Novel highly sensitive, specific, and straightforward strategy for comprehensive N-terminal proteomics reveals unknown substrates of the mitochondrial peptidase Icp55

J Proteome Res. 2013 Sep 6;12(9):3823-30. doi: 10.1021/pr400435d. Epub 2013 Aug 21.

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 μg of protein per condition and analyzing only 40% of the obtained fractions. Using ChaFRADIC we compared mitochondrial proteins from wild-type and icp55Δ yeast (30 μg 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Aminopeptidases / chemistry*
  • Aminopeptidases / physiology
  • Chromatography, Gel
  • Chromatography, Ion Exchange
  • Mitochondrial Proteins / chemistry
  • Mitochondrial Proteins / isolation & purification*
  • Mitochondrial Proteins / metabolism
  • Molecular Sequence Data
  • Peptide Fragments / chemistry
  • Peptide Fragments / isolation & purification*
  • Peptide Mapping
  • Protein Processing, Post-Translational
  • Proteolysis
  • Proteome / metabolism*
  • Proteomics
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / isolation & purification*
  • Saccharomyces cerevisiae Proteins / metabolism
  • Saccharomyces cerevisiae Proteins / physiology
  • Sensitivity and Specificity
  • Sequence Analysis, Protein
  • Substrate Specificity
  • Tandem Mass Spectrometry

Substances

  • ISA2 protein, S cerevisiae
  • Mitochondrial Proteins
  • Peptide Fragments
  • Proteome
  • Saccharomyces cerevisiae Proteins
  • Icp55 protein, S cerevisiae
  • Aminopeptidases