title: The Separation and Quantitation of Peptides with and without Oxidation of Methionine and Deamidation of Asparagine Using Hydrophilic Interaction Liquid Chromatography with Mass Spectrometry (HILIC-MS).
abstract: Peptides with deamidated asparagine residues and oxidized methionine residues are often not resolved sufficiently to allow quantitation of their native and modified forms using reversed phase (RP) chromatography. The accurate quantitation of these modifications is vital in protein biotherapeutic analysis because they can affect a protein's function, activity, and stability. We demonstrate here that hydrophilic interaction liquid chromatography (HILIC) adequately and predictably separates peptides with these modifications from their native counterparts. Furthermore, coefficients describing the extent of the hydrophilicity of these modifications have been derived and were incorporated into a previously made peptide retention prediction model that is capable of predicting the retention times of peptides with and without these modifications. Graphical Abstract ᅟ.
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia, Athens, GA, 30602, USA.
authors.forename: Majors J
authors.initials: MJ
authors.lastname: Badgett
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia, Athens, GA, 30602, USA.|Advanced Materials Technology, Wilmington, DE, 19810, USA.
authors.forename: Barry
authors.initials: B
authors.lastname: Boyes
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia, Athens, GA, 30602, USA. orlando@ccrc.uga.edu.
authors.forename: Ron
authors.initials: R
authors.lastname: Orlando
mesh_terms: D000577:Amides / Q000032:analysis; D000595:Amino Acid Sequence; D001216:Asparagine / Q000032:analysis; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007074:Immunoglobulin G / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008715:Methionine / Q000032:analysis; D010084:Oxidation-Reduction; D010455:Peptides / Q000032:analysis*
chemical_list: D000577:Amides; D007074:Immunoglobulin G; D010455:Peptides; D001216:Asparagine; D008715:Methionine
keywords: Deamidation of asparagine; Hydrophilic interaction liquid chromatography (HILIC); Mass spectrometry (MS); Modified peptides; Oxidation of methionine; Peptide prediction retention model; Protein biotherapeutics; Quantitation; Separation