Analysis of variable N-glycosylation site occupancy in glycoproteins by liquid chromatography electrospray ionization mass spectrometry

Nettleship, Joanne E., Aplin, Robin, Radu Aricescu, A., Evans, Edward J., Davis, Simon J., Crispin, Max and Owens, Raymond J. (2007) Analysis of variable N-glycosylation site occupancy in glycoproteins by liquid chromatography electrospray ionization mass spectrometry. Analytical Biochemistry, 361 (1), 149-151. (doi:10.1016/j.ab.2006.11.005).

Abstract

The homogeneity of a protein sample is one of the key factors contributing to successful crystallization and protein structure solution by X-ray crystallography. In the case of glycoproteins, the N-glycans are a source of both chemical and conformational heterogeneity, each of which can hinder crystallization [1]. An additional source of heterogeneity is variability in glycosylation site occupancy. These are important considerations for structural genomics consortia because more than 90% of proteins with posttranslational modifications are glycoproteins [2]. Procedures are now in place for reducing individual N-glycans to single N-acetylglucosamine residues for glycoproteins expressed in mammalian cells in a high-throughput manner (Chang et al., unpublished), but this does not solve the problem of variable site occupancy. The existence of variable occupancy at a particular site is strong evidence that site is not essential for protein folding [3–5]. In principle, therefore, if it is known which sites are variably occupied, the sequon can be deleted by mutagenesis without unduly affecting expression. Here, we describe a simple method for analysis of variably occupied N-glycosylation sites in glycoproteins secreted from mammalian cells.

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Contributors

Author: Max Crispin

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