Bailey Aaron

and 4 more

LC-MS intact mass analysis and LC-MS/MS peptide mapping are foundational assays for developing biologic drugs and other commercial protein products. Certain PTM types, such as truncation and oxidation, increase the difficulty of precise proteoform characterization owing to inherent limitations in peptide and intact protein analyses. Top-down MS (TDMS) can resolve this ambiguity via fragmentation of specific proteoforms. We optimized our existing flow-programmed denaturing online buffer exchange ((fp)dOBE) approach to improve ESI sensitivity and increase TDMS sampling time for industrial applications. Using bovine alpha-lactalbumin (αLac), we tested data-dependent (DDA) and targeted strategies with 14 different MS/MS scan types featuring combinations of collisional- and electron-based fragmentation as well as proton transfer charge reduction. This large dataset was processed using a new software platform, named TDAcquireX, that improves proteoform characterization through TDMS data aggregation. (fp)dOBE-based DDA-TDMS analysis readily identified truncated proteoforms. For targeted TDMS, we used Sliding Window fragment ion deconvolution to analyze composite proteoform (cPrSM) results. This strategy facilitates probability-based noise filtering of individual fragments, simultaneously increasing matched fragments while decreasing total fragment masses. Using this strategy, we characterized oxidation positional isomers on αLac, finding ETD fragmentation uniquely provided accurate relative occupancy ratios by oxidation-specific challenges.