Linkage-Specific in Situ Sialic Acid Derivatization for N-Glycan Mass Spectrometry Imaging of Formalin-Fixed Paraffin-Embedded Tissues

Abstract

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.

Addressing the glycan complexity by using mass spectrometry: In the pursuit of decoding glycologic

“…(Figure 1) Although this particular linkage is as labile as those of the other deoxy sugars, it becomes as stable as those of the other ordinary monosaccharides after esterification [60,61] or amidation [62,63] of a carboxyl group. Another important feature of sialic acid is that if linked to 3-position of adjacent galactose could form lactone structure, which can be used to distinguish the positional isomers of this particular monosaccharide [64].…”

Sensitive and Structure-Informative <i>N</i>-Glycosylation Analysis by MALDI-MS; Ionization, Fragmentation, and Derivatization

“…67) is instability can limit the downstream chemical and/or enzymatic treatments. 70,71) As pointed out by other researchers, lactone forms cannot survive enzymatic digestion, resulting in partial de-lactonization.…”

“…10). Although the speci city of conversion for the SALSA method was not yet perfect (95.1% for α2,6-linked disialylated N-glycan), SALSA showed a better conversion speci city as compared with the conventional double amidation method, 71) proving better reliability in quantifying the relative ratio of α2,3-/α2,6-linkages.…”

“…70,71) Li et al introduced two-step reactions for the linkage-speci c derivatization of sialic acids on glycoproteins xed to a solid support. 70) More recently, Holst et al Fig.…”

“…71) Our group has recently developed a similar but more reliable method for amidation-based sialic acid linkagespeci c derivatization, which we refer to as SALSA (sialic acid linkage-speci c alkylamidation) for released N-glycan analysis. 72) SALSA converts carboxyl groups (-COOH) on sialic acid residues into alkylamide forms (-CONHR) with di erent length alkyl chains in a linkage-speci c manner, allowing direct discrimination of sialyl linkages based on the di erence in m/z values (Fig.…”