Novel Highly Sensitive, Specific, and Straightforward Strategy for Comprehensive N-Terminal Proteomics Reveals Unknown Substrates of the Mitochondrial Peptidase Icp55

作者：Venne A Saskia; Voegtle F Nora; Meisinger Chris; Sickmann Albert; Zahedi Rene P^*

来源：Journal of Proteome Research, 2013, 12(9): 3823-3830.

DOI：10.1021/pr400435d

• degradomics
• N-terminal
• COFRADIC
• Icp55
• protease
• peptidase

We present a novel straightforward method for enrichment of N-terminal peptides, utilizing charge-based fractional diagonal chromatography (ChaFRADIC). Our method is robust, easy to operate, fast, specific, and more sensitive than existing methods, enabling the differential quantitation of 1459 nonredundant N-terminal peptides between two S. cerevisiae samples within 10 h of LC-MS, starting from only 50 mu g of protein per condition and analyzing only 40% of the obtained fractions. Using ChaFRADIC we compared mitochondrial proteins from wild-type and icp55 Delta yeast (30 mu g each). Icp55 is an intermediate cleaving peptidase, which, following mitochondrial processing peptidase (MPP)-dependent cleavage of signal sequences, removes a single amino acid from a specific set of proteins according to the N-end rule. Using ChaFRADIC we identified 36 icp55 substrates, 14 of which were previously unknown, expanding the set of known icp55 substrates to a total of 52 proteins. Interestingly, a novel substrate, Isa2, is likely processed by Icp55 in two consecutive steps and thus might represent the first example of a triple processing event in a mitochondrial precursor protein. Thus, ChaFRADIC is a powerful and practicable tool for protease and peptidase research, providing the sensitivity to characterize even samples that can be obtained only in small quantities.

• 出版日期2013-9