CSF N-Glycomics Using MALDI MS Techniques in Alzheimer’s Disease

Abstract

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.

Another building block in the plant cell wall: Barley xyloglucan xyloglucosyl transferases link covalently xyloglucan and anionic oligosaccharides derived from pectin

“…To release N‐glycans, samples were incubated with 1 unit of the peptide‐N‐glycosidase F (Roche, Basel, Switzerland) at 37°C for 16 h. N‐glycans were isolated by non‐porous graphitised carbon solid‐phase extraction (SPE; Supelclean ENVI‐Carb SPE), and desalted by reverse‐phase SPE (LiChroprep RP18, 25−40 µm; Hykollari et al ., 2017). To increase signal intensities, free N‐glycans were permethylated (Palmigiano et al ., 2018) and analysed by the UltrafleXtreme MALDI ToF/ToF mass spectrometer (Bruker Daltonics, Billerica, MA, USA) in the reflectron positive ion mode using as a matrix solution 2% (w/v) 2,3‐dihydroxybenzoic acid in 30% (v/v) acetonitrile with the addition of 10 m m NaOH to unify the adduct formation (Pažitná et al ., 2020). Raw data were processed by FlexAnalysis (Bruker Daltonics) and GlycoWork Bench software (Ceroni et al ., 2008).…”

Astrocytes Modify Migration of PBMCs Induced by β-Amyloid in a Blood-Brain Barrier in vitro Model

“…With the support of PNGase F, capable of enzymatically remove all N-glycosylated chains from proteins (Chen C. et al, 2012), we proved that the 85 and 75 kDa bands of ICAM-1, here described, are two differently N-glycosylated forms of the protein. According to type and position of attached sugars and branching, N-glycosylated proteins can be distinguished in complex, hybrid, and high-mannose types (Palmigiano et al, 2018). High mannose glycans seem to be critical for the response of innate immune system (van Kooyk and Rabinovich, 2008).…”

Aberrant sialylation in a patient with a HNF1α variant and liver adenomatosis

“…N-glycan preparation from whole serum (10 μl) or from serum purified glycoproteins (about 100 µL, as described above) consisted of glycoprotein denaturation by RapiGest™ SF Surfactant (Waters Corporation, Milford, Massachusetts) in 50 mM NH4HCO3 buffer, reduction in 5 mM Dithiothreitol (DTT, Sigma) at 56 °C for 30 min, alkylation in 15 mM iodoacetamide (IAA, Sigma) in the dark at room temperature for 45 min, and peptide-Nglycosydase F (PNGase F) digestion, (2-4 U, Roche, Molecular Biochemicals, Mannheim, Germany) overnight at 37°C. The released glycans were therefore purified from the resulting mixture by a 1 cc C18 Sep-Pak cartridge (Waters, Milford, MA), followed by a further purification by solid-phase-extraction (SPE) on HyperSep™ Hypercarb™ (Thermo Scientific™, Bellefonte, PA, USA) 50 mg/L cartridges (Palmigiano et al, 2018a;Messina et al, 2019). The obtained N-glycans were therefore permethylated in a DMSO/NaOH slurry followed by ICH3 addition, according to the method originally developed by Ciucanu and Kerek (1984) to enhance glycan detection sensitivity upon MALDI-MS. Mass spectra of permethylated N-glycans, dissolved in MeOH at an estimated concentration of about 10 pmol/µL, were performed using 5-chloro-2-mercaptobenzothiazole (CMBT, 10 mg/ml in 80:20 MeOH/H2O, v/v) as matrix (Palmigiano et al, 2018a;Messina et al, 2019).…”

“…The released glycans were therefore purified from the resulting mixture by a 1 cc C18 Sep-Pak cartridge (Waters, Milford, MA), followed by a further purification by solid-phase-extraction (SPE) on HyperSep™ Hypercarb™ (Thermo Scientific™, Bellefonte, PA, USA) 50 mg/L cartridges (Palmigiano et al, 2018a;Messina et al, 2019). The obtained N-glycans were therefore permethylated in a DMSO/NaOH slurry followed by ICH3 addition, according to the method originally developed by Ciucanu and Kerek (1984) to enhance glycan detection sensitivity upon MALDI-MS. Mass spectra of permethylated N-glycans, dissolved in MeOH at an estimated concentration of about 10 pmol/µL, were performed using 5-chloro-2-mercaptobenzothiazole (CMBT, 10 mg/ml in 80:20 MeOH/H2O, v/v) as matrix (Palmigiano et al, 2018a;Messina et al, 2019). MALDI TOF and MALDI TOF/TOF analyses were conducted on a 4800 Proteomic Analyzer (AB Sciex), equipped with a Nd:YAG laser operating at a wavelength of 255 nm with <500ps pulse and 200 Hz firing rate.…”