Research Spotlight: Characterization of Glycosphingolipids and Their Diverse Lipid Forms through Two-Stage Matching of LC-MS/MS Spectra

Posted on August 24, 2021


Glycosphingolipid Ms

Thank you to Dr. Kari Basso, University of Florida, for submitting this recent research article! This research utilized our glycosphingolipid GM3. Glycosphingolipids (GSL) play various roles in biological and pathological events. These lipids are known to play a role in signal transduction, cell division, recognition, adhesion, and apoptosis. Some GSLs are also correlated to diseases such cancer and Alzheimer’s Disease. GSL expression is an important area of study due to the significant role that these lipids play in various processes. Being able to detect abnormal GSL expression in cells and tissues could lead to a greater understanding of the functions and mechanisms of action that they have in these processes. Furthermore, being able to detect abnormal expression of GSLs could be useful biomarkers for disease diagnostics and therapeutics.

The advancement of highly sensitive mass-spectrometry (MS) tools makes MS analysis of glycolipids a promising detection method. However, there are several challenges faced when attempting to analyze glycolipids using MS methods including ion suppression of low-abundance species and the likelihood of isobaric overlaps. These challenges often mean that liquid chromatographic (LC) techniques must be used prior to MS detection. Apart from the challenges in the laboratory, there are even more logistical challenges that must be overcome.

To address these issues, Dr. Basso and her research group are working to develop synthetic strategies to rapidly access GSLs and different lipid forms, as well as developing new tandem MS/MS-based methods for high-throughput GSL analysis. The aforementioned research article explains their recent progress towards a MS/MS-based method for GSL analysis.

GSLs, unlike most other lipids, produce two very distinct, equally intense, species-specific product ions in MS. These are carbohydrate fragments (cleavage of the glycan with the charge remaining on the nonreducing side gives product ions without the ceramide moiety) and glycolipid fragments (glycan cleavage with the charge remaining on the reducing end retaining the ceramide moiety). A key observation of GSL cleavage in MS studies is that the lipid form essentially does not affect the fragmentation pattern of the GSL which means that the relative intensities of the carbohydrate fragments are independent of the lipid structure. Also, the glycolipid fragments show changing masses as dictated by lipid forms, while the ion-relative intensities remain unchanged.

Using these observations, Dr. Basso and her group proposed a two-stage matching process to characterize GSLs. The first step is to match the experimental spectra directly against the reference carbohydrate fragments to obtain GSL species identification. The second stage treats glycolipid fragments by the lipid rule-based matching method to identify ceramide composition. This allows all of the spectral data obtained from a MS study to be used to identify the glycolipid. This method, combined with an extensive database of spectral data, would also be conducive to high-throughput GSL analysis. This method should be applicable for both negative and positive mode spectra for both neutral and acidic GSLs. The current method primarily relies on low-energy dissociation via CID which provides limited lipid structure detail. Using a higher energy dissociation method such as electron-capture dissociation (ECD) or ultraviolet photodissociation (UVPD) could be used to capture more structural detail like double bond location and presence of various functional groups.

This two-stage method makes the full characterization of GSLs easily achievable. We are thankful for Dr. Basso and the work that her group is doing, and we are excited to play a small role in this research effort! We can’t wait to see what exciting achievements her group achieves in lipid research for years to come!

To check out this research in its entirety, click HERE! And don’t forget to go check out our Glycosphingolipid Products!

Image Credit: Graphical Abstract of Original Research Publication