Selective enrichment of glycopeptides from glycoprotein digests using ion-pairing normal-phase liquid chromatography.
Detailed structural analysis of glycoproteins requires methods capable of isolating glycopeptides from tryptic digests of purified glycoproteins and complex protein mixtures. Here, we describe the selective and reproducible isolation of glycopeptides from a peptide mixture using ion-pairing normal-phase chromatography (IP-NPLC). The addition of inorganic monovalent ions in normal-phase chromatography appears to increase the hydrophobicity difference between peptides and glycopeptides, allowing for more efficient separation. Our data show that IP-NPLC effectively enriches glycopeptides from a tryptic digest of ribonuclease B, bovine fetuin, and a complex mixture of glycoproteins, when compared with normal-phase chromatography alone. The results of the IP-NPLC experiments can be explained using the Wimley-White water/octanol free energy scale to illustrate the hydrophobicity difference of nonglycosylated peptides with and without ion-pairing. We believe that IP-NPLC will be an important tool in glycoprotein characterization and glycoproteomic studies.
Ding
Wen
W
Genomics and Proteomics Group, Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada K1A 0R6. Wen.Ding@nrc-cnrc.gc.ca
Hill
Jennifer J
JJ
Kelly
John
J
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006020:Glycopeptides / Q000302:isolation & purification*; D006023:Glycoproteins / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D014357:Trypsin / Q000737:chemistry
D006020:Glycopeptides; D006023:Glycoproteins; D014357:Trypsin