High Throughput Quantification of N-Glycans Using One-Pot Sialic Acid Modification and Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry

摘要

Appropriate glycosylation of recombinant therapeutic glycoproteinsnhas been emphasized in biopharmaceuticalnindustries because the carbohydrate component can affectnsafety, efficacy, and consistency of the glycoproteins.nReliable quantification methods are essential to ensurenconsistency of their products with respect to glycosylation,nparticularly sialylation. Mass spectrometry (MS) hasnbecome a popular tool to analyze glycan profiles andnstructures, showing high resolution and sensitivity withnstructure identification ability. However, quantification ofnsialylated glycans using MS is not as reliable because ofnthe different ionization efficiency between neutral andnacidic glycans. We report here that amidation in mildnacidic conditions can be used to neutralize acidic Nglycansnstill attached to the protein. The resulting amidatednN-glycans can then be released from the proteinnusing PNGase F, and labeled with permanent charges onnthe reducing end to avoid any modification and thenformation of metal adducts during MS analysis. ThenN-glycan modification, digestion, and desalting steps werenperformed using a single-pot method that can be done innmicrocentrifuge tubes or 96-well microfilter plates, enablingnhigh throughput glycan analysis. Using this methodnwe were able to perform quantitative MALDI-TOF MS ofna recombinant human glycoprotein to determine changesnin fucosylation and changes in sialylation that were in veryngood agreement with a normal phase HPLC oligosaccharidenmapping method.