Separating ganglioside isomers using HRIM–MS


In this article, Komal Kedia (Senior Scientist at Merck), Rena Zhang (Senior Scientist at Merck), Weixun Wang (Senior Scientist at Merck), Kevin Bateman (Scientific Associate VP at Merck) and Kelly Moser (Applications Manager at MOBILion Systems) show how ganglioside isomers with suspected roles in neurodegenerative diseases can be separated on the basis of their ion mobility, using a new high-resolution ion mobility–mass spectrometry (HRIM-MS) technique in conjunction with conventional HPLC.  

Function of gangliosides 

Gangliosides are a class of glycosphingolipids, which, together with glycoproteins and glycosaminoglycans, form the ‘glycocalyx’ that covers the surfaces of eukaryotic cells, and which in endothelial cells is believed to regulate vascular permeability, modulate the interaction with blood, and transmit physical forces to the cytoskeleton.1

The total mass of gangliosides varies significantly between tissues and cell types, but it is notable that the human brain contains 10 to 30 times more gangliosides than any other tissue or organ in the body, with particularly high concentrations being present in neurons and glial cells. This reflects the important role of gangliosides in the modulation of membrane proteins and ion channels, in cell signaling, and in inter-cellular communication.1

Accordingly, altered expression of gangliosides has been found to take place during the course of healthy aging, and it has been implicated in neurodegenerative disorders including Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease.2 This has made the role of gangliosides in the brain a topic of considerable research interest in recent years, from the perspectives of diagnosis, progression, and potential clinical interventions.  

Gangliosides consist of a ceramide lipid tail attached by a glycosidic linkage to a glycan headgroup containing one or more sialic acid residues (Figure 1).3 The great variety possible in the glycan headgroup is reflected in the fact that over 200 structures have been reported to date, although most gangliosides found in vertebrates fall into one of only a few major classes.2 Notably, over 90% of the mass of gangliosides in the human brain comprises just four gangliosides, known as GM1, GD1a, GD1b and GT1b, although the presence of isomers that cannot be separated using standard high-performance liquid chromatography (HPLC) techniques hinders the development of a more detailed understanding of their roles. 

In this article, we show a real-world example of how this problem can be overcome, using new technology that allows ions to be separated on the basis of their different mobilities in an inert carrier gas. This approach, known as high-resolution ion mobility–mass spectrometry (HRIM-MS), is based on a technology called structures for lossless ion manipulation (SLIM), which uses moving electric fields to transport ions along a 13-meter serpentine path in a lossless fashion within seconds, allowing them to be separated with very high resolution, while not compromising on sensitivity (Figure 2).4

In Figure 2, we couple HRIM-MS to HPLC to provide greatly improved separation of ganglioside isomers within a typical HPLC timeframe. 

Experimental data

Extraction: Gangliosides were extracted from cerebrospinal fluid using chloroform–methanol. The upper phase was collected, and additional cleanup was performed using C-18 solid-phase extraction. 

HPLC: 1290 Infinity ll LC (Agilent Technologies); HALO-HILIC column, 150 mm × 4.6 mm, 2.7 µm. Mobile phase A: 90% acetonitrile, 10% water + 15 mM ammonium acetate. Mobile phase B: Water + 15 mM ammonium acetate. 

HRIM: MOBIE (MOBILion Systems) 

QTOF MS: 6545XT (Agilent Technologies) 


The results of HPLC–HRIM–QTOF analysis of two gangliosides from the extract are shown in Figure 3 (showing GD1a and 1b) and Figure 4 (showing GT1). 

In each case, panels 2–5 show data relating to the peak of interest highlighted in the extracted-ion chromatogram (panel 1), with the corresponding mass spectra (panel 2) and isotopic distributions (panel 3) indicating the presence of just one species. However, adding the results from the ion mobility dimension (panel 4) shows that multiple species with different ion mobilities are actually present, and the distribution of these across the isotope peak set is shown by the heat map (panel 5). 

Conclusions and future work 

In this proof-of-concept study, we have shown how the addition of HRIM-MS technology to HPLC workflows enables separation of isomeric and isobaric ganglioside species that hitherto were unresolvable using HPLC alone. A feature of the analysis is also the high speed of the ion mobility separation, making this approach amenable to high-throughput studies. 

Future work will focus on validating this approach for characterising gangliosides, with a focus on accuracy, precision, and limits of detection and quantitation. It is hoped that we will be able to use this validated method to profile gangliosides in the brain and cerebrospinal fluid, to identify biomarkers for target engagement and pharmacodynamics. Based on the preliminary results we have reported here, we expect that the use of HRIM-MS will enable the characterisation of additional isomeric gangliosides previously ‘invisible’ with traditional HPLC platforms. A further line of investigation will be semi-targeted lipidomics covering other major classes of lipids.  

Figure 1: Structures of gangliosides
Figure 2: Schematic of the SLIM-based HRIM device connected to an Agilent 654XT Q-ToF
Figure 3: HPLC–HRIM–QTOF analysis of a ganglioside extract, showing GD1a and GD1b (m/z 917.47 (2H–)). For a description of panels 1–5, see the main text
Figure 4: HPLC–HRIM–QTOF analysis of a ganglioside extract, showing GT1 (m/z 1063.03 (2H–)). For a description of panels 1–5, see the body text


  1. J. Jin, F. Fang, W. Gao, H. Chen, J. Wen, X. Wen and J. Chen, The structure and function of the glycocalyx and its connection with blood-brain barrier, Frontiers in Cellular Neuroscience, 2021, published online,    
  2. S. Sipione, J. Monyror, D. Galeguillos, N. Steinberg and V. Kadam, Gangliosides in the brain: Physiology, pathophysiology and therapeutic applications, Frontiers in Neuroscience, 2020, 14: 572965. 
  3. T. Kolter, Ganglioside Biochemistry, ISRN Biochemistry, 2012, 506160. 
  4. L. Deng, Y.M. Ibrahim, A.M. Hamid, S.V.B. Garimella, I.K. Webb, X. Zheng, S.A. Prost, J.A. Sandoval, R.V. Norheim, G.A. Anderson, A.V. Tolmachev, E.S. Baker, and R.D. Smith, Ultra-high resolution ion mobility separations utilizing traveling waves in a 13 m serpentine path length structures for lossless ion manipulations module, Analytical Chemistry, 2016, 88: 8957–8964. 

Related Articles

Join FREE today and become a member
of Drug Discovery World

Membership includes:

  • Full access to the website including free and gated premium content in news, articles, business, regulatory, cancer research, intelligence and more.
  • Unlimited App access: current and archived digital issues of DDW magazine with search functionality, special in App only content and links to the latest industry news and information.
  • Weekly e-newsletter, a round-up of the most interesting and pertinent industry news and developments.
  • Whitepapers, eBooks and information from trusted third parties.
Join For Free