title: High throughput quantification of N-glycans using one-pot sialic acid modification and matrix assisted laser desorption ionization time-of-flight mass spectrometry.
abstract: Appropriate glycosylation of recombinant therapeutic glycoproteins has been emphasized in biopharmaceutical industries because the carbohydrate component can affect safety, efficacy, and consistency of the glycoproteins. Reliable quantification methods are essential to ensure consistency of their products with respect to glycosylation, particularly sialylation. Mass spectrometry (MS) has become a popular tool to analyze glycan profiles and structures, showing high resolution and sensitivity with structure identification ability. However, quantification of sialylated glycans using MS is not as reliable because of the different ionization efficiency between neutral and acidic glycans. We report here that amidation in mild acidic conditions can be used to neutralize acidic N-glycans still attached to the protein. The resulting amidated N-glycans can then be released from the protein using PNGase F, and labeled with permanent charges on the reducing end to avoid any modification and the formation of metal adducts during MS analysis. The N-glycan modification, digestion, and desalting steps were performed using a single-pot method that can be done in microcentrifuge tubes or 96-well microfilter plates, enabling high throughput glycan analysis. Using this method we were able to perform quantitative MALDI-TOF MS of a recombinant human glycoprotein to determine changes in fucosylation and changes in sialylation that were in very good agreement with a normal phase HPLC oligosaccharide mapping method.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Nebraska-Lincoln, Lincoln, Nebraska 68588, USA.
authors.forename: Geun-Cheol
authors.initials: GC
authors.lastname: Gil
authors.forename: Bryce
authors.initials: B
authors.lastname: Iliff
authors.forename: Ron
authors.initials: R
authors.lastname: Cerny
authors.forename: William H
authors.initials: WH
authors.lastname: Velander
authors.forename: Kevin E
authors.initials: KE
authors.lastname: Van Cott
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006031:Glycosylation; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism; D019158:N-Acetylneuraminic Acid / Q000737:chemistry*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011134:Polysaccharides / Q000032:analysis*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D007074:Immunoglobulin G; D011134:Polysaccharides; D011994:Recombinant Proteins; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D019158:N-Acetylneuraminic Acid