Abstract
Glycosphingolipids (GSLs) are crucially important components of the cellular membrane, where they comprise microdomains with many critical biological functions. Despite this fact, qualitative and quantitative techniques for the analysis of GSLs still lag behind the needs of researchers. In this study, a reliable procedure for the elucidation of cellular GSL-glycomes was established based on (a) enzymatic glycan cleavage by endoglycosylceramidases derived from Rhodococcus sp. in combination with (b) glycoblotting-assisted sample preparation. The mixture of endoglycosylceramidase I and II was employed to maximize the release of glycan moieties from the major classes of GSLs (i.e. ganglio-, (neo)lacto- and globo-series GSLs). The glycoblotting technique enabled the quantitative detection of GSL-glycans using as few as 2 × 10(5) cells. Thirty-seven different kinds of cellular GSL glycans were successfully observed in 11 kinds of cells, including Chinese hamster ovary cells and their lectin-resistant mutants as well as murine and human embryonic carcinoma cells. Furthermore, in-depth structural clarification in terms of discrimination of isomers was achieved by MALDI-TOF/TOF mass spectrometry analysis and/or linkage-specific glycosidase digestion. These novel analytical techniques were shown to be capable of delineating cell-specific GSL-glycomes. Thus, they are anticipated to have a broad range of applications for the characterization, description, and comparison of various cellular/tissue samples in the fields of drug discovery and regenerative medicine.
Highlights
Given the biological importance of glycosphingolipids (GSLs), a widespread need exists for sensitive, rapid, and quantitative GSL-glycome analysis
Glycoblotting-assisted Sample Preparation for Cellular GSL-glycomics—To employ cellular GSL-glycans as substrates for the optimization of rhodococcal EGCase I and II-assisted glycan cleavage for cellular GSL glycomics, a glycoblotting procedure followed by MALDI-TOF(/TOF) mass spectrometric (MS) analysis was adopted (Fig. 1)
GSLs were extracted from cells by homogenization via sonication and lysis with methanol and chloroform followed by centrifugation [10]
Summary
Given the biological importance of glycosphingolipids (GSLs), a widespread need exists for sensitive, rapid, and quantitative GSL-glycome analysis. In-depth structural clarification in terms of discrimination of isomers was achieved by MALDI-TOF/TOF mass spectrometry analysis and/or linkage-specific glycosidase digestion These novel analytical techniques were shown to be capable of delineating cell-specific GSLglycomes. Qualitative and Quantitative Cellular GSL-glycomics ubiquitously distributed glycoconjugates They are the major component of biologically functional microdomains on ectoplasmic membranes and play crucial roles in neuritogenesis, cell proliferation, and receptors for certain bacterial toxins and viruses [1, 2]. The feasibility of quantitatively delineating cell state- and property-specific GSL-glycomes based on rhodococcal EGCase-assisted glycan cleavage and glycoblotting-assisted sample preparation followed by MALDI-TOF/TOF MS analysis was demonstrated by applying the optimized protocol to various cell lines including the Chinese hamster ovary (CHO) cell line and its lectin-resistant mutants as well as murine and human embryonic carcinoma cell lines
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
