NMR Method Detects Glycosylations On Intact Proteins

Glycosylation is the most abundant type of protein posttranslational modification. However, there’s no direct method for characterizing the chemical changes. Mass spectrometry methods, which require enzymatic digestion of the protein, can reveal the location and composition of a glycosylation, but they can’t determine the exact stereochemistry or glycosidic linkage. Mario Schubert, Michal J. Walczak, and coworkers at ETH Zurich have reported a simple and direct nuclear magnetic resonance spectroscopy method for detecting and characterizing intact glycosylated proteins (Angew. Chem. Int. Ed. 2015, DOI: 10.1002/anie.201502093). For each protein, the researchers collect a pair of two-dimensional NMR spectra—¹H-¹H and ¹H-¹³C. The distinctive patterns reveal the saccharide types and linkages in each glycosylation. The analysis takes place under denaturing conditions, so the chemical shifts for all the amino acids reflect random coil structures. Any deviations from the random coil pattern indicate modifications. The team used the method to analyze glycosylated proteins from bacteria, fungi, plants, and animals. The approach works well for glycans containing up to about 20 saccharides. Because the new method can’t be used to assign a modification’s location on the protein sequence, it will be complementary to mass spec techniques.