Application of a combined weak cation-exchange/crown ether column: first demonstrations of a versatile tool for proteome subselection.
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.
Tuytten
Robin
R
Pronota NV, VIB Bioincubator, Technologiepark 4, B-9052 Zwijnaarde Ghent, Belgium. robin.tuytten@pronota.com
Ruttens
Bart
B
Gheysen
Katelijne
K
Sandra
Koen
K
De Cremer
Koen
K
Vlieghe
Dominique
D
Van Landuyt
Natalie
N
Thomas
Grégoire
G
Martins
José C
JC
Sandra
Pat
P
Kas
Koen
K
Verleysen
Katleen
K
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*
D043844:Crown Ethers; D006020:Glycopeptides; D020543:Proteome; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase