Analytical Sciences, Short talk

Determination of the primary structure of glycans by enzymatic cleavage and cryogenic IR-spectroscopy

I. Diukova1, T. Rizzo1*
1EPFL, Laboratory of Molecular Physical Chemistry

Glycans, or oligosaccharides, play critical roles in numerous physiological processes and are involved in some way or another in most major diseases. In addition, their characterization is essential for monitoring the quality of biotherapeutics. Because the function of glycans is directly related to their structure, glycan structural characterisation has become an exponentially growing field. Despite their importance, glycan characterization is challenging because of their isomeric complexity.

One of the most powerful techniques for glycan investigation is the combination of liquid chromatography (LC) with mass spectrometry (MS), however even this method cannot distinguish all the various forms of isomerism. In our laboratory, we have recently demonstrated that cryogenic messenger-tagging IR-spectroscopy provides unique vibrational fingerprints of glycans that are extremely sensitive to the slightest structural differences.

In the present work, we monitor the step-wise enzymatic degradation of glycans using liquid chromatography and then measure cryogenic IR spectra of the resulting smaller glycans. This approach will be applied for creating and then using a glycan database. The main principle of the identification of an unknown glycan involves the repetition of these two steps until the spectra that we measure are those of glycans already present in our database. By knowing the isomeric specificity of the applied enzymes, we hope to reconstruct the primary structure of the unknown glycan and then add its spectrum to our database. In this way, we define the structure of the unknown oligosaccharide and expand our database.

We chose the N-linked glycans Man-1, Man-3, NGA2 and NA2 as starting examples and performed two different approaches of glycosidase digestion. Protocols of single exoenzyme cleavage for Man-3, NGA2, NA2 and multi-exoenzyme cleavage for NGA2 and NA2 were developed. After digestion, we studied and cleaned all samples by hydrophilic interaction liquid chromatography (HILIC) using an AQUITY UPLC H-Class Plus System (Waters) with a Q-TOF mass spectrometer (Waters Premier) as a detector. The glycan fragments were separately collected and prepared for spectroscopy. We used a home-built, cryogenic, tandem mass spectrometer together with an IR OPO to measure the infrared spectra of the glycan reference compounds and the glycan fragments after enzymatic cleavage.

The results we have obtained confirm the possibility of combining these two complementary analytical techniques and serve as a starting point for constructing a glycan database. In the future, this approach will be extended to more complex unknown sugars as the database expands.