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.

Glycopeptide enrichment and separation for protein glycosylation analysis

“…In addition, a weak cation exchange/crown ether column, originally proposed for the analysis of inorganic ions was employed for the separation of peptides with free α‐NH 2 or ε‐NH 2 groups from lysine side chains by Tuytten et al. . Interestingly, this also led to the co‐elution of glycopeptides .…”

“…. Interestingly, this also led to the co‐elution of glycopeptides . Unfortunately, the investigation of this interesting column for proteomics purposes was not continued and the mechanism of separation remains unclear.…”