Periodic Reporting for period 4 - GLYCANAL (High-Throughput Cryogenic Spectroscopy for Glycan Analysis)
Período documentado: 2023-03-01 hasta 2023-08-31
Glycans, also known as oligosaccharides or polysaccharides, are polymers consisting of individual sugar molecules (i.e. monosaccharides) linked to one another. They are ubiquitous in nature and represent one of the most important classes of molecules in living systems. Nearly every cell is coated with a layer of glycans that plays a central role in cell-to-cell communication, the recognition of pathogens, and the transport of molecules into the cell. As a result, they are involved in one way or another in virtually every human disease.
Glycans are central to the functioning of biotherapeutics, for example. The pattern of glycans attached to a protein drug affects its effectiveness, lifetime, and cytotoxicity, and this must be carefully controlled during their biosynthesis. Viruses often use glycans to shield themselves from detection, making analysis of the glycans coating the viral surface a key step in developing effective vaccines. Changes in the glycans attached to proteins are often associated with certain diseases, and thus they can be used as biomarkers for early disease detection. Free glycans found in human milk play an important role in the immune development of infants.
Given the undeniable importance of glycans, one needs a fast and reliable way to detect and analyze them, akin to the powerful tools used for protein analysis. The problem is that glycans are notoriously difficult to analyze. Because many of the monosaccharide building blocks have the same mass (i.e. they are isomers), glycans with different monosaccharide content can also be isomeric and thus cannot easily be distinguished by mass spectrometry, which is the most sensitive tool used for biomolecular analysis. In addition, there are a variety of ways that the building blocks can be connected to one another, giving rise to an additional level of isomeric complexity.
The goal of the GLYCANAL project has therefore been to develop a completely new approach to glycan analysis that is sensitive, fast, and accurate. The approach that we have taken has been to measure the manner in which each molecule absorbs infrared radiation, which provides a unique, identifying fingerprint of the molecule, and we do this in combination with ultrahigh-resolution ion mobility selection of different isomers, which first sorts them by their shape. These measurements are made inside a mass spectrometer, which makes them extremely sensitive.
The ultimate objectives are: (1) to develop an extensive spectroscopic database for glycan isomers that can be used to identify them rapidly; and (2) to make this technique widely available to researchers in companies and academia.
Glycans are central to the functioning of biotherapeutics, for example. The pattern of glycans attached to a protein drug affects its effectiveness, lifetime, and cytotoxicity, and this must be carefully controlled during their biosynthesis. Viruses often use glycans to shield themselves from detection, making analysis of the glycans coating the viral surface a key step in developing effective vaccines. Changes in the glycans attached to proteins are often associated with certain diseases, and thus they can be used as biomarkers for early disease detection. Free glycans found in human milk play an important role in the immune development of infants.
Given the undeniable importance of glycans, one needs a fast and reliable way to detect and analyze them, akin to the powerful tools used for protein analysis. The problem is that glycans are notoriously difficult to analyze. Because many of the monosaccharide building blocks have the same mass (i.e. they are isomers), glycans with different monosaccharide content can also be isomeric and thus cannot easily be distinguished by mass spectrometry, which is the most sensitive tool used for biomolecular analysis. In addition, there are a variety of ways that the building blocks can be connected to one another, giving rise to an additional level of isomeric complexity.
The goal of the GLYCANAL project has therefore been to develop a completely new approach to glycan analysis that is sensitive, fast, and accurate. The approach that we have taken has been to measure the manner in which each molecule absorbs infrared radiation, which provides a unique, identifying fingerprint of the molecule, and we do this in combination with ultrahigh-resolution ion mobility selection of different isomers, which first sorts them by their shape. These measurements are made inside a mass spectrometer, which makes them extremely sensitive.
The ultimate objectives are: (1) to develop an extensive spectroscopic database for glycan isomers that can be used to identify them rapidly; and (2) to make this technique widely available to researchers in companies and academia.
The work performed under this project can be divided into three main areas:
1) The design, construction, and optimization of an instrument in which to combine ultrahigh-resolution ion mobility, cryogenic vibrational spectroscopy, and mass spectrometry for glycan analysis.
2) The development of protocols to construct a database of glycan infrared spectra that could be used for glycan analysis. This included developing schemes by which to measure infrared spectra in cases where no standards exist.
3) The gradual population of this spectral database for human milk oligosaccharides (HMOs) and N-linked glycans.
4) The demonstration of coupling IR spectroscopy inside a mass spectrometer with liquid chromatography (LC) separation of isomeric species, since LC is so central to existing workflows for biomolecule analysis. This paves the way for incorporating our technology onto commercial machines, making it widely accessible to the scientific community.
These achievements have been disseminated at the most important meeting in our field (the Annual Meeting of the American Society for Mass Spectrometry (ASMS)), as well as at annual meetings of the International Mass Spectrometry Conference (IMSC), the British Mass Spectrometry Society (BMSS), and the Swiss Group for Mass Spectrometry (SGMS). Moreover, we have reported our results in 20 peer-reviewed publications in top-level journals. Finally, this technology has led to several patent applications and has formed the basis of a startup called Isospec Analytics SA.
1) The design, construction, and optimization of an instrument in which to combine ultrahigh-resolution ion mobility, cryogenic vibrational spectroscopy, and mass spectrometry for glycan analysis.
2) The development of protocols to construct a database of glycan infrared spectra that could be used for glycan analysis. This included developing schemes by which to measure infrared spectra in cases where no standards exist.
3) The gradual population of this spectral database for human milk oligosaccharides (HMOs) and N-linked glycans.
4) The demonstration of coupling IR spectroscopy inside a mass spectrometer with liquid chromatography (LC) separation of isomeric species, since LC is so central to existing workflows for biomolecule analysis. This paves the way for incorporating our technology onto commercial machines, making it widely accessible to the scientific community.
These achievements have been disseminated at the most important meeting in our field (the Annual Meeting of the American Society for Mass Spectrometry (ASMS)), as well as at annual meetings of the International Mass Spectrometry Conference (IMSC), the British Mass Spectrometry Society (BMSS), and the Swiss Group for Mass Spectrometry (SGMS). Moreover, we have reported our results in 20 peer-reviewed publications in top-level journals. Finally, this technology has led to several patent applications and has formed the basis of a startup called Isospec Analytics SA.
Our new instrument for glycan analysis is unique, having capabilities that are not found anywhere else in the world. It combines ultrahigh-resolution ion mobility with cryogenic ion spectroscopy. This instrument has a number of features that goes beyond the state of the art.
• The path-lengths used for ion mobility can extend to the order of 1 km in a region of that is only 1 m x 1 m. It accomplishes this by pushing the glycan molecules along a labyrinthine 10 m path through which they can be cycled up to 100 times. This results in the ability to separate isomers with the minutest in difference in their shape.
• We have the ability to manipulate molecules during the separation process. We can store them, break them into pieces, and then and analyze the pieces, both by ion mobility and cryogenic vibrational spectroscopy.
• We developed the ability to multiplex the measurement of vibrational spectra – that is, to measure spectra of several mobility-separated species simultaneously. We do this using an ion trap containing several compartments where we can store ionized glycans separately without mixing.
• Using this new machine, we have demonstrated the ability to detect and identify a complex glycan in a sample in as little as 10 seconds, a process that previously would take hours. This has allowed the direct coupling of our method with liquid chromatography in real time.
• The path-lengths used for ion mobility can extend to the order of 1 km in a region of that is only 1 m x 1 m. It accomplishes this by pushing the glycan molecules along a labyrinthine 10 m path through which they can be cycled up to 100 times. This results in the ability to separate isomers with the minutest in difference in their shape.
• We have the ability to manipulate molecules during the separation process. We can store them, break them into pieces, and then and analyze the pieces, both by ion mobility and cryogenic vibrational spectroscopy.
• We developed the ability to multiplex the measurement of vibrational spectra – that is, to measure spectra of several mobility-separated species simultaneously. We do this using an ion trap containing several compartments where we can store ionized glycans separately without mixing.
• Using this new machine, we have demonstrated the ability to detect and identify a complex glycan in a sample in as little as 10 seconds, a process that previously would take hours. This has allowed the direct coupling of our method with liquid chromatography in real time.