Comprehensive analysis of glycosphingolipid glycans by lectin microarrays and MALDI-TOF mass spectrometry.

Nature Protocols
Haoqi DuZheng Li

Abstract

Glycosphingolipids (GSLs) are ubiquitous glycoconjugates present on the cell membrane; they play significant roles in many bioprocesses such as cell adhesion, embryonic development, signal transduction and carcinogenesis. Analyzing such amphiphilic molecules is a major challenge in the field of glycosphingolipidomics. We provide a step-by-step protocol that uses a lectin microarray to analyze GSL glycans from cultured cells. The procedure describes (i) extraction of GSLs from cell pellets, (ii) N-monodeacylation using sphingolipid ceramide N-deacylase digestion to form lyso-GSLs, (iii) fluorescence labeling at the newly exposed amine group, (iv) preparation of a lectin microarray, (v) GSL-glycan analysis by a lectin microarray, (vi) complementary mass spectrometry analysis and (vii) data acquisition and analysis. This method is high-throughput, low cost and easy to conduct, and it provides detailed information about glycan linkages. This protocol takes ~10 d.

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Citations

Oct 14, 2021·Mass Spectrometry Reviews·Natalija Topić PopovićRozelindra Čož-Rakovac

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Software Mentioned

FlexControl
GenePix Pro
EXPANDER
LIPID MAPS
Excel
GlycoWorkbench
SPSS
FlexAnalysis

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