Glycan Visualization - Single-Molecule Imaging of Glycoconjugates
On October 12, 2023, the team of Klaus Kern, Rebecca L. Miller and Thomas Ziegler published an article in Science titled "Direct observation of glycans bonded to proteins and lipids at the single-molecule level". The study developed a novel single-molecule imaging method that can directly "see" the structure and attachment points of each glycan in the Glycoconjugate, thus breaking through the limitations of current analytical techniques.
Glycans modified on proteins and lipids are ubiquitous in various biological systems and participate in a variety of physiological and pathological processes. The analysis of glycoconjugates mainly relies on ensemble-averaged methods, which makes it difficult to reveal the specific location and structure of each glycan on a single molecule, especially in glycoproteins. This article demonstrates a single-molecule glycoconjugate imaging technique based on low-temperature scanning tunneling microscopy (LT STM), which can directly observe single glycoconjugate molecules. The intact glycoconjugate ions obtained by electrospray ionization are gently deposited on the surface to achieve single-molecule imaging. With the help of quantum mechanical modeling, this submolecular imaging resolution can reveal the complete structure of glycans in glycopeptides, Glycolipids, N-glycoproteins and O-glycoproteins and their attachment sites.
Carbohydrate compounds are one of the four basic organic components of life systems. They are widely present in all organisms and participate in various processes such as cell function, growth and development, recognition, and energy storage. In biological systems, glycans often exist in the form of "glycoconjugates", that is, glycans are attached to proteins or lipids through Enzymatic Glycosylation. This modification is the most common and complex type of post-translational modification of proteins, which greatly expands the functions of proteins and lipids. The importance of glycoconjugates in health and disease makes them a key target in basic and translational research, especially in the development of new therapeutic and diagnostic strategies.
However, the complexity of glycoconjugate structures brings research challenges. Not only do they have high structural heterogeneity and structural isomerism, but current analytical methods mostly rely on Chemical Labeling, enzymatic hydrolysis, and ensemble-averaged strategies. In other words, we can often only infer the glycan structure of a single molecule based on the overall characteristics of the sample, but cannot really observe where and how the glycans on each glycoconjugate are attached, especially in proteins with multiple glycosylation sites. Therefore, in order to reveal the structural and functional relationship of glycans, direct observation at the single-molecule level is urgently needed.
This study developed a new method to achieve direct imaging and analysis of single glycoconjugate molecules. Using a LT STM in combination with electrospray ion soft landing technology, the complete glycoconjugate molecules were gently deposited on the metal surface and then imaged one by one. The imaging results combined with quantum mechanical simulations revealed the complete structure and precise attachment sites of different glycans on proteins or lipids. The study successfully performed single-molecule observations on a variety of samples such as Glycopeptides, glycolipids, N-glycoproteins, and O-glycoproteins, and could even identify each monosaccharide structure and its spatial configuration and side groups.
To break through this bottleneck, the research team developed an imaging method based on LT STM. STM is an instrument that can "see" individual atoms and molecules at the sub-nanometer scale. In the past, it has been used to image small molecules, carbon nanotubes, etc., but it is the first time to apply it to complex biomolecules, especially glycoconjugates.
The key steps of the method are as follows:
With this combined strategy, the research team can not only distinguish the connection structure between glycans and proteins/lipids, but also identify the slight differences between different monosaccharides (such as Glucose, galactose, sialic acid), and even observe the spatial position of substituents (such as sulfate groups). This ability far exceeds traditional methods and can be called a microscope in the glycan world.
Fig. 1 Key steps of low-temperature scanning tunneling microscopy. (Anggara, et al., 2023)
To verify the applicability of this technology, the researchers selected a series of representative glycoconjugates for single-molecule imaging, covering multiple levels from simple to complex:
In particular, the distribution pattern of O-glycans observed in MUC1 verified the previous speculation on its enzymatic specificity, and provided an intuitive basis for studying the mechanism of tumor-related O-glycosylation.
This study demonstrated for the first time the feasibility of single-molecule, non-labeled, in situ, high-resolution imaging of complex glycoconjugates. Its groundbreaking contributions include:
Although current STM imaging still relies on the auxiliary analysis of known protein sequences and molecular conformations, with the development of technologies such as probe functionalization, tunneling spectroscopy, nuclear spin detection and AI automatic structure decoding in the future, STM is expected to achieve direct identification and imaging of unknown proteins and glycoconjugates, greatly enhancing the depth and breadth of glycoproteomics and glycolipidomics research.
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