N-glycan profiling in Crohn’s Disease: a MALDI-TIMS approach

N-glycosylation is a tightly regulated post-translational modification that plays essential roles in protein regulation, cell-cell interactions, inflammation, and immune responses. Altered N- glycosylation has been associated with several diseases, including Crohn’s disease (CD) [1], with reported reduced levels of galactosylation and fucosylation, together with increased sialylation and highly branched and complex glycan structures, as tri- and tetra-antennary N-glycans [2]. We applied MALDI-TIMS profiling to investigate N-glycan profiles from 5 healthy individuals (N), 5 CD patients in active treatment (AT), and 5 pre-treatment CD patients (PT), aiming to explore their potential in discriminating the 3 conditions. Coupled PRM-PASEF tandem mass spectrometry was used to confirm the composition of N-glycans of interest. Plasma samples were reduced, alkylated, digested with PNGase F and purified using HILIC SPE cartridges. Samples were spotted with double layer using CHCA matrix. MALDI-TIMS profiling was performed in positive ion mode, over a mass range of 1000–3200 m/z and an ion mobility range of 1.20–2.60 1/K0. Raw peak lists from one patient per condition were extracted from DataAnalysis and filtered using mMass. GlycoWorkbench was then used for N-glycan annotation and comparison in the three groups (N, CD-AT and CD-PT). The preliminary results suggest not only disease-related but also treatment-related modulation of N- glycan profiles. As an example, Figure 1 displays distinct patterns: Hex3HexNAcdHex1 (m/z 1485.53) showed higher expression in CD-PT, compared to CD-AT patients and N subjects, while an opposite pattern was observed for Hex5HexNAc4dHex1 (m/z 1809.64). Further analysis by MZmine will integrate TIMSCCS and ion mobility data to improve N-glycans isomer characterization. To complete the analytical workflow, an HILIC-TIMS-PASEF approach will be applied, providing additional information on N-glycan heterogeneity and condition-specific glycosylation patterns.