期刊
JOURNAL OF PROTEOME RESEARCH
卷 12, 期 9, 页码 3823-3830出版社
AMER CHEMICAL SOC
DOI: 10.1021/pr400435d
关键词
degradomics; N-terminal; COFRADIC; Icp55; protease; peptidase
资金
- Ministerium fur Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen
- Bundesministerium fur Bildung und Forschung (DYNAMO)
- Deutsche Forschungsgemeinschaft [DFG ZA 639/1-1]
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.
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