Publication Information
Venne et al., 2015
Abstract
Proteomics. 2015 Jul;15(14):2458-69. doi: 10.1002/pmic.201500014.
An improved workflow for quantitative N-terminal charge-based fractional
diagonal chromatography (ChaFRADIC) to study proteolytic events in Arabidopsis
thaliana.
Venne AS(1), Solari FA(1), Faden F(2)(3), Paretti T(1)(4), Dissmeyer N(2)(3),
Zahedi RP(1).
Author information:
(1)Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V, Dortmund,
Germany.
(2)Leibniz Institute of Plant Biochemistry (IPB), Halle (Saale), Germany.
(3)ScienceCampus Halle - Plant-Based Bioeconomy, Halle (Saale), Germany.
(4)Department of Molecular Medicine, Institute of Biochemistry, University of
Pavia, Italy.
We applied an extended charge-based fractional diagonal chromatography
(ChaFRADIC) workflow to analyze the N-terminal proteome of Arabidopsis thaliana
seedlings. Using iTRAQ protein labeling and a multi-enzyme digestion approach
including trypsin, GluC, and subtilisin, a total of 200 μg per enzyme, and
measuring only one third of each ChaFRADIC-enriched fraction by LC-MS, we
quantified a total of 2791 unique N-terminal peptides corresponding to 2249
different unique N-termini from 1270 Arabidopsis proteins. Our data indicate the
power, reproducibility, and sensitivity of the applied strategy that might be
applicable to quantify proteolytic events from as little as 20 μg of protein per
condition across up to eight different samples. Furthermore, our data clearly
reflect the methionine excision dogma as well as the N-end rule degradation
pathway (NERP) discriminating into a stabilizing or destabilizing function of
N-terminal amino acid residues. We found bona fide NERP destabilizing residues
underrepresented, and the list of neo N-termini from wild type samples may
represent a helpful resource during the evaluation of NERP substrate candidates.
All MS data have been deposited in the ProteomeXchange with identifier PXD001855
(http://proteomecentral.proteomexchange.org/dataset/PXD001855).
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
DOI: 10.1002/pmic.201500014
PMID: 26010716 [Indexed for MEDLINE]
