title: Online coupling of reverse-phase and hydrophilic interaction liquid chromatography for protein and glycoprotein characterization.
abstract: We have developed a novel system for coupling reverse-phase (RP) and hydrophilic interaction liquid chromatography (HILIC) online in a micro-flow scheme. In this approach, the inherent solvent incompatibility between RP and HILIC is overcome through the use of constant-pressure online solvent mixing, which allows our system to perform efficient separations of both hydrophilic and hydrophobic compounds for mass spectrometry-based proteomics applications. When analyzing the tryptic digests of bovine serum albumin, ribonuclease B, and horseradish peroxidase, we observed near-identical coverage of peptides and glycopeptides when using online RP-HILIC--with only a single sample injection event--as we did from two separate RP and HILIC analyses. The coupled system was also capable of concurrently characterizing the peptide and glycan portions of deglycosylated glycoproteins from one injection event, as confirmed, for example, through our detection of 23 novel glycans from turkey ovalbumin. Finally, we validated the applicability of using RP-HILIC for the analysis of highly complex biological samples (mouse chondrocyte lysate, deglycosylated human serum). The enhanced coverage and efficiency of online RP-HILIC makes it a viable technique for the comprehensive separation of components displaying dramatically different hydrophobicities, such as peptides, glycopeptides, and glycans.
authors.affiliation: Department of Chemistry, The University of Hong Kong, Hong Kong, China.
authors.forename: Maggie P Y
authors.initials: MP
authors.lastname: Lam
authors.forename: S O
authors.initials: SO
authors.lastname: Siu
authors.forename: Edward
authors.initials: E
authors.lastname: Lau
authors.forename: Xiuli
authors.initials: X
authors.lastname: Mao
authors.forename: H Z
authors.initials: HZ
authors.lastname: Sun
authors.forename: Philip C N
authors.initials: PC
authors.lastname: Chiu
authors.forename: William S B
authors.initials: WS
authors.lastname: Yeung
authors.forename: David M
authors.initials: DM
authors.lastname: Cox
authors.forename: Ivan K
authors.initials: IK
authors.lastname: Chu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods; D056148:Chromatography, Reverse-Phase / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D006023:Glycoproteins / Q000032:analysis* / Q000302:isolation & purification; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000032:analysis / Q000302:isolation & purification; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification; D040901:Proteomics / Q000295:instrumentation* / Q000379:methods; D044967:Serum / Q000737:chemistry
chemical_list: D006023:Glycoproteins; D011134:Polysaccharides; D011506:Proteins