Glycosylation is one of the posttranslational modifications of proteins in eukaryotes. This step is thought to modulate a wide range of protein functions both on the cellular surfaces and within the cells. [1] This highly dynamic process in which rapid changes in the carbohydrate structures occur in response to cellular signals or cellular stages result in the key informational markers of some serious human diseases. For example, it is known that carbohydrates of IgG can be drastically altered to form unusual structures in patients with rheumatoid arthritis and specific carbohydrates are used as tumor-associated markers in pancreatic and colon cancers. Recently, it has also been reported that the oligosaccharides of the therapeutic human IgG play a critical role of enhancing antibody-dependent cellular cytotoxicity. Glycosylation of nuclear and cytoplasmic proteins with a GlcNAc residue is ubiquitous in nearly all eukaryotes and is a crucial event in posttranslational modifications for regulating protein functions within the cells. [1b] Dynamic perturbations in O-GlcNAc regulation have been implicated in the etiology of type II diabetes, cancer, and neurodegenerative diseases. Although there have been substantial advances in our understanding of the effects of glycosylation on some biological systems, we still do not fully understand the specific functional roles of carbohydrates and the relationship between their structures and functions. The major difficulty in carbohydrate sequencing is a consequence of the fact that the purification of trace amounts of oligosaccharides requires extremely tedious multistep processes. This is because crude sample mixtures prepared by enzymatic digestion from cells, organs, serum, etc. usually contain large amounts of impurities such as peptides, lipids, and salts. These technical problems in the sequencing of carbohydrates make it impossible to achieve high-throughput protein glycomics. We report herein that the combined use of two novel techniques, chemoselective glycoblotting and MALDI-TOF/ TOF mass spectrometry, [6] allows for both facile purification and precise analysis of common oligosaccharides and glycopeptides from native glycoproteins.
The concept of the "sugar family tree," constructed by Fischer, [7] motivated us to use the chemoselective "glycoblotting" technique, which has become a key feature in the efficient isolation of carbohydrates in this study. Fischer found that the reaction of glucose, mannose, and major oligosaccharides with phenylhydrazine proceeds smoothly to give the corresponding stable phenylhydrazone derivatives under mild basic and aqueous conditions. Once oligosaccharides are released from glycoconjugates, they can be regarded as a general class of the compound library which have an aldehyde or ketone group at each reducing terminal. As indicated in Scheme 1 a, aldehydes preferentially react with reagents bearing hydrazine-like functional groups. Fischer-type reagents do not need any catalyst or reducing agent to accelerate the reaction with sugars and the reactions usually proceed under mild conditions. However, the reactions of aldehydes with primary amino groups require some activating reagent for the formation of stable products. As a result, carbohydrates preferentially react with Fischer-type reagents even in the presence of large amounts of peptides or amino acids with primary amino groups.
The unique chemical characteristics of the sugar family encouraged us to design novel polymers for capturing only carbohydrates from crude samples on the basis of the [*] Prof.