title: A novel HPLC-UV-MS method for quantitative analysis of protein glycosylation.
abstract: Monoclonal antibody samples derived from transgenic plants (plantibodies) may often contain significant amounts of aglycosylated variants. Because glycosylated and non-/de-glycosylated proteins exhibit different functional and pharmacokinetic properties, accurate measurement of non- and de-glycosylated glycoprotein abundances is important. Glycosylation of plant-derived glycoproteins presents specific challenges. Here we describe a novel method to accurately measure relative and absolute amounts of non-glycosylated, de-glycosylated, and total glycosylated protein using an HPLC-UV-MS methodology. Additionally, these results were compared with glycopeptide profiling by MALDI MS. Our studies demonstrated that the quantitative aspect of HPLC-UV method was superior to MALDI MS profiling, which significantly overestimated the relative amounts of aglycosylated species in the isolated glycopeptide fractions.
authors.affiliation: The Dow Chemical Company, Analytical Sciences, BioAnalytical, 1897 Building, Midland, MI 48667, USA. ASKarnoup@dow.com
authors.forename: Anton S
authors.initials: AS
authors.lastname: Karnoup
authors.forename: Krishna
authors.initials: K
authors.lastname: Kuppannan
authors.forename: Scott A
authors.initials: SA
authors.lastname: Young
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000032:analysis; D061067:Antibodies, Monoclonal, Humanized; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006023:Glycoproteins / Q000032:analysis*; D006031:Glycosylation; D007074:Immunoglobulin G / Q000032:analysis; D010446:Peptide Fragments / Q000032:analysis; D011506:Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000068878:Trastuzumab; D014466:Ultraviolet Rays
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D006023:Glycoproteins; D007074:Immunoglobulin G; D010446:Peptide Fragments; D011506:Proteins; D000068878:Trastuzumab