title: Enrichment of protein N-termini by charge reversal of internal peptides.
abstract: Protein N-termini provide useful information for the understanding of posttranslational processing of proteins. The majority of proteins undergo N-terminal processing, such as proteolytic truncation or modifications like acetylation. Multiple methods currently exist for the enrichment of N-terminal peptides for proteomic analyses. Here, we report a novel, simple, and straightforward N-terminomic strategy, based on charge reversal of internal peptides followed by their removal through strong cation exchange chromatography. Our initial proof-of-concept study shows the feasibility of this technique, yielding over 3000 identifications of protein N-termini. We further show the application of this strategy in investigating the N-terminome of mouse embryonic fibroblasts cells deficient for both cathepsin B and L in comparison to wild type) control cells. Finally, we demonstrate that this workflow can be used in combination with a gel-based strategy, allowing preseparation of proteins and thus providing an estimate of the molecular weight of the identified cleavage products.
authors.affiliation: Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany.
authors.forename: Zon W
authors.initials: ZW
authors.lastname: Lai
authors.affiliation: Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany.|Faculty of Biology, University of Freiburg, Freiburg, Germany.|Spemann Graduate School of Biology and Medicine, University of Freiburg, Freiburg, Germany.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Gomez-Auli
authors.affiliation: Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany.
authors.forename: Eva J
authors.initials: EJ
authors.lastname: Keller
authors.affiliation: Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany.
authors.forename: Bettina
authors.initials: B
authors.lastname: Mayer
authors.affiliation: Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany.
authors.forename: Martin L
authors.initials: ML
authors.lastname: Biniossek
authors.affiliation: Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany.|Spemann Graduate School of Biology and Medicine, University of Freiburg, Freiburg, Germany.|BIOSS Centre for Biological Signaling Studies, University of Freiburg, Freiburg, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Schilling
mesh_terms: D000818:Animals; D002401:Cathepsin B / Q000235:genetics; D056668:Cathepsin L / Q000235:genetics; D002460:Cell Line; D002852:Chromatography, Ion Exchange / Q000379:methods*; D005347:Fibroblasts / Q000737:chemistry / Q000378:metabolism; D017353:Gene Deletion; D051379:Mice; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D055672:Static Electricity; D053719:Tandem Mass Spectrometry
chemical_list: D010455:Peptides; D011506:Proteins; D002401:Cathepsin B; D056668:Cathepsin L
keywords: N-terminomics; Proteases; Proteolysis; TAILS; Technology