An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides

Minyong Chen, Xiaofeng Shi, Rebecca M. Duke, Cristian I. Ruse, Nan Dai, Christopher H. Taron and James C. Samuelson ()
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Minyong Chen: New England Biolabs Inc.
Xiaofeng Shi: New England Biolabs Inc.
Rebecca M. Duke: New England Biolabs Inc.
Cristian I. Ruse: New England Biolabs Inc.
Nan Dai: New England Biolabs Inc.
Christopher H. Taron: New England Biolabs Inc.
James C. Samuelson: New England Biolabs Inc.

Nature Communications, 2017, vol. 8, issue 1, 1-15

Abstract: Abstract A method for selective and comprehensive enrichment of N-linked glycopeptides was developed to facilitate detection of micro-heterogeneity of N-glycosylation. The method takes advantage of the inherent properties of Fbs1, which functions within the ubiquitin-mediated degradation system to recognize the common core pentasaccharide motif (Man3GlcNAc2) of N-linked glycoproteins. We show that Fbs1 is able to bind diverse types of N-linked glycomolecules; however, wild-type Fbs1 preferentially binds high-mannose-containing glycans. We identified Fbs1 variants through mutagenesis and plasmid display selection, which possess higher affinity and improved recovery of complex N-glycomolecules. In particular, we demonstrate that the Fbs1 GYR variant may be employed for substantially unbiased enrichment of N-linked glycopeptides from human serum. Most importantly, this highly efficient N-glycopeptide enrichment method enables the simultaneous determination of N-glycan composition and N-glycosites with a deeper coverage (compared to lectin enrichment) and improves large-scale N-glycoproteomics studies due to greatly reduced sample complexity.

Date: 2017
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DOI: 10.1038/ncomms15487

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