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

Nat Commun. 2017 May 23:8:15487. doi: 10.1038/ncomms15487.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Cycle Proteins / chemistry*
  • Electrophoresis, Polyacrylamide Gel
  • F-Box Proteins / chemistry*
  • Fetuins / chemistry
  • Genetic Variation
  • Glycopeptides / chemistry*
  • Glycoproteins / chemistry
  • Glycosylation
  • Humans
  • Immunoglobulin G / chemistry
  • Lectins / chemistry
  • Mannose / chemistry
  • Mutagenesis
  • Mutation
  • Nerve Tissue Proteins / chemistry*
  • Plasmids / metabolism
  • Polysaccharides / chemistry*
  • Protein Binding
  • Proteomics
  • Ribonucleases / chemistry
  • Salts / chemistry
  • Tandem Mass Spectrometry
  • Trypsin / chemistry

Substances

  • Cell Cycle Proteins
  • F-Box Proteins
  • FBXO2 protein, human
  • Fetuins
  • Glycopeptides
  • Glycoproteins
  • Immunoglobulin G
  • Lectins
  • Nerve Tissue Proteins
  • Polysaccharides
  • Salts
  • Ribonucleases
  • ribonuclease B
  • Trypsin
  • Mannose