Analysis of Serum N-Glycans of Gene-KO Mouse Using MALDI-7090 and SialoCapper-ID Kit

Significance of the Topic

Glycan sialylation at the nonreducing terminus governs critical biological processes including pathogen recognition, immune modulation, and tumor progression. Discriminating between α2,3- and α2,6-linked sialic acids is essential to understand their distinct roles in disease and to develop targeted diagnostics and therapeutics.

Study Objectives and Overview

This work demonstrates a streamlined mass spectrometry–based approach for linkage-specific profiling of serum N-glycans from sialyltransferase knockout mice. The objectives were to validate the SialoCapper™-ID Kit combined with BlotGlyco® beads for one-pot linkage derivatization and to apply MALDI-7090 MS1 and high-energy CID MS2 to assign α2,3/α2,6 sialylation without liquid chromatography or enzymatic cleavage.

Methodology and Instrumentation

Glycoprotein Preparation and Glycan Release

• Denaturation and reduction of serum glycoproteins with SDS/DTT.
• Addition of NP-40 and PNGase F digestion at 37 °C overnight to release N-glycans.

One-Pot Purification and Linkage-Specific Derivatization

• Capture of released glycans on BlotGlyco® beads targeting the reducing end.
• Sequential SALSA alkylamidation: isopropylamine for α2,6 and methylamine for α2,3 sialic acids, using the SialoCapper™-ID Kit.

Labeling and MS Analysis

• 2-Aminobenzoic acid tagging at the reducing end; cleanup via HILIC-SPE chip.
• Spotting with DHB matrix on MALDI plate.
• MALDI-7090 acquisition in negative-ion reflectron mode for MS1 profiling.
• High-energy CID with ASDF enabled MS2 sequencing of selected ions.

Main Results and Discussion

MS1 spectra exhibited pairs of peaks differing by 28 Da, corresponding to α2,3 versus α2,6 linkage patterns. Software support tools assigned monosaccharide compositions, revealing the absence of α2,6 sialylation in ST6Gal1-KO and double-KO samples, confirming ST6Gal1 as the primary α2,6 transferase. MS2 of the m/z 2749 ion from the double KO showed a characteristic 348 Da neutral loss (isopropylamidated NeuGc), indicating residual α2,6 sialylation by an alternate enzyme.

Benefits and Practical Applications

• Linkage assignment directly by MS without chromatography or enzyme digestion.
• One-pot workflow reduces handling and sample loss.
• High throughput and sensitivity compatible with complex biological samples.
• MS2 confirmation improves confidence in detecting unexpected linkages.

Future Trends and Applications

Emerging directions include integrating spatial glycomics by imaging MALDI, expanding the SALSA kit to O-glycans and glycolipids, and coupling real-time derivatization with ambient MS techniques. Advances in software for automated glycan structure prediction and the development of novel linkers may further streamline clinical glycomics.

Conclusion

The SialoCapper™-ID Kit with BlotGlyco® beads and MALDI-7090 offers a rapid, reliable platform for discriminating sialic acid linkages in N-glycans through a single-pot derivatization and MS workflow. MS2 validation ensures accurate detection of both anticipated and novel sialylation patterns.

Reference

1. Ohmi Y, et al. Majority of alpha2,6-sialylated glycans in the adult mouse brain exist in O-glycans: SALSA-MS analysis for knockout mice of alpha2,6-sialyltransferase genes. Glycobiology, in press (2020).
2. Nishikaze T, et al. Differentiation of Sialyl Linkage Isomers by One-Pot Sialic Acid Derivatization for Mass Spectrometry-Based Glycan Profiling. Analytical Chemistry. 89: 2353–2360 (2017).
3. Hanamatsu H, et al. Sialic Acid Linkage Specific Derivatization of Glycosphingolipid Glycans by Ring-Opening Aminolysis of Lactones. Analytical Chemistry. 90: 13193–13199 (2018).