title: Quantitation and Identification of Thousands of Human Proteoforms below 30 kDa.
abstract: 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.
authors.affiliation: Departments of Chemistry and Molecular Biosciences, Northwestern University , 2170 Campus Drive, Evanston, Illinois 60208, United States.
authors.forename: Kenneth R
authors.initials: KR
authors.lastname: Durbin
authors.affiliation: Departments of Chemistry and Molecular Biosciences, Northwestern University , 2170 Campus Drive, Evanston, Illinois 60208, United States.
authors.forename: Luca
authors.initials: L
authors.lastname: Fornelli
authors.affiliation: Departments of Chemistry and Molecular Biosciences, Northwestern University , 2170 Campus Drive, Evanston, Illinois 60208, United States.
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Fellers
authors.affiliation: Departments of Chemistry and Molecular Biosciences, Northwestern University , 2170 Campus Drive, Evanston, Illinois 60208, United States.
authors.forename: Peter F
authors.initials: PF
authors.lastname: Doubleday
authors.affiliation: Cancer Research UK Cambridge Institute, Li Ka Shing Centre, University of Cambridge , Robinson Way, Cambridge CB2 0RE, U.K.
authors.forename: Masashi
authors.initials: M
authors.lastname: Narita
authors.affiliation: Departments of Chemistry and Molecular Biosciences, Northwestern University , 2170 Campus Drive, Evanston, Illinois 60208, United States.
authors.forename: Neil L
authors.initials: NL
authors.lastname: Kelleher
mesh_terms: D002478:Cells, Cultured; D016922:Cellular Senescence; D056148:Chromatography, Reverse-Phase; D006801:Humans; D020033:Protein Isoforms / Q000378:metabolism; D020543:Proteome / Q000378:metabolism*; D012984:Software; D053719:Tandem Mass Spectrometry
chemical_list: D020033:Protein Isoforms; D020543:Proteome
keywords: Fourier transform mass spectrometry; GELFrEE; cellular senescence; differential mass spectrometry; label-free; proteoform; quantitative proteomics; top-down proteomics