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Resolving Lipid Isomers with Novel Ion Mobility Mass Spectrometry Methods

IM-MS is particularly useful for lipid structural analysis by combining high sensitivity with high specificity

Lipids are a class of biomolecules essential to the structure and function of living organisms, contributing to cell membrane structure, cell activity regulation, and energy storage. Understanding lipid structure, biochemistry, and metabolism is fundamental to basic science and clinical research, so scientists need methods to analyze lipids and establish their roles in health and disease.

However, lipids are structurally diverse biomolecules that can vary in headgroup type (the part of the lipid that makes up the hydrophilic backbone of membrane phospholipids), acyl chain length, position of attachment, degree of unsaturation, and stereochemistry.1 In addition, since many lipids are isomeric, being able to separate and characterize individual isomers is beneficial for ongoing lipidomics studies.

Mass spectrometry (MS) has been used extensively to elucidate lipid structural details and is now routinely coupled with ion mobility (IM-MS) to provide an additional dimension for spatially resolved separations and characterization. IM-MS also provides faster separations than traditional liquid chromatography mass spectrometry (LC-MS) methods, which can be time consuming and often lack sufficient resolution to fully separate and differentiate all crucial isomers. IM-MS is particularly useful for lipid structural analysis by combining high sensitivity with high specificity.

An additional dimension of separation

The key benefit of IM-MS is the ability to measure the gas-phase mobility of ionized molecules and calculate a two-dimensional collision cross section (CCS). By measuring the CCS value, as well as the conventional mass-to-charge ratio (m/z), IM-MS separations account for a molecule’s size, charge, shape, and structure. As a result, analytes that have the same molecular mass can be separated more effectively by their shape, charge, and CCS. 

This comprehensive 2D separation allows scientists to differentiate lipid isomers into their respective classes. Subtle structural motifs of lipid CCS structure can reveal more granular detail about the chemical class being observed, such as cerebrosides, sphingomyelins, phosphatidylcholines, or other lipid classes and sub-classes.2 

High resolution IM (HRIM) is emerging as a useful technique for the analysis of complex lipids, providing separations with more than four times the resolution than those achieved with conventional IM-MS. 

Recent research has shown that HRIM-MS produces highly reproducible separation and quantification of ganglioside isomers – a sub-lipidome in the brain that contains complex sphingolipid species whose dysregulation has been linked to neurodegenerative diseases.3 Using this technology to advance the knowledge of the lipid content in preclinical models and patient samples provides the opportunity to develop new therapeutic targets and biomarkers, to tackle neurodegenerative diseases, and many more conditions.

Unparalleled resolving power 

A study carried out by researchers at Vanderbilt University’s Department of Chemistry explored the separation of lipids originating from complex total lipid fractions using HRIM-MS.4 

The group investigated glucosylceramide (GlcCer), a sphingolipid, and the phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Lipid identifications were made based on the accurate mass measurement and IM-derived CCS values via database (lipid Atlas)1 comparison as available and, for previously unreported features, based on mass and IM-MS trend analyses. CCS values for these tentatively identified lipid features were determined for both conventional Drift Tube Ion Mobility-MS (DTIM-MS) and HRIM-MS.

Using HRIM-MS, some lipid features previously observed with DTIM-MS were shown to separate in their drift spectra. Of the three total lipid extracts studied, there were a variety of features that were resolved only via HRIM-MS. On average, both the PC and PE total lipid extracts showed two lipid isomer features being resolved for each lipid mass. Surprisingly, the GlcCer total lipid extract displayed lipid features being resolved into two, three, and even four features for each lipid mass.

Figure 1 shows lipid isomer separations of different features from the PC total lipid extract. HRIM-MS provides a higher resolution separation, resolving peaks not identified using a conventional ion mobility approach.

Chart showing ion mobility arrival time distributions at conventional and high resolutions.
Figure 1. Ion mobility arrival time distributions for PC 36:01 at conventional resolution (ca. 70 top) and high resolution ion mobility (ca. 250 bottom). Bottom panel clearly shows the baseline separation of a second lipid conformation or species and the potential for resolving a third species at even higher resolving powers with future developments.
MOBILion Systems, Inc.

Looking ahead

Different lipid classes have a range of biological functions which makes their separation and identification important. The ability to accurately identify lipid profiles in a high-throughput manner permits the rapid separation of isomeric lipids without the need for extensive sample preparation or LC, opening potential new avenues in lipidomics research. The methods demonstrated in this study could, in the future, allow verification of lipid extract trends once CCS values are calculated after calibration, and expand the range of lipid extracts that can be studied such as phosphatidylserine, sphingomyelin, phosphatidylglycerol, phosphatidylinositol, and sulfatide.

The high peak capacity achievable with HRIM-MS allows researchers to overcome isotopic interferences associated with lower resolution IMS separations. This, together with the use of fragmentation on HRIM-MS to determine higher specificities of each lipid extract subclass, is enabling many new applications in lipidomics and can be transferred to other biomolecular research fields.

References  

1. Leaptrot, K.L., May, J.C., Dodds, J.N. et al. Ion mobility conformational lipid atlas for high confidence lipidomics. Nat Commun. 2019; 10: 985.

2. May, J., Goodwin, C. R., Lareau, N., et al. Conformational ordering of biomolecules in the gas phase: nitrogen collision cross sections measured on a prototype high resolution drift tube ion mobility-mass spectrometer, Analytical Chemistry, 2014; 86, 22107-2116.

3. Wormwood Moser, K. L., Van Aken, G., DeBord, D., et al. High-defined quantitative snapshots of the ganglioside lipidome using high resolution ion mobility SLIM assisted shotgun lipidomics, Analytica Chimica Acta, 2021; 1146: 77-87.

4. Reardon, A. R., Leaptrot, K. L., Rose, B. S., et al. Improved Separation of Isomers from Total Lipid Fractions via High Resolution Ion Mobility (HRIM) Incorporating Structures for Lossless Ion Manipulation, Poster, ASMS 2021.