on 12-01-201612:17 AM - edited on 10-15-202106:30 AM by AnalyteGuru
Luca Fornelli, Kenneth Robert Durbin, Ryan T. Fellers, Bryan P. Early, Joseph B. Greer, Richard D. LeDuc, Philip D. Compton, and Neil L. Kelleher
Over the past decade, developments in high resolution mass spectrometry have enabled the high throughput analysis of intact proteins from complex proteomes, leading to the identification of thousands of proteoforms. Several previous reports on top-down proteomics (TDP) relied on hybrid ion trap – Fourier transform mass spectrometers combined with data-dependent acquisition strategies. To further reduce TDP to practice, we use a quadrupole – Orbitrap instrument coupled with software for proteoform-dependent data acquisition to identify and characterize nearly 2,000 proteoforms at a 1% false discovery rate from human fibroblasts. By combining a 3 m/z isolation window with short transients to improve specificity and signal-to-noise for proteoforms >30 kDa, we demonstrate improving proteome coverage by capturing 439 proteoforms in the 30 to 60 kDa range. Three different data acquisition strategies were compared and resulted in the identification of many proteoforms not observed in replicate data-dependent experiments. Notably, the dataset is reported with updated metrics and tools, including a new viewer and assignment of permanent Proteoform Record identifiers for inclusion of highly-characterized proteoforms (i.e., those with Cscores >40) in a repository curated by the Consortium for Top Down Proteomics.