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Reference: Venne AS, et al. (2013) Novel highly sensitive, specific, and straightforward strategy for comprehensive N-terminal proteomics reveals unknown substrates of the mitochondrial peptidase icp55. J Proteome Res 12(9):3823-30

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Abstract


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 mug of protein per condition and analyzing only 40% of the obtained fractions. Using ChaFRADIC we compared mitochondrial proteins from wild-type and icp55Delta yeast (30 mug 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.

Reference Type
Journal Article
Authors
Venne AS, Vogtle FN, Meisinger C, Sickmann A, Zahedi RP
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