Discrimination of cellular developmental states focusing on glycan transformation and membrane dynamics by using BODIPY-tagged lactosyl ceramides.

Glycosphingolipids (GSLs) are a group of molecules composed of a hydrophilic glycan part and a hydrophobic ceramide creating a diverse family. GSLs are de novo synthesised from ceramides at the endoplasmic reticulum and Golgi apparatus, and transported to the outer surface of the plasma membrane. It has been known that the glycan structures of GSLs change reflecting disease states.

Reactivation of hyperglycemia-induced hypocretin (HCRT) gene silencing by N-acetyl-d-mannosamine in the orexin neurons derived from human iPS cells.

Orexin neurons regulate critical brain activities for controlling sleep, eating, emotions, and metabolism, and impaired orexin neuron function results in several neurologic disorders. Therefore, restoring normal orexin function and understanding the mechanisms of loss or impairment of orexin neurons represent important goals. As a step toward that end, we generated human orexin neurons from induced pluripotent stem cells (hiPSCs) by treatment with N-acetyl-d-mannosamine (ManNAc) and its derivatives.

Syntheses of lactosyl ceramide analogues carrying novel bifunctional BODIPY dyes directed towards the differential analysis of multiplexed glycosphingolipids by MS/MS using iTRAQ.

Lactosyl ceramide analogues carrying novel bifunctional BODIPY-based fluorescent tags were designed and synthesised for live cell imaging. Addition of azide functionality on the fluorophore facilitated isobaric tagging for quantitative multiplexed analysis of biomolecules based on tandem mass spectrometry.

Analysis of the cellular dynamics of fluorescently tagged glycosphingolipids by using a nanoliquid chromatography-tandem mass spectrometry platform.

Despite the increasing biological interests on glycoconjugates, the synthetic mechanism of oligosaccharides has not yet been revealed except for the enzymes involved. To clarify the synthetic events that occur inside cells, spatiotemporal analysis of fluorescently tagged glycosphingolipids was carried out. Transformation of the incorporated lactosylceramide analogue carrying 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl group (BODIPY fluorophore) was analyzed using nanoLC-fluorescence detection-nanoelectrospray ionization-mass spectrometry.

Synthesis of a fluorescently tagged sialic acid analogue useful for live-cell imaging.

A cytidine 5'-monophosphate (CMP)-sialic acid analogue carrying a fluorescent reporter group, an inhibitor of sialyltransferase, was synthesised in order to investigate glycan synthesis events in cells. The compound was found to be a substrate of a CMP-sialic acid transporter, and specific Golgi vesicles were visualised in the cells.

Fluorescence-monitored zero dead-volume nanoLC-microESI-QIT-TOF MS for analysis of fluorescently tagged glycosphingolipids.

An analysis of the glycan processing event is of particular importance to understand the nontemplate dependent synthetic mechanism of the multiple glycosylation reactions taking place in the Golgi apparatus in connection with the post-translational modification of biomolecules. In our efforts to address the issue, we constructed an analysis platform using nano-liquid chromatography (LC), which also worked as a spray tip, with an optical-fiber-based blue (470 nm) light emitting diode (LED)-induced fluorescence (520 nm) detector coupled with a microelectrospray ionization (ESI)-quadrupole ion trap (QIT)-time of flight (TOF) mass spectrometer (MS). This system was designed to enable both quantitative and qualitative analyses of fluorescently tagged molecules such as BODIPY-tagged lactosylceramide.

Analysis of behavior of sodiated sugar hemiacetals under low-energy collision-induced dissociation conditions and application to investigating mutarotation and mechanism of a glycosidase.

Analysis of anomericity is one of the most important issues in the structure elucidation of carbohydrates. Mass spectrometry (MS)-based methods are of particular interest and important to address the issue related to resolving anomericity of monosaccharide units in a glycan. However, direct analysis of hemiacetals has not been possible by MS because of the nonavailability of information regarding the gas-phase behavior of such ion species.

Ion-trap mass spectrometry unveils the presence of isomeric oligosaccharides in an analyte: stage-discriminated correlation of energy-resolved mass spectrometry.

Mass spectrometry, especially tandem mass spectrometry, has been widely used in the field of analytical sciences for handling biological and chemical samples. The technique resolves molecular and fragment ions based on the mass to charge ratio. Energy-resolved mass spectrometry (ERMS) further provides an activation energy-related factor in the dissociation reaction.

Energy-resolved structural details obtained from gangliosides.

Gangliosides, a family of glycosphingolipids (GSLs) that comprise sialic acid residue(s), are an important class of molecules that exist on the outer surface of the plasma membrane. To assess the functions of a particular series of gangliosides that play important roles in brain functions, their structures and localizations need to be investigated. We studied the structures of these gangliosides by collision-induced dissociation using quadrupole ion-trap mass spectrometry.

High-yielding and controlled dissociation of glycosides producing B- and C-ion species under collision-induced dissociation MS/MS conditions and use in structural determination.

Collision-induced dissociation (CID) in mass spectrometry is a powerful technique with which to understand gas-phase chemical reactions. A mass spectrometer is used to carry out the reaction, isolation, and analysis. On the other hand, structural analysis of glycan structures is of extreme importance in the analysis of biomolecules, such as glycoproteins and glycolipids.