Combining Glucose Units, m/z, and Collision Cross Section Values: Multiattribute Data for Increased Accuracy in Automated Glycosphingolipid Glycan Identifications and Its Application in Triple Negative Breast Cancer

Abstract

Glycan head-groups attached to glycosphingolipids (GSLs) found in the cell membrane bilayer can alter in response to external stimuli and disease, making them potential markers and/or targets for cellular disease states. To identify such markers, comprehensive analyses of glycan structures must be undertaken. Conventional analyses of fluorescently labeled glycans using hydrophilic interaction high-performance liquid chromatography (HILIC) coupled with mass spectrometry (MS) provides relative quantitation and has the ability to perform automated glycan assignments using glucose unit (GU) and mass matching. The use of ion mobility (IM) as an additional level of separation can aid the characterization of closely related or isomeric structures through the generation of glycan collision cross section (CCS) identifiers. Here, we present a workflow for the analysis of procainamide-labeled GSL glycans using HILIC-IM-MS and a new, automated glycan identification strategy whereby multiple glycan attributes are combined to increase accuracy in automated structural assignments. For glycan matching and identification, an experimental reference database of GSL glycans containing GU, mass, and CCS values for each glycan was created. To assess the accuracy of glycan assignments, a distance-based confidence metric was used. The assignment accuracy was significantly better compared to conventional HILIC-MS approaches (using mass and GU only). This workflow was applied to the study of two Triple Negative Breast Cancer (TNBC) cell lines and revealed potential GSL glycosylation signatures characteristic of different TNBC subtypes.

Utility of Ion-Mobility Spectrometry for Deducing Branching of Multiply Charged Glycans and Glycopeptides in a High-Throughput Positive ion LC-FLR-IMS-MS Workflow

“…For the [M + H + Na] 2+ , the CCSDs for each glycan isomer were significantly different with peak apexes at ∼476 ± 0.3 Å 2 for F(6)­A2G1 and ∼509 ± 0.2 Å 2 for F(6)­A2G1’ (Figure S2c and Figure d). This shows that intact IMS could indeed be used in a high-throughput LC-FLR-IMS-MS positive ion mode to distinguish between F(6)­A2G1 and F(6)­AG1’ using the most abundant [M + 2H] 2+ species and also the [M + H + Na] 2+ species …”

“…This shows that intact IMS could indeed be used in a high-throughput LC-FLR-IMS-MS positive ion mode to distinguish between F(6)A2G1 and F(6)AG1' using the most abundant [M + 2H] 2+ species and also the [M + H + Na] 2+ species. 30 The second IMS approach that we applied to deduce glycan branching was a bottom-up fragment-based IMS-MS/MS glycan sequencing analysis (Scheme 1.2). 26−28,38−44 For this approach, the glycans or glycopeptides are fragmented prior to IMS analysis to generate unique product ions or "blocks" with unique m/z and CCS values.…”

“…Interestingly, the majority of IMS analysis of glycans has been obtained through direct infusion analysis, i.e., IMS-MS analysis without LC separation. Little IMS data on N -glycans and glycopeptides exists where glycans are separated on an HILIC LC column with FLR detection prior to IMS analysis in positive ion mode. , Therefore, we examined the usefulness of extending IMS to the already well-established LC-FLR-MS high-throughput positive ion workflows for both RFMS-labeled N -glycans and also glycopeptides, in the form of a LC-FLR-IMS-MS workflow. Of particular focus was to see if in this workflow IMS alone could differentiate the branching of isomeric glycans and glycopeptides in positive ion mode without the need for onerous modification of the already existing and well-established LC-FLR-MS methodology.…”

Ion-Mobility Mass Spectrometry of Glycans and Glycopeptides Using Both Intact and Glycan Sequencing Approaches In Positive Ion Mode Facilitates Glycan Isomer and Glycan Motif Identification

“…Analysis of the alkali metal containing species also provides an additional data point for identification which in turn should help improve ID accuracy. 30 Although unique CCS values could be determined for the F(6)A2G1 isomers using an IMS-MS intact approach, we were interested to see if the glycan sequencing IMS-MS/MS approach could be used to differentiate F(6)A2G1 isomers. We released and analyzed the glycans as described previously however this time a voltage was applied in the collision cell during the IMS-MS analysis to generate glycan product ions through fragmentation.…”

“…LC, IMS and MS can be combined into a single analytical technique for glycan analysis, known as LC-IMS-MS. LC-IMS-MS analysis allows for a multi-dimensional analysis, by combining m/z, GU and CCS data to improve glycan characterization and can be used for both intact and fragment based glycan sequencing IMS analysis. This has been described by Wongtrakul-Kish et al 30 , who demonstrated that through exploiting the aforementioned orthogonal attributes for intact glycans (for glycans from glycosphingolipids) that glycan isomer identification can be increased up to ~90%. Whilst this is a promising methodology, substantial improvement to the identification of specific glycan epitopes and isomers is possible by including the attributes of the glycans that can be derived from the fragmentation of the glycans from the same methodology.…”