Applying high-throughput Top-Down MS to an entire proteome requires a 
yet-to-be-established model for data processing. Since Top-Down is becoming 
possible on a large scale, we report our latest software pipeline dedicated to 
capturing the full value of intact protein data in automated fashion. For intact 
mass detection, we combine algorithms for processing MS1 data from both 
isotopically resolved (FT) and charge-state resolved (ion trap) LC-MS data, 
which are then linked to their fragment ions for database searching using 
ProSight. Automated determination of human keratin and tubulin isoforms is one 
result. Optimized for the intricacies of whole proteins, new software modules 
visualize proteome-scale data based on the LC retention time and intensity of 
intact masses and enable selective detection of PTMs to automatically screen for 
acetylation, phosphorylation, and methylation. Software functionality was 
demonstrated using comparative LC-MS data from yeast strains in addition to 
human cells undergoing chemical stress. We further these advances as a key 
aspect of realizing Top-Down MS on a proteomic scale.