System Metaglycomes: Mapping Dynamic Cell Surface N‐glycome, O‐glycome and Glycolipidome by Mass Spectrometry
Authors: Barboza Mariana , Wong Maurice , Luke Jonathan , Cheng Zhi , Xu Gege , Gareau Melanie , Raybould Helen , Lebrilla Carlito B. 
Published: June 2021

Mammalian cell surface membranes are coated with a vast repertoire of glycan chains attached to proteins and lipids that play key roles in almost all biological processes involving cell‐cell adhesion, recognition, signaling, and growth. Aberrant expression of glycan moieties has been implicated in numerous congenital and acquired human diseases. Despite its potential for biomarker discovery, analysis of cell surface glycomes is challenging owing to the structural complexity and variety of glycan structures. Moreover, glycan structures and their spatial and temporal distribution of normal human and animal model tissues (e.g.: rodents) remain largely unknown. In this work, we developed a mass spectrometry‐based multimethod platform to obtain detailed profiles and characterize N‐glycans (N‐glycome), O‐glycans (O‐glycome), and glycolipids (Glycolipidome) from neonatal and adult mouse organs and tissues.

Methods

Selected organs and tissues were collected from neonatal and adult C57bl/6J mice. Brain, heart, lungs, liver, spleen, kidneys, muscle, intestine and adipose tissues were dissected and homogenized. Membrane fractions (MF) containing cell surface glycoproteins and glycolipids were obtained by a series of centrifugation and ultracentrifugation steps. N‐Glycans were enzymatically released from plasma, isolated MF and purified by solid phase extraction (SPE). O‐Glycans were released from the remaining protein pellet using β‐elimination followed by SPE. Purified N‐glycans and O‐glycans were analyzed separately by nano‐LC‐Chip/Q‐TOF MS and were annotated using Mass Hunter and in‐house N‐glycan and O‐glycan libraries. Glycolipids were extracted from deglycosylated MF through a modified Folsh procedure, purified and analyzed by nano‐LC‐Chip/Q‐TOF MS. Tandem mass spectrometry was utilized for structural characterization of both the glycan moieties and the ceramide portion of GSLs.

Results

We obtained unique cell surface metaglycomes for each organ and tissue analyzed allowing for the identification of and the mapping of tissue‐specific glycan signatures. For instance, the N‐glycome of specific functional brain areas was dominated by fucosylated and sialylated glycans in adult mice, while in neonatal mice neutral fucosylated species were predominant. Glycolipidome fingerprints of the same brain areas showed to be dominated by sialylated glycoshingolipids, with no fucose detected. Transition from neonatal to adulthood was marked by an increase of sialylation levels and variations in the composition of the ceramide portion, including the number of hydroxyl groups, degree of unsaturation, and chain length of the sphingoid and the fatty acid type. With the exception of plasma and kidneys, non‐neural organ N‐glycomes were dominated by high mannose glycans. Sialylated and sialylated‐fucosylated glycans were the predominant species in plasma, while fucosylated glycans were the most abundant species in kidneys N‐glycome.

To date, this is the most comprehensive glycomic platform developed towards a systematic approach for the concurrent identification of tissue and organ metaglycomes, including N‐glycome, O‐glycome and Glycosphigolipidome and to monitoring glycomic changes that organs undergo from neonatal to adulthood stages.

Novelty

A high‐throughput platform to concurrently assess cell surface metaglycomes of tissues including N‐glycome, O‐glycome and Glycosphigolipidome.

Support or Funding Information

NIH NIGMS

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.