Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging is a rapidly evolving field in which mass spectrometry techniques are applied directly on tissues to characterize the spatial distribution of various molecules such as lipids, protein/peptides, and recently also N-glycans. Glycans are involved in many biological processes and several glycan changes have been associated with different kinds of cancer, making them an interesting target group to study. An important analytical challenge for the study of glycans by MALDI mass spectrometry is the labile character of sialic acid groups which are prone to in-source/postsource decay, thereby biasing the recorded glycan profile. We therefore developed a linkage-specific sialic acid derivatization by dimethylamidation and subsequent amidation and transferred this onto formalin-fixed paraffin-embedded (FFPE) tissues for MALDI imaging of N-glycans. Our results show (i) the successful stabilization of sialic acids in a linkage specific manner, thereby not only increasing the detection range, but also adding biological meaning, (ii) that no noticeable lateral diffusion is induced during to sample preparation, (iii) the potential of mass spectrometry imaging to spatially characterize the N-glycan expression within heterogeneous tissues.

Matrix-assisted laser desorption/ionization combined with laser-induced postionization (MALDI-2) is a recently introduced method for enhanced mass spectrometry imaging of numerous classes of biomolecules, including phospho- and glycolipids in tissue sections at high lateral resolution. Here we describe the first adaptation of the technology to a Bruker timsTOF fleX mass spectrometer. Upon use of a 1 kHz postionization laser, MALDI-2 produces a sizable increase in the number of detected features as well as in ion signal intensities. This enhancement is similar to that described previously for low repetition rate MALDI-2 systems, but now enables substantially enhanced measurement speeds. In our proof-of-concept study, we furthermore demonstrate, on examples of rat brain and testis tissue sections, that the combination of MALDI-2 with the trapped ion mobility spectrometry (TIMS) functionality of the instrument can crucially support unravelling the complex molecular composition of the lipidome. Numerous isomeric/isobaric ion species are successfully separated upon using the collisional cross section (CCS) as additional specific physical property. With the possibilities of high data acquisition speed or high separation powers in combination with the increased sensitivity of MALDI-2 available in one instrument, the described methodology could be a valuable tool in many areas of biological and medical research.

On-tissue digestion matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can be used to record spatially correlated molecular information from formalin-fixed, paraffin-embedded (FFPE) tissue sections. In this work, we present the in situ multimodal analysis of N-linked glycans and proteins from the same FFPE tissue section. The robustness and applicability of the method are demonstrated for several tumors, including epithelial and mesenchymal tumor types. Major analytical aspects, such as lateral diffusion of the analyte molecules and differences in measurement sensitivity due to the additional sample preparation methods, have been investigated for both N-glycans and proteolytic peptides. By combining the MSI approach with extract analysis, we were also able to assess which mass spectral peaks generated by MALDI-MSI could be assigned to unique N-glycan and peptide identities.