CSF N-Glycoproteomics Using MALDI MS Techniques in Neurodegenerative Diseases

Tear N‐glycomics in vernal and atopic keratoconjunctivitis

“…7 More recently, an increasingly growing interest in glycomic studies has emerged, since qualitative and quantitative glycan alterations are often crucial to afford key information on pathological mechanisms in a large variety of disorders and severe diseases such as rheumatoid arthritis, 8,9 diabetes, 10 inflammation, 11 tumors, 12 and Alzheimer's disease. [13][14][15][16][17][18][19] Glycosylation is a complex post-translational modification of proteins, very sensitive to the biochemical environment, depending on the action of several glyco-enzymes. Even though it is not under direct genetic control, glycosylation affects several biological processes.…”

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.…”

Advances in mass spectrometry‐based glycoproteomics: An update covering the period 2017–2021

“…The most common MS-based analytical technique used for the analysis of glycoproteins is liquid chromatography electrospray-ionization MS (LC-ESI-MS) [38,39]. Other MS-based approaches that have made important contributions to the glycoproteomics field include capillary electrophoresis-MS (CE-MS) [40,41], ion mobility-MS (IMS) [42,43], and matrix-assisted laser desorption ionization time-of-flight MS (MALDI-TOF-MS) [44,45]. The above-mentioned analytical techniques have been useful for the investigation of site-specific aberrations of glycoproteins associated with a large number of diseases [21,42,46,47].…”