Highlights
- •Butyrate induces changes in the CaCo-2 O-glycome and proteome upon differentiation.
- •Differentiated cells show a higher expression of sialylated O-glycan structures.
- •Terminal fucosylation shows lower expression in the differentiated cells.
- •Butyrate induces changes in specific proteins involved in cell metabolism, monosaccharide precursor biosynthesis and cell adhesion.
ABSTRACT
Graphical abstract

Keywords
Abbreviations:
(ALP) (Alkaline phosphatase), (BSM) (bovine submaxillary mucin), CRC (colorectal cancer), CaCo-2 (Cancer coli-2), DTT (DL-dithiothreitol), F (deoxyhexose), Gal (galactose), GalNAc (N-acetylgalactosamine), GalNAcol (reduced reducing end of GalNAc), GlcNAc (N-acetylglucosamine), GI (Gastrointestinal), H (hexose), m/z (mass to charge ratio), MS (mass spectrometry), N (N-acetylhexosamine), Neu5Ac (N-acetylneuraminic acid), PCA (principal component analysis), PGC-LC (porous graphitized carbon liquid chromatography), RT (room temperature), S (sialic acid), sLeA (sialyl-Lewis A), sLeX (sialyl-Lewis X), Su (sulfate), (TMT) (tandem mass tag)INTRODUCTION
EXPERIMENTAL PROCEDURES
Chemicals and reagents
Cell culture
Phase-contrast microscopy
Alkaline phosphatase activity
Cell lysis and O-glycan release
PGC-LC-MS/MS analysis
Quantitative Proteomics Using TMT Labeling
Experimental Design and Statistical Rationale
RESULTS
Cell differentiation assay

Glycomic analysis


Structural identification of glycan species
Proteomic analysis


Data integration


DISCUSSION
CONCLUSIONS
AUTHOR CONTRIBUTIONS
DATA AVAILABILITY
ACKNOWLEDGEMENTS
Supplementary data
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Bacterial butyrate is beneficial for intestinal homeostasis as the preferred energy source of intestinal epithelia, capable of inducing differentiation. Here we describe glycomic and proteomic signatures of butyrate-induced epithelial differentiation of the intestinal cell line CaCo-2. We identified an upregulation of specific O-glycan signatures, as well as specific proteins involved in cell metabolism, monosaccharide precursor biosynthesis and cell adhesion. Using an integrative approach, we generated hypotheses about the origin of the glycosylation changes with differentiation.
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