Quantitative Comparison of Global Carbohydrate Structures of Glycoproteins Using LC−MS and In-Source Fragmentation

• Published in: Analytical chemistry (Washington) 1997-07, Vol.69 (13), p.2517-2524
• Main Authors: Mazsaroff, Istvan, Yu, Wen, Kelley, Brian D, Vath, James E
• Format: Article
• Language: English
• Subjects: Amidohydrolases - metabolism; Analytical, structural and metabolic biochemistry; Biochemistry; Biological and medical sciences; Carbohydrate Conformation; Carbohydrates; Chromatography, High Pressure Liquid - methods; Factor VIII - chemistry; Factor VIII - metabolism; Fundamental and applied biological sciences. Psychology; Glycoproteins; Glycoproteins - chemistry; Glycoproteins - metabolism; Humans; Ions; Mass Spectrometry - methods; Peptide Fragments - chemistry; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Proteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Thrombin - metabolism
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac961116+

A comparative method for the quantitative analysis of the ratio of oxonium fragment (reporter) ions derived from sialic acid and N-acetylhexosamine residues on a large intact glycoprotein, the B domain of recombinant human factor VIII (rhFVIII), was developed. The method utilized liquid chromatography−electrospray ionization mass spectrometry (LC−ESI MS) on a single-quadrupole instrument. During development, systematic approaches such as full-matrix and simplex strategies were used for the optimization of the signal-to-noise ratio by controlling source temperature and cone voltage. The method was found to be precise (RSD = 0.84%), sensitive (capable of differentiating 1 sialic acid residue change among at least 29 sialic acids on a 103-kDa glycoprotein that is 38% carbohydrate), applicable to a wide range of loading (11.6−372 μg of FVIII), and accurate according to a comparison to matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Combining the method with enzymatic removal of N-glycans, selective O-glycan analysis was also performed leading to differential fragment ion analysis ascribed to N- and O-glycans. Quantitative ESI in-source dissociation MS combined with LC can generally be used for glycoproteins, as one of the indicators, to compare the distribution of carbohydrate residues over N- and O-glycans, to investigate their isoforms, and compare batch-to-batch characteristics of biopharmaceuticals.