Direct Isoform Analysis of High-Mannose-Containing Glycoproteins by On-Line Capillary Electrophoresis Electrospray Mass Spectrometry

• Published in: Analytical chemistry (Washington) 1997-07, Vol.69 (13), p.2510-2516
• Main Authors: Yeung, Bernice, Porter, Thomas J, Vath, James E
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins - analysis; Bone Morphogenetic Proteins - chemistry; Chemistry; Chromatography, Ion Exchange; Electrophoresis, Capillary - methods; Fundamental and applied biological sciences. Psychology; Glycoproteins; Glycoproteins - analysis; Glycoproteins - chemistry; Humans; Mannose - analysis; Mass Spectrometry - methods; Molecular Conformation; Molecular Sequence Data; Molecular Weight; Proteins; Recombinant Proteins - analysis; Recombinant Proteins - chemistry; Ribonucleases - analysis; Ribonucleases - chemistry; Scientific imaging; Signal Processing, Computer-Assisted; Transforming Growth Factor beta
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac9611172

A method for the analysis of high-mannose glycoproteins based on capillary electrophoresis and electrospray mass spectrometry (CE−ESI MS) was developed. The combination of UV and MS data allowed for the determination of the identities of glycoform peaks separated by CE, using molecular weight information obtained by ESI MS. The method does not require oligosaccharide release or derivatization, and is applicable for neutral glycans such as high-mannose structures. Two high mannose-containing proteins, ribonuclease B (RNase B) and recombinant human bone morphogenetic protein-2 (rhBMP-2), were used as examples to demonstrate the utility of this technique. Microheterogeneity observed in the CE−UV separation of glycoforms was accounted for by the reconstructed ion chromatograms in ESI MS. Carbohydrate-specific reporter ions generated by in-source fragmentation of the intact proteins during ESI was compared to the chromatographic UV results. This analysis may prove to be a useful qualitative or semiquantitative tool for comparing carbohydrate contents among different glycoproteins, among isoforms of a given protein, or in batch-to-batch comparisons of biopharmaceuticals.