Project Details
Projekt
On-surface glycan sequencing – combining preparative mass spectrometry with scanning probe microscopy to decipher the glycocode
Applicant
Marko Grabarics
Subject Area
Analytical Chemistry
Term
since 2022
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 509729094
Biological information and function are encoded in the sequence of biopolymers, such as nucleic acids, proteins, and glycans. Whereas a range of automated, high-throughput methods are available for DNA and protein sequencing, the de novo structure elucidation of glycans is still an unresolved challenge in biochemistry. In contrast to linear biopolymers, glycans tend to form branched structures with complex regio- and stereochemistry, resulting in a vast number of potential isomers. Since isomers have identical mass, conventional tandem MS-based methods often struggle to distinguish them and fail to reveal the unique underlying glycan structure. Thus, there is a clear need for novel analytical techniques that can overcome this limitation and provide all regio- and stereochemical information required for the unambiguous sequence assignment of glycans.The aim of this project is to develop such a de novo glycan sequencing method based on the direct imaging of chemically selected, individual molecules with atomic detail. For this purpose, we will combine preparative MS with state-of-the-art scanning probe microscopy (SPM) techniques under ultrahigh vacuum conditions, and obtain real-space images of mass-selected glycans deposited onto atomically defined surfaces. Employing a unique preparative MS apparatus, we will implement the on-surface glycan sequencing workflow using a set of human milk oligosaccharide (HMO) standards as model compounds. These molecules display all key aspects of glycan structural complexity, providing an ideal platform to develop and optimize the method.Put together, SPM imaging with chemically selective deposition will enable a range of real-world applications that could previously not be tackled. Building on proof-of-concept experiments on HMOs, we will next integrate preparative MS and SPM imaging into established glycomic workflows in order to study protein O-glycosylation. The O-glycan chains released from glycoproteins will be enriched and fractionated by liquid chromatography, and deposited onto clean crystal surfaces by preparative MS to allow for their sequencing using high-resolution SPM.Finally, on-surface glycan sequencing will be extended to study chemical modifications of the carbohydrate backbone. In particular, we will focus on imaging the sulfation pattern of glycosaminoglycans (GAGs), a family of highly acidic polysaccharides. Since the arrangement of sulfate groups governs the binding of bioactive GAG sequences to proteins, deciphering the sulfation code is essential to understanding the structure–function relationships of these molecules. To this end, we will utilize chemically modified SPM probes which, through increasing spatial resolution and chemical selectivity, will facilitate the distinction of functional groups and thus help us determine the position of sulfate modifications in GAGs.
DFG Programme
WBP Fellowship
International Connection
United Kingdom
