One-pot solid-phase glycoblotting and probing by transoximization for high-throughput glycomics and glycoproteomics.
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.
Shimaoka
Hideyuki
H
Graduate School of Advanced Life Science, Frontier Research Center for the Post-Genome Science and Technology, Hokkaido University, N21, W11 Sapporo 001-0021, Japan.
Kuramoto
Hiromitsu
H
Furukawa
Jun-ichi
J
Miura
Yoshiaki
Y
Kurogochi
Masaki
M
Kita
Yoko
Y
Hinou
Hiroshi
H
Shinohara
Yasuro
Y
Nishimura
Shin-Ichiro
S
D000178:Acrylamides / Q000138:chemical synthesis*; D000447:Aldehydes / Q000737:chemistry; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D019075:Electrophoresis, Capillary; D005692:Galactose Oxidase / Q000378:metabolism*; D006020:Glycopeptides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007659:Ketones / Q000737:chemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010072:Oxamic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D010091:Oximes / Q000737:chemistry*; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics
D000178:Acrylamides; D000447:Aldehydes; D006020:Glycopeptides; D007659:Ketones; C519888:N-(2-(2-(2-tert-butoxycarbonylaminooxyacetylamino-ethoxy)ethoxy)ethyl)-2-methacrylamide; D010091:Oximes; D011134:Polysaccharides; D005692:Galactose Oxidase; D010072:Oxamic Acid; C008728:oxamide