title: Application of a combined weak cation-exchange/crown ether column: first demonstrations of a versatile tool for proteome subselection.
abstract: The present paper introduces the use of a weak cation-exchange/crown ether column in the proteomics field. The 18-crown-6 ether functionality is well-known to selectively complex ammonium and monoalkylammonium ions, which should make this column highly suitable to trap peptides with free alpha-NH(2) or free epsilon-NH(2) groups from lysine side chains. This unique selection mechanism was put to the test in an N-teromics setup which aims for the enrichment of deliberately acetylated protein N-terminal peptides from a serum digest. It was demonstrated that peptides with free alpha-NH(2) groups and peptides with alpha-amino-acetylated groups can be separated from each other using this weak cation-exchange/crown ether column. The peptides of interest, bearing no free primary amines, were found to be significantly enriched in the column's flow through. At the same time a favorable coenrichment of N-glycosylated peptides was observed. To obtain more insight in the contributions of the two distinct column functionalities, i.e., the weak cation exchanger and the crown ether, the experimental data were checked against a theoretical prediction of the outcome.
authors.affiliation: Pronota NV, VIB Bioincubator, Technologiepark 4, B-9052 Zwijnaarde Ghent, Belgium. robin.tuytten@pronota.com
authors.forename: Robin
authors.initials: R
authors.lastname: Tuytten
authors.forename: Bart
authors.initials: B
authors.lastname: Ruttens
authors.forename: Katelijne
authors.initials: K
authors.lastname: Gheysen
authors.forename: Koen
authors.initials: K
authors.lastname: Sandra
authors.forename: Koen
authors.initials: K
authors.lastname: De Cremer
authors.forename: Dominique
authors.initials: D
authors.lastname: Vlieghe
authors.forename: Natalie
authors.initials: N
authors.lastname: Van Landuyt
authors.forename: Grégoire
authors.initials: G
authors.lastname: Thomas
authors.forename: José C
authors.initials: JC
authors.lastname: Martins
authors.forename: Pat
authors.initials: P
authors.lastname: Sandra
authors.forename: Koen
authors.initials: K
authors.lastname: Kas
authors.forename: Katleen
authors.initials: K
authors.lastname: Verleysen
mesh_terms: D000595:Amino Acid Sequence; D016477:Artifacts; D043844:Crown Ethers; D006020:Glycopeptides / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D010865:Pilot Projects; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000295:instrumentation* / Q000379:methods*
chemical_list: D043844:Crown Ethers; D006020:Glycopeptides; D020543:Proteome; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase