Liquid chromatography-mass spectrometry (LC-MS) is still the laboratory workhorse for many applications, but in recent years, there has been growing interest in alternative separations techniques that can offer benefits where LC has limitations. One area where reversed phase LC struggles is glycoproteomics. Glycoforms of peptides tend to co-elute because the retention properties of different glycan structures are similar. As a result, this doesn’t give the MS much time to acquire tandem mass spectra, which is necessary for identifying the different glycoforms. In turn, this can limit how thoroughly the glycoproteins are characterized.
So how can this be improved? Enter ZipChip™.
The ZipChip platform is based on microchip zone electrophoresis (MZE). This is a totally different separations mechanism than reversed phase LC as the separations are based on charge, size and conformation in solution, not retention on a stationary phase. So, what about those glycan structures that interact similarly with the LC column you may ask? Well, they have different branching structures and sometimes even different charges. (See where we’re going with this?)
Researchers at Boston University recently demonstrated how MZE-MS are a valuable technique for characterizing glycopeptides, oliogosaccharides and monosaccharides in biological samples. Using the ZipChip system for separations, they observed that the glycosylated peptides are cleanly resolved in the separation space from their nonglycosylated counterparts. This is due to the impact the glycan structures have on the size and conformation of the peptides. Even more impressive is that different glycoforms of the glycopeptides – including some isoforms – were easily resolved. Remember that with MZE, the size and conformation of analytes impacts how they separate, so changes in the number of sugar groups branched together, or how they are branched together, can have a big effect on the results. This provided another layer of information about the glycan structures in addition to tandem MS characterization. The authors also point out that MZE is orthogonal to reversed phase LC and could be combined with LC fraction collection to create a powerful 2D separation system. Can’t wait to see that work hit the presses!
In addition to the glycopeptide work, the team at BU also demonstrated MZE-MS characterization of released and labeled oliogosaccharides and monosaccharides using the same analysis conditions. That’s three types of characterization using one method!
Overall the MZE-MS method described by BU demonstrates the potential to more thoroughly probe site-specific glycan microheterogeneity. This could be a major asset to the fields of glycomics and glycoproteomics where researchers are attempting deep characterization of exceedingly complex samples. These studies are vital to learning more about factors affecting cellular mechanisms and impacting disease states. This is great work coming out of Boston University – we can’t wait to see what else they are working on!
For more information about how this technology and the worked performed by Boston University, we invite you to read this recently published technical paper in Analytical Chemistry. The paper, titled “Microfluidic capillary electrophoresis-mass spectrometry for analysis of monosaccharides, oligosaccharides and glycopeptides,”
By: Erin Redman, Senior Research Scientist