High Throughput Quantitative Glycomics and Glycoform-focused Proteomics of Murine Dermis and Epidermis

Abstract

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...

A ratiometric lectin microarray approach to analysis of the dynamic mammalian glycome

“…Recent efforts toward decoding the glycome have focused on the use of mass spectrometry (4)(5)(6)(7)(8)(9). Although great strides have been made in the use of this technique, mass spectrometry has difficulty in discriminating between the many structural isomers presented by glycans.…”

N-Linked neutral oligosaccharides in the stratum corneum of normal and ichthyotic skin

“…These high-mannose type oligosaccharides are located in lysosomes, lamellar granules and cell-cell connection desmosome sites. Desmocollins and desmogleins are also assigned as carriers of high-mannose type oligosaccharides, and it was suggested that high-mannose type oligosaccharides control desquamation [24]. However, in the present samples of hyperkeratotic skin, the profile of the high-mannose type oligosaccharides was not changed.…”

“…Alteration of N-glycan profiles in diseases have been reported in many human disorders, and these have been used for disease diagnosis and for elucidation of disease pathomechanisms [4,8]. Focusing on glycoproteins N-glycans, it was demonstrated that the mouse epidermis had abundant high-mannose oligosaccharides [24]. In addition, the oligosaccharides binding proteins were also determined [24].…”

“…Focusing on glycoproteins N-glycans, it was demonstrated that the mouse epidermis had abundant high-mannose oligosaccharides [24]. In addition, the oligosaccharides binding proteins were also determined [24]. However, there are no reports of studies on N-glycans in human epidermis.…”

“…Epidermal proteins which comprise high levels of mannose type oligosaccharides were analyzed in mouse [24]. These high-mannose type oligosaccharides are located in lysosomes, lamellar granules and cell-cell connection desmosome sites.…”