Despite recent advances in our understanding of the significance of the protein glycosylation, the throughput of protein glycosylation analysis is still too low to be applied to the exhaustive glycoproteomic analysis. Aiming to elucidate the N-glycosylation of murine epidermis and dermis glycoproteins, here we used a novel approach for focused proteomics. A gross N-glycan profiling (glycomics) of epidermis and dermis was first elucidated both qualitatively and quantitatively upon N-glycan derivatization with novel, stable isotope-coded derivatization reagents followed by MALDI-TOF(/TOF) analysis. This analysis revealed distinct features of the N-glycosylation profile of epidermis and dermis for the first time. A high abundance of high mannose type oligosaccharides was found to be characteristic of murine epidermis glycoproteins. Based on this observation, we performed high mannose type glycoform-focused proteomics by direct tryptic digestion of protein mixtures and affinity enrichment. We The rapid progress in the sequence analysis of genomes of a variety of living organisms is accelerating the investigation of various related proteins involved in biological processes and disorders. Carbohydrate modifications can profoundly affect protein function. Their importance in disease is evident from a growing number of embryonic lethal phenotypes seen in knock-out mice with defects in glycoconjugate assembly or processing (1), and hence a large scale protein glycosylation analysis has become important.The accurate identification of protein glycosylation is challenging because of the complex structures, labile nature, and microheterogeneity of glycoproteins. The presence of nonglycopeptides in a sample also limits the sensitivity for analysis of glycopeptides on mass spectrometry due to ion suppression. Although several new techniques enabling the large scale identification of glycoproteins have recently been developed (2-4), they cannot provide information about oligosaccharide moieties because the analysis is performed on peptides of which such moieties are enzymatically removed prior to the analysis. It is also well known that glycosylation is cell type-specific, so a single glycoprotein can have a different spectrum of glycan structures when expressed in different cells. Recent progress in mass spectrometry (e.g. electron capture-induced dissociation using Fourier transform mass spectrometry (5, 6) and MALDI-LIFT-TOF/TOF (7)) has demonstrated the ability to provide information both on peptide sequence and glycan structure for the analysis of glycopeptides. However, the throughput of these techniques is not high enough to apply to large scale protein glycosylation analyses. Therefore, unveiling the significance of protein glycosylation in an efficient manner requires further thought. One solution is to develop a focused approach based on function and information content.In this study, we describe a glycomic approach to rationalize the focusing process using murine dermis and epidermis as models. A gross N-glycan profi...

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We have developed an effective and practical trap-and-release method based on chemoselective ligation of carbohydrates with reactive aminooxyl groups attached to the surface of nanoparticles (referred to as glycoblotting nanoparticles). These glycoblotting nanoparticles were synthesized by UV irradiation of diacetylene-functionalized lipids that contain the aminooxyl group. The glycoblotting nanoparticles captured carbohydrates in aqueous solution under mild conditions and were collected by simple centrifugation. The trapped carbohydrates were effectively released from the nanoparticles under acidic conditions to give pure oligosaccharides. This glycoblotting process reduced the time required for the purification process of carbohydrates to less than 6 h, compared to the several days needed for conventional chromatographic techniques. The oligosaccharides (N-glycan) were released from ovalbumin (glycoprotein) by PNGase F after tryptic digestion. MALDI-TOF mass spectra before purification did not show any significant signals corresponding to N-glycans because these signals were hidden by the large signals of the abundant peptides. However, after purification with the glycoblotting nanoparticles, only signals corresponding to oligosaccharides appeared. We also demonstrated a clear analysis of the oligosaccharides contained in the mice dermis by means of glycoblotting.

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Glycosylation of proteins greatly affects their structure and function, but traditional genomics and transcriptomics are not able to precisely capture tissue-or speciesspecific glycosylation patterns. We describe here a novel approach to link different "omics" data based on exhaustive quantitative glycomics of murine dermis and epidermis. We first examined the dermal and epidermal N-glycome of mouse by a recently established glycoblotting technique. We found that the Gal␣1-3Gal epitope was solely expressed in epidermis tissue and was preferentially attached to adhesion molecules in a glycosylation site-specific manner. Clarified glycomic and protemic information combined with publicly available microarray data sets allowed us to identify galectin-3 as a receptor of Gal␣1-3Gal epitope. These findings provide mechanistic insight into the causal connection between the genotype and the phenotype seen in ␣3GalT-1-deficient mice and transgenic mice expressing endo-␤-galactosidase C. Because humans do not possess the Gal␣1-3Gal structure on their tissues, we further examined the human dermal and epidermal N-glycome. Comparative glycomics revealed that the GalNAc␤1-4GlcNAc (N,N-diacetyllactosediamine) epitope, instead of the Gal␣1-3Gal epitope, was highly expressed in human epidermis. Posttranslational protein glycosylation changes the biological and physical properties of glycoconjugates, which include functions as signals or ligands to control their distribution, antigenicity, metabolic fate, stability, and solubility (1). Cells in the epidermis, which forms a major part of the epithelial barrier, undergo desquamation and are continuously being renewed (2), a process that requires changes in adhesion. Because glycoproteins are often involved in adhesion between cells and their extracellular matrices, the glycoproteome of the epidermis may therefore provide new insight into the functional roles of protein glycosylation. Mammalian epidermal glycoconjugates have mostly been studied histochemically using lectins (3) or monoclonal antibodies (4). Although these studies revealed that the cell surfaces of keratinocytes in the epidermis contain numerous glycoconjugates, these approaches cannot provide detailed structural information about the oligosaccharides or their carrier proteins.We previously clarified the quantitative glycomic profile of murine dermis and epidermis using novel aminooxy-based isotope tags and MALDI-TOF MS analysis (5), which revealed distinct features of the N-glycosylation profile of dermis and epidermis. We found that murine epidermal glycoproteins have a high abundance of high mannose-type oligosaccharides, and the striking roles of lysosomal enzymes in epidermis during lipid remodeling and desquamation were further discussed. This study was the first to demonstrate the usefulness of quantitative gross N-glycan profiling for performing systematic glycoform-focused proteomics. To advance and accelerate this approach, we recently established a novel technology platform for large scale quantitative glyc...