Imaging Single Glycoconjugates

Información del proyecto

GlycoX

Identificador del acuerdo de subvención: 101075996

DOI

10.3030/101075996

Fecha de la firma de la CE 30 Noviembre 2022

Fecha de inicio 1 Mayo 2023

Fecha de finalización 30 Abril 2028

Financiado con arreglo a

• European Research Council (ERC)

Coste total

€ 2 000 000,00

Aportación de la UE

€ 2 000 000,00

Coordinado por

MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN EV
Germany

Periodic Reporting for period 1 - GlycoX (Imaging Single Glycoconjugates)

Período documentado: 2023-05-01 hasta 2025-10-31

Resumen del contexto y de los objetivos generales del proyecto

Project GlycoX aims to develop a single molecule analytical tool to access molecular structures of glycan-decorated biomolecules, also known as glycoconjugates, such as glycolipids (e.g. the bacterial lipopolysaccharides that coat and protect every bacteria) and glycoproteins (e.g. the famous SARS Cov2 spike glycoproteins that allow the virus to invade our healthy cells). These biomolecules are notorious for possessing vast structural diversity (i.e. more than one structures exist in nature) that are intractable by today’s analytical techniques, as a result of these techniques studying molecular structures by averaging information from millions of molecules at a time (also known as ensemble averaging). To address this technological gap, Project GlycoX is set to develop a technology to access individual molecular structures of glycoconjugates. To do so, we aim to develop tools to transfer molecules from solutions onto surfaces in ultrahigh vacuum so that these adsorbed molecules could be visualized one-at-a-time at the submolecular level by low temperature scanning probe microscopy and interpreted by ab initio quantum mechanical calculations. The project is set to (i) reveal for the first time individual structures of many important glycoconjugates relevant in many key biological functions and diseases; (ii) develop single molecule sequencing workflow based on single molecule spectroscopy enabled by scanning probe microscopy technology; and (iii) develop a new deposition technology to prepare hydrated biomolecules on surface such that the folding states of glycoconjugates could be studied for the first time. Altogether, Project GlycoX marks the first step toward unveiling the exciting and mysterious roles of glycans in human health as well as in sustainable materials and therapeutics.

Trabajo realizado desde el comienzo del proyecto hasta el final del período abarcado por el informe y los principales resultados hasta la fecha

Project GlycoX has successfully demonstrated the proof-of-concept single molecule structural analysis of glycoconjugates by direct imaging on surface. This was achieved by transferring glycoconjugates molecules in solution into gas phase by electrospray ionization, followed by their purification in gas-phase by mass selection, their soft landing on cold surfaces in vacuum, and their direct imaging by scanning tunneling microscopy (STM) at cryogenic temperatures. The work demonstrates the ability of STM to resolve the stereoconfiguration of individual monosaccharides (sugar subunits) in the glycan moiety of glycoconjugates, as well as to distinguish and locate common modifications to glycan chains such as acetyls and sulfates, all at the ultimate single molecule level. In addition, through studies of a model protein, RNase B, STM was found to reveal the attachment point and molecular structure of every glycan that is connected to the protein backbone. Building upon these findings, the work subsequently shows the perspective of the method by determining individual structures of glycoconjugates found in nature such as GM3 and GD3 gangliosides glycolipids and MUC1 glycoproteins that both could be found on surfaces of cancer cells. In overall, this proof-of-concept work marks an exciting milestone in single molecule analysis of biomolecules.

Avances que van más allá del estado de la técnica e impacto potencial esperado (incluida la repercusión socioeconómica y las implicaciones sociales más amplias del proyecto hasta la fecha)

Project GlycoX accomplishments to date has marked an important milestone in single molecule analysis of biomolecules. From the STM viewpoint, this marks the first time low temperature STM imaging has been used as an analytical tool to elucidate individual primary structures of structurally diverse biomolecules isolated from biological systems, here exemplified by the ganglioside glycolipids and mucin glycoproteins. From the glycoscience viewpoint, this marks the first structural elucidation of heavily glycosylated molecule, such as mucin glycoproteins, at the ultimate single molecule level. The structural information obtained would be key to understand the roles of glycans in biology, in therapeutics, as well as in development of sustainable materials. The technology developed in Project GlycoX however is still at the very early stage with many limitations that need to be tackled before one could take full advantage of the technology. The most important limitation concerns the low data throughput of single molecule studies, which calls for automation works to eliminate many manual steps in STM imaging and STM image analysis. To realize this, building bridges toward computational science and engineering would be one of the key needs of Project GlycoX in near future. In addition to improvements in data throughput, the sensitivity of direct imaging toward atomic resolution should be envisioned by taking advantage of imaging with functionalized tip as well as advanced tip-based spectroscopy to access atomic-level spatial and chemical resolution.