Direct observation of glycans bonded to proteins and lipids at the single-molecule level

Proteins and lipids decorated with glycans are found throughout biological entities, playing roles in biological functions and dysfunctions. Current analytical strategies for these glycan-decorated biomolecules, termed glycoconjugates, rely on ensemble-averaged methods that do not provide a full view of positions and structures of glycans attached at individual sites in a given molecule, especially for glycoproteins. We show single-molecule analysis of glycoconjugates by direct imaging of individual glycoconjugate molecules using low-temperature scanning tunneling microscopy. Intact glycoconjugate ions from electrospray are soft-landed on a surface for their direct single-molecule imaging. The submolecular imaging resolution corroborated by quantum mechanical modeling unveils whole structures and attachment sites of glycans in glycopeptides, glycolipids, N-glycoproteins, and O-glycoproteins densely decorated with glycans. Many proteins, especially those that are secreted from eukaryotic cells, have sugar chains attached to facilitate quality control or mediate protein-protein or cell-cell interactions. These sugars are often complex and heterogeneous and can be challenging to study by conventional structural or biophysical methods. Anggara et al . show that glycans attached to peptides and lipids can be imaged directly using single-molecule scanning tunneling microscopy. These biomolecules can be applied to a surface by a gentle electrospray deposition and, if necessary, manipulated to stretch out structured regions. The authors observed distinct glycan configurations and imaged large fragments of proteins, including a densely glycosylated mucin. —Michael A. Funk Single-molecule imaging of glycan-decorated proteins and lipids was achieved.