title: Novel highly sensitive, specific, and straightforward strategy for comprehensive N-terminal proteomics reveals unknown substrates of the mitochondrial peptidase Icp55.
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.
authors: Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., Dortmund, Germany.
authors: A Saskia
authors: AS
authors: Venne
authors: F-Nora
authors: FN
authors: Vögtle
authors: Chris
authors: C
authors: Meisinger
authors: Albert
authors: A
authors: Sickmann
authors: René P
authors: RP
authors: Zahedi
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry* / Q000502:physiology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification*; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology; D012680:Sensitivity and Specificity; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: C410064:ISA2 protein, S cerevisiae; D024101:Mitochondrial Proteins; D010446:Peptide Fragments; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; C545440:Icp55 protein, S cerevisiae; D000626:Aminopeptidases