In this chapter, we present the methodology currently applied in our laboratory for the structural elucidation of the cerebrospinal fluid (CSF) N-glycome. N-glycans are released from denatured carboxymethylated glycoproteins by digestion with peptide-N-glycosidase F (PNGase F) and purified using both C18 Sep-Pak® and porous graphitized carbon (PGC) HyperSep™ Hypercarb™ solid-phase extraction (SPE) cartridges. The glycan pool is subsequently permethylated to increase mass spectrometry sensitivity. Molecular assignments are performed through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) analysis considering either the protein N-linked glycosylation pathway or MALDI TOF MS/MS data. Each stage has been optimized to obtain high-quality mass spectra in reflector mode with an optimal signal-to-noise ratio up to m/z 4800. This method has been successfully adopted to associate specific N-glycome profiles to the early and the advanced phases of Alzheimer’s disease.
CITATION STYLE
Palmigiano, A., Messina, A., Bua, R. O., Barone, R., Sturiale, L., Zappia, M., & Garozzo, D. (2018). CSF N-glycomics using MALDI MS techniques in Alzheimer’s disease. In Methods in Molecular Biology (Vol. 1750, pp. 75–91). Humana Press Inc. https://doi.org/10.1007/978-1-4939-7704-8_5
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