5 Looking Ahead
Glycan analysis, or glycomics, in which scientists elucidate the specific glycosylation patterns of biomolecules, has become an essential aspect of biomedical research, drug development, and biopharma quality control. Compared to traditional methods, HRIM excels in ease of use, generalisability of one method to multiple analyte classes, resolution, and speed of analysis. These attributes permit researchers to tackle the complexity of glycan structures and their heterogeneity on timescales that enable high-throughput analysis. The HRIM analyses discussed here are nearly 100-fold faster than LC analyses, but are only the beginning. More rapid sampling - a subject of ongoing development - could speed analysis times by additional orders of magnitude.
References
Figure 3A: Extracted high mannose reduced permethylated N-linked glycans separated by 180-minute LC workflow.
In HRIM, as with other ion mobility technologies, an ion’s migration time is determined by its mass-to-charge ratio and its size and shape. As ions are driven along the separation path; the collision with an inert buffer gas slows them down to a degree proportional to their size. The length of the ion separation path is crucial to achieving the high degree of separation required to gain adequate resolution of glycans with challenging structural diversity. MOBILion’s first HRIM product has an ion path of 13 meters (42 feet). The unique serpentine path design of the printed circuit boards allows for the 13m ion path to fit into a device the size of a briefcase allowing for unparalleled separation of isomeric structures.
HRIM set-up is straightforward because it occurs in the gas phase under nearly ideal conditions. There is no need to optimise columns, flow rates, or liquid components as with LC. In addition, HRIM achieves rapid separations with data collection in the order of milliseconds to seconds. Much has been written about how the most advanced HRIM approaches boost resolving power by expanding the pathlength [21] and when combined with MS (HRIM-MS, Figure 2) offers tremendous potential for seamless separation of glycans followed by structural determination in biomedical and clinical research [22,23].
HRIM-MS in Practice
Recent work at the Complex Carbohydrates Research Center (CCRC) at the University of Georgia, Athens, Georgia, USA, has demonstrated the performance of HRIM-MS. Experiments compared this next-generation ion mobility technology, with an optimised LC-MS protocol [24].
The experimental set-up at CCRC used the MOBILion HRIM instrument coupled to an Agilent 6545 Quadrupole Time of Flight (Q-ToF). Permethylated N-glycans released from Fetuin and RNaseB were analysed by 180-minute reverse phase nanoflow LC-MS/ MS, typical of a traditional LC N-glycan analysis workflow. The same glycan species were analysed using HRIM-MS. In 2 minutes, all glycans identified using the 180-minute LC separation were separated and identified using the HRIM-MS workflow. This 90-fold time savings allows, for example, a three- month LC analysis to be compressed to a one-day analysis. Moreover, the HRIM-MS analysis resolved the structural isomers of a biantennary, disialylated complex glycan that could not be resolved with LC (Figure 3B, G5).
Being able to rapidly run hundreds of samples enables the establishment of a ‘normal range’ of glycan heterogeneity. That advantage allows greater insight into the behaviour of glycans in a wide range of illnesses, in a time frame compatible with the fast pace of biopharmaceutical development.
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