title: Applying label-free quantitation to top down proteomics.
abstract: With the prospect of resolving whole protein molecules into their myriad proteoforms on a proteomic scale, the question of their quantitative analysis in discovery mode comes to the fore. Here, we demonstrate a robust pipeline for the identification and stringent scoring of abundance changes of whole protein forms <30 kDa in a complex system. The input is ~100-400 μg of total protein for each biological replicate, and the outputs are graphical displays depicting statistical confidence metrics for each proteoform (i.e., a volcano plot and representations of the technical and biological variation). A key part of the pipeline is the hierarchical linear model that is tailored to the original design of the study. Here, we apply this new pipeline to measure the proteoform-level effects of deleting a histone deacetylase (rpd3) in S. cerevisiae. Over 100 proteoform changes were detected above a 5% false positive threshold in WT vs the Δrpd3 mutant, including the validating observation of hyperacetylation of histone H4 and both H2B isoforms. Ultimately, this approach to label-free top down proteomics in discovery mode is a critical technical advance for testing the hypothesis that whole proteoforms can link more tightly to complex phenotypes in cell and disease biology than do peptides created in shotgun proteomics.
authors.affiliation: Departments of Chemistry, Molecular Biosciences and the Proteomics Center of Excellence , 2145 N. Sheridan Road, Evanston, Illinois 60208, United States.
authors.forename: Ioanna
authors.initials: I
authors.lastname: Ntai
authors.forename: Kyunggon
authors.initials: K
authors.lastname: Kim
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Fellers
authors.forename: Owen S
authors.initials: OS
authors.lastname: Skinner
authors.forename: Archer D
authors.initials: AD
authors.lastname: Smith
authors.forename: Bryan P
authors.initials: BP
authors.lastname: Early
authors.forename: John P
authors.initials: JP
authors.lastname: Savaryn
authors.forename: Richard D
authors.initials: RD
authors.lastname: LeDuc
authors.forename: Paul M
authors.initials: PM
authors.lastname: Thomas
authors.forename: Neil L
authors.initials: NL
authors.lastname: Kelleher
mesh_terms: D006655:Histone Deacetylases / Q000032:analysis / Q000235:genetics; D009154:Mutation / Q000235:genetics; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics
chemical_list: D011506:Proteins; D006655:Histone Deacetylases