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

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.