Qualitative and Quantitative Cellular Glycomics of Glycosphingolipids Based on Rhodococcal Endoglycosylceramidase-assisted Glycan Cleavage, Glycoblotting-assisted Sample Preparation, and Matrix-assisted Laser Desorption Ionization Tandem Time-of-flight Mass Spectrometry Analysis

Abstract

Background: Given the biological importance of glycosphingolipids (GSLs), a widespread need exists for sensitive, rapid, and quantitative GSL-glycome analysis. Results: Rhodococcal endoglycosylceramidase (EGCase)-assisted glycan cleavage was optimized for the major GSL classes and combined with glycoblotting to unveil cellular glycomic profiles. Conclusion: Cellular GSL-glycomes were quantitatively and qualitatively characterized by newly established technique. Significance: GSL-glycomics provides a unique way to delineate/characterize cells.

Quantitative GSL-glycome analysis of human whole serum based on an EGCase digestion and glycoblotting method

“…We recently established a procedure for quantitative and qualitative cellular glycomic analysis of GSLs based on rhodococcal endoglycoceramidase (EGCase)-assisted glycan cleavage, glycoblotting-assisted purifi cation, and MALDI-TOF MS analysis ( 19 ). The aim of this study was to apply this method to quantitative analysis of GSL-glycans and to clarify the GSL-glycomic profi le of human serum.…”

“…The GSL extraction procedures were essentially the same as previously described ( 19 ). Briefl y, the lipid fraction was extracted by adding C/M solution (2:1, v/v; 450 l) to oxidized human serum (1.4-44 l), followed by sonication using an ultrasonic homogenizer (Taitec Corp., Saitama, Japan) at room temperature.…”

“…The resulting extracts were centrifuged at 15,000 rpm for 10 min. Supernatants containing GSLs were concentrated with a centrifugal evaporator and resuspended in 50 mM acetate buffer, pH 5.5 (48 l), containing 0.2% Triton X-100 (Sigma-Aldrich Japan, Tokyo, Japan) as a surfactant, followed by the addition of 2 l of EGCase I (25 mU) to release intact glycans from GSLs, as previously described ( 19 ). Enzymatic digestion was performed at 37°C for 16 h. To distinguish GSLglycans from contaminating free oligosaccharides, crude gangliosides were also suspended in the absence of EGCases, which served as a negative control.…”

“…Purifi ed GSL-glycan solutions were mixed with 2,5-dihydrobenzoic acid solution (10 mg/ml prepared in 30% acetonitrile) and subjected to MALDI-TOF MS analysis as previously described ( 19 ), with minor modifi cations. Briefl y, all measurements were performed using an Ultrafl ex II TOF/TOF mass spectrometer equipped with a refl ector and controlled by the FlexControl 3.0 software package (Bruker Daltonics, Bremen, Germany) according to general protocols.…”

Total Cellular Glycomics: A Glycomic Approach to Describe Cells and Streamline the Discovery Process for Cellular Biomarkers

“…We recently established a procedure for quantitative structural analysis of cellular GSL-associated glycans using rhodococcal endoglycoceramidases (EGCases) and the aforementioned glycoblotting procedure (22). Total lipids are extracted from cell pellets and resuspended in chloroform-methanol solution by sonication, and oligosaccharides are subsequently released from GSLs by EGCases.…”