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Anal Chem. 2014 May 20;86(10):4961-8. doi: 10.1021/ac500395k. Epub 2014 May 7.

Applying label-free quantitation to top down proteomics.

Author information

1
Departments of Chemistry, Molecular Biosciences and the Proteomics Center of Excellence , 2145 N. Sheridan Road, Evanston, Illinois 60208, United States.

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.

PMID:
24807621
PMCID:
PMC4033644
DOI:
10.1021/ac500395k
[Indexed for MEDLINE]
Free PMC Article

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