quantified different forms of studied proteins by both bottom-up and top down approaches. The quantitative results produced by intact protein analysis strategy were more consistent. In general, the intact/top-down method outperformed the bottom up approach in these studies in terms of both efficiency and accuracy. Overall, a hybrid approach that combines the advantages of both bottom up and intact/top-down methods would be the most optimal for this type of experiment.
1Thermo Fisher Scientific, San Jose, CA 2Thermo Fisher Scientific, South San Francisco, CA 3Thermo Fisher Scientific, Rockford, IL