An improved workflow for quantitative N-terminal charge-based fractional diagonal chromatography (ChaFRADIC) to study proteolytic events in Arabidopsis thaliana

作者：Venne A Saskia; Solari Fiorella A; Faden Frederik; Paretti Tomasso; Dissmeyer Nico; Zahedi Rene P^*

来源：Proteomics, 2015, 15(14): 2458-2469.

DOI：10.1002/pmic.201500014

• NERD
• N-End rule pathway
• N-Terminal methionine excision
• Protein degradation
• Technology
• Two-dimensional chromatography

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 g 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 g 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 ().

• 出版日期2015-7