Iodoacetic acid

Red pitaya fruits (Hylocereus polyrhizus) were purchased from Pasar Borong (Selangor, Malaysia). Ripened pitaya fruits were selected based on the size uniformity at the same stage of ripening free of visual defects. The fruits were stored in a cold room at 4°C until use for the extraction procedure. All chemicals and reagent were in analytical or electrophoresis grade. SP-Sepharose, Sephacryl S-200, Bradford Reagent, BSA, DTNB, PMSF, EDTA, ovomucoid, iodoacetic acid, bestatin, β-mercaptoethanol, PMSF, and trichloroacetic acid (TCA) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Tris-HCL, Triton X-100, Tween-80, SDS, casein, haemoglobin, acetone, ethanol, isopropanol, and methanol were obtained from Merck (Darmstadt, Germany).

Bovine serum albumin (BSA), ammonium sulfate, p-amino benzoic acid (PABA), p-nitro phenyl acetate (PNPA), trizma base (tris), iodo acetic acid, hexamethonium bromide, decamethonium bromide and protein estimation kit (Bradford Method) were obtained from Sigma. Phenyl methyl sulfonyl fluoride (USB, Switzerland) and DTT (SRL, India). All other chemicals and reagents were of analytical grade (Merck, India), unless or otherwise mentioned.

N-glycomic analysis was performed according to a protocol described previously59 . Briefly, 50 μg of each sample was reduced by dithiothreitol (Sigma, Aldrich) and then carboxymethylated by iodoacetic acid (Sigma Aldrich). Samples were subsequently dialyzed, freeze-dried and digested by trypsin (Sigma Aldrich). The peptides/glycopeptides were purified using Oasis HLB Plus Short cartridges (Waters). N-glycans were released from glycopeptides by PNGase F (Roche Applied Science) and isolated from peptides using Sep-Pak C18 catridges (Waters). The released N-glycans were permethylated, purified by Sep-Pak C18 cartridges again, freeze-dried and dissolved in 10 μl 1,5-dihydroxybenzoic acid in 70% (v/v) aqueous methanol; for MS/MS, 20 mg/ml 3,4-diaminobenzophenone in 75% (v/v) aqueous acetonitrile). MALDI-TOF MS analysis using a Voyager-DETM STR mass spectrometer (Applied Biosystems). The data were analyzed using Data Explorer (Applied Biosystems) and Glycoworkbench60 .

Protein quantification was performed by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, USA), and high abundant protein depletion was achieved by Aurum™ Serum Protein Mini Kit (Bio-Rad). Trypsin (Promega gold standard), Guanidine Hydrochloride (GnHCl), Dithiothreitol (DTT), iodoacetic acid (IAA), and trifluoroacetic acid (TFA) were procured from Sigma-Aldrich while LC–MS Grade water and Pierce™ C18 Spin columns (Cat. 89870) were procured from Thermo Fisher Scientific Inc, USA. Protein ladder 10–250 kDa (Precision Plus Protein™ Dual Color Standards) was purchased from BioRad.

The brain lysate prepared as above (100 µg) was reduced with 1 M DTT (Sigma-Aldrich) and alkylated with 1 M iodoacetic acid (Sigma-Aldrich) for 30 min each at room temperature followed by acetone precipitation for 1 h at −80°C. Samples were centrifuged at 13,684 g for 15 min to pellet down the precipitate, which was washed with chilled acetone. Ammonium bicarbonate (100 mM, Sigma-Aldrich) with trypsin (V511A, Promega; enzyme to protein ratio 1:100) was added to resuspend the precipitate, mixed at 34 g for 1 min, then incubated at 37°C for 18 h. The digested peptides were cleaned up by OASIS SPE cartridge at 14,000 g for 10 min. Peptides were eluted with Ammonium bicarbonate (100 mM), acidified with 0.1% formic acid and concentrated to 10 μl by speed vac. Liquid chromatography with tandem mass spectrometry was done using an AB SCIEX Triple TOF 5600. A spectral data-dependent acquisition library was generated for peptide identification, and identified proteins were quantitated using SWATH AB SCIEX software. Proteins were selected for further study based on a 5% false-discovery rate cut-off and a minimum of two peptides per protein.