Abstract
Glycosphingolipids (GSLs) are found in cellular membranes of most organisms and play important roles in cell-cell recognition, signaling, growth, and adhesion, among others. A method based on nanoflow high performance liquid chromatography-chip-quadrupole-time-of-flight mass spectrometry (nanoHPLC Chip-Q-TOF MS) was applied towards identifying and quantifying intact GSLs from a variety of samples, including cultured cell lines and animal tissue. The method provides the composition and sequence of the glycan, as well as variations in the ceramide portion of the GSL. It was used to profile the changes in the glycolipidome of Caco-2 cells as they undergo differentiation. A total of 226 unique GSLs were found among Caco-2 samples from five differentiation time-points. The method provided a comprehensive glycolipidomic profile of a cell during differentiation to yield the dynamic variation of intact GSL structures.
Highlights
Glycosphingolipids (GSLs) are found in cellular membranes of most organisms and play important roles in cell-cell recognition, signaling, growth, and adhesion, among others
The cell line comes from colorectal cancer, differentiated Caco-2 cells acquire morphological and functional features that resemble intestinal epithelial cells
The cell surface glycosylation of proteins was previously reported to change during the differentiation process, based on the analysis of released N-glycans[12]
Summary
OPEN Intact glycosphingolipidomic analysis of the cell membrane during differentiation yields. Glycosphingolipids (GSLs) are a group of complex lipids that are comprised of a ceramide (N-acylsphingosine) glycosidically linked to a glycan moiety These molecules are ubiquitous in cell membranes, where they are known to participate in cellular processes such as signaling, adhesion, and cell differentiation, among other functions[1]. We employ a nanoflow high performance liquid chromatography chip-quadrupole-time-of-flight mass spectrometry (nanoHPLC Chip-Q-TOF MS) method to comprehensively identify and quantitate over 220 intact cell-surface GSLs of Caco-2 during differentiation. The method effectively covered 15 different glycan headgroups, including acidic GSLs with sulfation and sialylation, as well as fucosylated and non-fucosylated neutral GSLs. The glycans of each identified GSL were characterized by their composition and connectivity, while the ceramides were distinguished by their lipid chain length, number of hydroxyl groups, and degrees of unsaturation. It further yields highly reproducible and quantitative information allowing measurement of distinct changes during cellular progression
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