1Rosa Viner, 2Nan Liu, 1Seema Sharma, 3Sergei Snovida, 3Krishna Vattern, 1David Horn,
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
United States
