Differentiation of Sialyl Linkage Isomers by One-Pot Sialic Acid Derivatization for Mass Spectrometry-Based Glycan Profiling

Abstract

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used for high-throughput glycan profiling analysis. In spite of the biological importance of sialic acids on nonreducing ends of glycans, it is still difficult to analyze glycans containing sialic acid residues due to their instability and the presence of linkage isomers. In this Article, we describe a one-pot glycan purification/derivatization method employing a newly developed linkage-specific sialic acid derivatization for MS-based glycan profiling with differentiation of sialyl linkage isomer. The derivatization, termed sialic acid linkage specific alkylamidation (SALSA), consists of sequential two-step alkylamidations. As a result of the reactions, α2,6- and α2,3-linked sialic acids are selectively amidated with different length of alkyl chains, allowing distinction of α2,3-/α2,6-linkage isomers from given mass spectra. Our studies using N-glycan standards with known sialyl linkages proved high suitability of SALSA for reliable relative quantification of α2,3-/α2,6-linked sialic acids compared with existing sialic acid derivatization approaches. SALSA fully stabilizes both α2,3- and α2,6-linked sialic acids by alkylamidation; thereby, it became possible to combine SALSA with existing glycan analysis/preparation methods as follows. The combination of SALSA and chemoselective glycan purification using hydrazide beads allows easy one-pot purification of glycans from complex biological samples, together with linkage-specific sialic acid stabilization. Moreover, SALSA-derivatized glycans can be labeled via reductive amination without causing byproducts such as amide decomposition. This solid-phase SALSA followed by glycan labeling has been successfully applied to human plasma N-glycome profiling.

Mass spectrometry for protein sialoglycosylation

“…However, the generated lactones suffer from the instability in water solution, resulting in poor reproducibility. In recent years, two‐step derivatization protocols have been applied to derivatize α2,3‐linked sialic acids to amides or esters instead of lactones . In the first step, α2,6‐linked sialic acids are efficiently derivatized by ammonium chloride, dimethylamine, or ethanol; whereas α2,3‐linked sialic acids form lactones.…”

“…In the second step, the lactones are regenerated and subsequently converted to amides or esters. In particular, a high‐specificity protocol based on double alkylamidation has been developed recently . The first alkylamidation aims at converting α2,6‐ and α2,3‐linked sialic acids into amide and lactone forms, respectively.…”

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

“…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. 9).…”

Electrochemical lactonization of trisialic acid

“…Terminal sialic acid with the carboxyl group is most frequently attached to a galactose via the α2,3 or α2,6 glyosidic linkage and studied with modern mass spectrometry. Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) analysis has been used for high‐throughput glycan profiling analysis . Sialyl lactonization within living cells is a challenge for glycobiologists and glycochemists .…”

“…Sialyl lactonization within living cells is a challenge for glycobiologists and glycochemists . In the lactonization of sialyl conjugates, a condensation occurs between the carboxyl group of sialic acid and the hydroxy group from an adjacent pyranoside of GD/GM gangliosides, or a sialyl residue of oligo/polysialic acid (OSA/PSA) . The sialyl gangliosides with intramolecular lactones have been suggested to be more immunogenic and tumor‐specific compared to their native forms, and those sialyl lactones have potential as immunogens in the development of cancer vaccines .…”