title: An improved workflow for quantitative N-terminal charge-based fractional diagonal chromatography (ChaFRADIC) to study proteolytic events in Arabidopsis thaliana.
abstract: 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).
authors.affiliation: Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V, Dortmund, Germany.
authors.forename: A Saskia
authors.initials: AS
authors.lastname: Venne
authors.affiliation: Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V, Dortmund, Germany.
authors.forename: Fiorella A
authors.initials: FA
authors.lastname: Solari
authors.affiliation: Leibniz Institute of Plant Biochemistry (IPB), Halle (Saale), Germany.|ScienceCampus Halle - Plant-Based Bioeconomy, Halle (Saale), Germany.
authors.forename: Frederik
authors.initials: F
authors.lastname: Faden
authors.affiliation: Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V, Dortmund, Germany.|Department of Molecular Medicine, Institute of Biochemistry, University of Pavia, Italy.
authors.forename: Tomasso
authors.initials: T
authors.lastname: Paretti
authors.affiliation: Leibniz Institute of Plant Biochemistry (IPB), Halle (Saale), Germany.|ScienceCampus Halle - Plant-Based Bioeconomy, Halle (Saale), Germany.
authors.forename: Nico
authors.initials: N
authors.lastname: Dissmeyer
authors.affiliation: Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V, Dortmund, Germany.
authors.forename: René P
authors.initials: RP
authors.lastname: Zahedi
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods; D008715:Methionine / Q000032:analysis / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D059748:Proteolysis; D040901:Proteomics / Q000379:methods; D057188:Workflow
chemical_list: D029681:Arabidopsis Proteins; D010455:Peptides; D008715:Methionine
keywords: N-End rule pathway; N-Terminal methionine excision; NERD; Protein degradation; Technology; Two-dimensional chromatography