This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Release of N-linked glycans from glycopeptide The native mouse BChE and recombinant mouse BChE samples were dissolved in ammonium bicarbonate buffer (50 mM, pH 8.4), and denatured immediately by boiling at 100 oC for 5min prior to trypsin digestion at 37 oC for 20 hours respectively. After trypsin digestion, the samples were heated at 100 oC for 5 min to deactivate the enzyme. The samples were passed through a C18 reverse phase cartridge. And then samples were treated with a second enzyme, peptide N-glycosidase F (New England BioLabs) and incubated at 37 oC for 20 hours to release the N-linked glycans. After enzymatic digestion, the samples were passed through a C18 reversed phase cartridge to separate the N-linked glycans and O-linked glycopeptides/peptides. The N-linked glycan fractions of the samples were eluted with 5% acetic acid and then lyophilized. The O-linked glycopeptide/peptide fractions were eluted with 2-propanol and dried under the nitrogen stream. ?-Elimination, Desalting, Borate removal O-linked carbohydrate fractions were cleaved from the glycopeptides by ?-elimination procedures. Briefly, 250 ?L of 50 mM NaOH were added to the sample (400 ?g) and then checked for pH. Upon determination that the pH was basic, another 250 ?L of 50 mM NaOH containing 19 mg of sodium borohydride were added to the sample and voltexed and incubated overnight at 45 ?C. The incubated sample then was neutralized with 10% acetic acid and desalted by passing through a packed column of DOWEXTM resins (50W x 8 ?100, Sigma Aldrich) and then were lyophilized. Dried sample was cleaned of borate with methanol: acetic acid (9:1) solution under a stream of nitrogen gas. Preparation of the per-O-methylated carbohydrates The lyophilized carbohydrate fraction was dissolved in dimethylsulfoxide and then methylated with NaOH and methyl iodide (Analytical Biochemistry 203, 101-108 (1992)). The reaction was quenched by addition of water, and O- per-methylated carbohydrates were extracted with dichloromethane. The organic phase was concentrated to dryness and then the glycans were passed through a C18 Sep-Pak, eluted with 85 % acetonitrile, dried under a stream of nitrogen, and dissolved in methanol prior to analysis by mass spectrometry. Matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI/TOF-MS) Profiling of N-linked and O-linked glycans was performed initially using MALDI/TOF-MS on a 4700 Proteomics analyzer (Applied Biosystems). Permethylated glycans (~1 ?L) were crystallized on a MALDI plate with 1 ?L of 2, 3-dihydroxybenzoic acid (DHBA, 20 mg/mL solution in 50 % methanol: water) as matrix. All spectra were acquired in the reflector positive ion mode and averaged spectra of 50 laser shots. Composition analysis by HPAEC Aliquots of the samples were obtained (native, ~70 ?g each for neutral and amino sugars, and sialic acid analyses;recombinant, ~100 ?g each for neutral and amino sugars, and sialic acid analyses) for monosaccharide composition analysis of N-linked and O-linked glycans of the two glycoproteins. The released N-and O-linked glycans intended for neutral and amino sugars were hydrolyzed with 400 ?L of 2 N trifluoroacetic acid (TFA) at 100?C for 4 h, whereas aliquots for sialic acids were hydrolyzed with 2M acetic acid at 80?C for 3 h. The hydrolysates were lyophilized, resuspended in H2O, sonicated for 7 min in ice and transferred to an injection vial. A mix of standards for neutral and amino sugars, and for sialic acids with a known number of moles was hydrolyzed in the same manner and at the same time as the samples. Four concentration of standards (0.5, 1.0, 2.0, and 4.0 nmoles per injection) were prepared to establish a calibration equation. The number of moles of each sugar in the sample was quantified by linear interpolation from the calibration equation. The neutral and amino sugars and sialic acids were analyzed by HPAEC using a Dionex DX500 system equipped with a GP40 gradient pump, an ED40 electrochemical detector, and a Thermo-Separation AS3500 autosampler containing a stainless steel needle. The individual neutral and amino sugars were separated by a Dionex CarboPac PA20 (3 x 150 mm) analytical column with an amino trap. The gradient programs used eluents A, degassed nanopure water;B, 200 mM NaOH for the neutral and amino sugars;C, 100 mM NaOH;and D, 1 M sodium acetate in 100 mM NaOH for the sialic acids. Injections were made every 40 minutes for the neutral and amino sugar determinations and every 35 minutes for the sialic acid determinations. All methods were based on protocols described by Hardy and Townsend (Hardy, M. R., and Townsend, R. R., """"""""High-pH anion-exchange chromatography of glycoprotein-derived carbohydrates"""""""", 1994, Methods Enzymol. 230: 208-225). Instrument control and data acquisition were accomplished using Dionex PeakNet software, version 5.01.
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