One-pot solid-phase glycoblotting and probing by transoximization for high-throughput glycomics and glycoproteomics

Chemistry. 2007;13(6):1664-73. doi: 10.1002/chem.200601613.


The development of rapid and efficient methods for high-throughput protein glycomics is of growing importance because the glycoform-focused reverse proteomics/genomics strategy will greatly contribute to the discovery of novel biomarkers closely related to cellular development, differentiation, growth, and aging as well as a variety of diseases such as cancers and viral infection. Recently, we communicated that rapid and efficient purification of carbohydrates can be achieved by employing sugar-specific chemical ligation with aminooxy-functionalized polymers, which we termed "glycoblotting" (see S.-I. Nishimura et al., Angew. Chem. 2005, 117, 93-98; Angew. Chem. Int. Ed. 2005, 44, 91-96). The chemoselective blotting of oligosaccharides present in crude biological materials onto synthetic polymers relies on the unique oxime-bond formation between aminooxy group displayed on the supporting materials and aldehyde/ketone group at the reducing terminal of all oligosaccharides, thus enabling highly selective and rapid oligosaccharide purification. Aiming to improve the detection sensitivity of the released oligosaccharides, we introduce here a novel strategy for one-pot solid-phase glycoblotting and probing by transoximization. We found that oligosaccharides captured by the polymer supports via the oxime bond can be released in the presence of excess O-substituted aminooxy derivatives in a weakly acidic condition. The released oligosaccharides could be recovered as newly formed oxime derivatives of the O-substituted aminooxy compound added, thus demonstrating the simultaneous releasing and probing. In addition, we synthesized a novel aminooxy-functionalized monomer, N-[2-[2-(2-tert-butoxycarbonylaminooxyacetylamino-ethoxy)ethoxy]ethyl]-2-methacrylamide, which allows for the large-scale preparation of a versatile polymer characterized by its high stability, high blotting capacity, and easy use. The one-pot protocol allowed to profile 23 kinds of N-glycan chains of human serum glycoproteins. This concept was further applied for the glycopeptides analysis in a crude mixture followed by galactose oxidase treatment to generate free aldehyde group at the non-reducing terminal of oligosaccharide moiety of glycopeptides. Our technique may be implemented in existing biochemistry and molecular diagnostics laboratories because enriched oligosaccharides and glycopeptides by solid-phase transoximization with high-sensitive labeling reagents are widely applicable in a variety of common analytical methods using two-dimensional HPLC, LC/MS, and capillary electrophoresis as well as modern mass spectrometry.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acrylamides / chemical synthesis*
  • Aldehydes / chemistry
  • Amino Acid Sequence
  • Chromatography, High Pressure Liquid
  • Electrophoresis, Capillary
  • Galactose Oxidase / metabolism*
  • Glycopeptides / analysis*
  • Glycopeptides / chemistry
  • Glycopeptides / metabolism
  • Glycosylation
  • Humans
  • Hydrogen-Ion Concentration
  • Ketones / chemistry
  • Mass Spectrometry
  • Molecular Sequence Data
  • Oxamic Acid / analogs & derivatives
  • Oxamic Acid / chemistry
  • Oximes / chemistry*
  • Polysaccharides / analysis*
  • Polysaccharides / chemistry
  • Polysaccharides / metabolism
  • Proteomics*


  • Acrylamides
  • Aldehydes
  • Glycopeptides
  • Ketones
  • N-(2-(2-(2-tert-butoxycarbonylaminooxyacetylamino-ethoxy)ethoxy)ethyl)-2-methacrylamide
  • Oximes
  • Polysaccharides
  • Galactose Oxidase
  • Oxamic Acid
  • oxamide