CSF N-Glycomics Using MALDI MS Techniques in Alzheimer’s Disease
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.
Key wordsAD CSF N-glycans MALDI MS MCI
Generous donation from Stellalucente Trust is gratefully acknowledged. This chapter is dedicated to our dear friend and colleague Francesco Le Pira, University of Catania, who too early passed away.
- 7.Stanley P, Schachter H, Taniguchi N (2009) N-glycans. In: Varki A, Cummings R, Esko J, Freeze H, Stanley P, Bertozzi CR, Hart GW, Etzler ME (eds) Essentials of glycobiology, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
- 14.Ikeda Y, Ihara H, Tsukamoto H et al (2014) Mannosyl (beta-1,4-)-glycoprotein beta-1,4-N-acetylglucosaminyltransferase (MGAT3); β1,4-N-acetylglucosaminyltransferase III (GnT-III, GlcNAcT-III). In: Taniguchi N et al (eds) Handbook of glycosyltransferase and related genes, 2nd edn. Springer, TokyoGoogle Scholar