Quantitation and Identification of Thousands of Human Proteoforms below 30 kDa

Top-down proteomics is capable of
identifying and quantitating
unique proteoforms through the analysis of intact proteins. We extended
the coverage of the label-free technique, achieving differential analysis
of whole proteins <30 kDa from the proteomes of growing and senescent
human fibroblasts. By integrating improved control software with more
instrument time allocated for quantitation of intact ions, we were
able to collect protein data between the two cell states, confidently
comparing 1577 proteoform levels. To then identify and characterize
proteoforms, our advanced acquisition software, named Autopilot, employed enhanced identification efficiency in identifying 1180
unique Swiss-Prot accession numbers at 1% false-discovery rate. This
coverage of the low mass proteome is equivalent to the largest previously
reported but was accomplished in 23% of the total acquisition time.
By maximizing both the number of quantified proteoforms and their
identification rate in an integrated software environment, this work
significantly advances proteoform-resolved analyses of complex systems.