Membrane microdomains (lipid rafts) are cholesterol- and glycosphingolipid-enriched lipid domains, which are involved in various cellular responses connecting the intracellular and extracellular environments. We investigated the neutrophilic function focusing on lactosylceramide-enriched membrane microdomains on the neutrophilic cell plasma membrane. Although membrane microdomains in neutrophils are known to be involved in neutrophilic functions, such as phagocytosis, the molecular mechanisms of action remain to be elucidated. In this review, strategies for comprehensive analysis of the microdomain-associated proteins for investigating the molecular mechanisms of action of these domains are described.
Expression patterns of glycoforms are known to be dependent on the various individual states, such as development, ageing, and disease. Transcriptomics and proteomics have been generally standardized to analyze the expression of certain DNAs and proteins, facilitating potential applications for diagnosis and disease treatment. However, no such convenient methods capable of analyzing glycomics are available currently. Glycobiology is one of the most important subjects in post-genomic era, and methods for glycan analyses have been steadily improved by advancement in mass spectrometry (MS). Nevertheless, a practical procedure for large number of glycan samples that fulfills the requirements of the high speed and high yield was unavailable, thus, limiting development of useful glycomics of certain biological materials, e.g. serum and tissue biopsy. To alleviate this problem, it is highly desirable to develop a standardized methodology to achieve high-speed quantitative and qualitative profiling of glycan expression patterns in the biological materials. Recently, a chemo-selective glycan enrichment technology, termed glycoblotting, has been developed to purify oligosaccharides derived from glycoproteins in highly effective quantitative manner, enabling profiling of serum glycans in a simple manner. This method is also applicable to automated analysis of multiple samples simultaneously. It flawlessly combines isolation and labeling of oligosaccharides suited for conventional analytical methods including MS analysis. We believe that the glycoblotting technique and the beads specifically designed for it (BlotGlyco) is an innovative approach in the “real world” glycan preparation. Glycoblotting and its related technologies would make a great contribution not only to the glycobiology community, but also for glycan-related biomarker search as well as for glycan-related drug development.
Rapid and sensitive technology for the analysis of protein glycosylation is a major requirement in glycomics and for the discovery of disease-associated glycan and glycoprotein biomarkers. Recently, we have developed an automated apparatus, AutoGlycoCutter (AGC), which enables rapid release of O-linked glycans from core protein as a reactive reducing oligosaccharide (Matsuno et al., Anal. Biochem., 362, 2007, 245-257. Yamada et al., Anal. Biochem., 371, 2007, 52-61). In this review, performance of the AGC and its usefulness were demonstrated based on the analytical results of O-linked glycans in some standard mucin-type glycoproteins or proteoglycan-type glycans. In addition, we also show two applications of the present method. One is the rapid and sensitive analysis of mucin-type glycan profiles in various cultured cancer cell lines, and the other is the development of a device for high-throughput analysis of multiple samples by hyphenation of AGC to the auto spotter for MALDI-TOF MS.
Remarkable advances in performance of recent mass spectrometers promote dramatically the proteome analysis as well as the application of mass spectrometry to glycan analysis, and mass spectrometry has become one of the main analytical methods in glycan study. However, although many researchers can operate mass spectrometers easily these days due to the increase of user-friendliness of the instruments, mass spectrometry is still developing in the field of glycan analysis, and it is very important not only to understand the principle of mass spectrometers and the related technologies but also to interpret obtained mass spectrum properly. In this paper, the glycan analysis using MALDI and ESI mass spectrometers is explained with some actual examples and useful notes.