Large volume injection of aqueous peptide samples on a monolithic silica based zwitterionic-hydrophilic interaction liquid chromatography system for characterization of posttranslational modifications.
Zwitterionic-hydrophilic interaction liquid chromatography (HILIC) has been found very appropriate for separation of polar compounds and peptides with post-translational modifications (PTMs) such as phosphorylation and glycosylation. In this study, a column switching system based on zwitterionic-HILIC silica based monolith columns was used for enrichment and separation of peptides and characterization of N-linked glycosylation by higher-energy collisional dissociation (HCD) Orbitrap mass spectrometry (MS). Peptides were found to be retained on a zwitterionic-HILIC precolumn, even in an aqueous buffer due to electrostatic interactions. Thus, a novel approach of using a zwitterionic-HILIC precolumn, for introduction of an aqueous sample such as a tryptic digest, followed by HILIC separation of the peptides is presented. The repeatability and loadability of the zwitterionic-HILIC-zwitterionic-HILIC column switching system were explored using a tryptic digest of transferrin and a mixture of six proteins. The column switching system was furthermore used to enrich and separate a tryptic digested rat liver extract gel fraction, where in total 48 peptides corresponding to 14 proteins were identified. N-linked glycoforms were also identified, both in the standard test proteins (transferrin and six protein mixture digest) and the rat liver extract fraction. In all cases, the identified N-linked glycoforms were identified at the end of the gradient, at high aqueous buffer content in the mobile phase, showing the suitability of the developed method for characterization of glycosylated peptides in aqueous samples.
Department of Chemistry, University of Oslo, P.O. Box 1033, Blindern, 0315 Oslo, Norway. Electronic address: helle.malerod@kjemi.uio.no.
Helle
H
Malerod
Magnus
M
Rogeberg
Nobuo
N
Tanaka
Tyge
T
Greibrokk
Elsa
E
Lundanes
D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid / Q000295:instrumentation* / Q000379:methods*; D006020:Glycopeptides / Q000032:analysis*; D006031:Glycosylation; D057927:Hydrophobic and Hydrophilic Interactions; D008099:Liver / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D051381:Rats; D015203:Reproducibility of Results; D012822:Silicon Dioxide / Q000737:chemistry; D014357:Trypsin
D006020:Glycopeptides; D010446:Peptide Fragments; D012822:Silicon Dioxide; D014357:Trypsin
Aqueous sample; Large volume injection; N-linked glycoforms; Silica based monolith; Zwitterionic-HILIC