Filling the Gaps in Peptide Maps with a Platform Assay for Top-Down
Characterization of Purified Protein Samples
Abstract
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.