Isospec Analytics will be present at the GlycoBioTec conference in Berlin. Be sure to follow Isospec’s CTO and Postdoctoral researcher at the Swiss Federal Institute of Technology Lausanne (EPFL), Dr. Stephan Warnke who will talk on January 18th (12:30). He will  present new data on the identification of glycans present in human milk, with a reduced need for analytical standards.

While glycans, play a fundamental role in many biological processes, their intrinsic isomeric heterogeneity presents a significant challenge to their analysis. This is further complicated by paucity of isomerically pure analytical standards. Glycans found in human milk illustrate this isomeric complexity and hence the difficulty to differentiate and identify isomeric structures with confidence. Cryogenic infrared (IR) fingerprinting is an emerging technique that promises unparalleled structural sensitivity. It can unambiguously identify glycans for which reference fingerprints have previously been recorded to a database. We demonstrate how the combination of ion mobility spectrometry (IMS) with mass spectrometry (MS) and IR fingerprinting can be used to identify isomers with a reduced need for isomerically pure standards. This is made possible through an IMS2 technique, where fragments of separated glycan isomers are identified using IR fingerprints, allowing us to reconstruct the isomeric form of the parent molecule in a bottom-up approach.

After separation, we fragment the parent molecules by collision-induced dissociation (CID). We then send the fragments through a second stage of mobility separation before directing them to an ion trap, where we perform cryogenic IR spectroscopy to identify them. By identification of fragments diagnostic of the isomeric form of the parent molecule, we determine the structure of latter using just a few standards to construct a fragment IR database. The IR spectrum of the parent is then added to our database for identification of fragments from still larger glycans.

We demonstrate our approach by the identification of isomeric Galacto-oligosaccharides (GOS) and human milk oligosaccharides (HMOs). Following the outlined strategy, we present proof-of-principle data to assign isomers GOS trisaccharides, the hexasaccharide Lacto-N-difucohexaose (LNDFH), as well as of the heptasaccharide MFLNH and the octasaccharide DFLNH. In each case, a small database of smaller standards and fragments thereof, is sufficient to identify the glycan backbone as well as the position of fucose residues. As we grow the database by adding isomer-selective IR fingerprints of the identified parent molecules, we can use it to identify isomers of still larger human milk oligosaccharides.