This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.N-linked oligosaccharide profiling by MALDI- and ESI-MSRelease of N-linked glycansAn aliquot (to provide about 2.0 mg) of the sample (1317042) was placed in microcentrifuge tube and lyophilized. The dried sample was dissolved in ammonium bicarbonate buffer (50 mM, pH 8.4) and denatured immediately by boiling at 100oC for 5min prior to trypsin digestion at 37oC for 20 hours. After trypsin digestion, the sample was heated at 100oC for 5 min to deactivate the enzyme, centrifuged for at 4oC for 15 min and washed with nanopure water and re-centrifuged. The sample was dried down in a speed vac. The sample was passed through a C18 reversed phase cartridge. A second enzyme, peptide N-glycosidase F (New England BioLabs) was added to the tryptic digest and incubated at 37oC for 20 hours to release the N-linked glycans. After enzymatic digestions, sample was passed through a C18 reversed phase cartridge to separate the N-linked glycans from the glycopeptides and peptides. The N-linked glycan fraction of the sample was eluted with 5% acetic acid and then lyophilized.Preparation of the per-O-methylated carbohydratesThe lyophilized N-linked fractions was dissolved in dimethylsulfoxide and then methylated with NaOH and methyl iodide (Ciucanu and Kerek, 1984). The reaction was quenched by addition of water, and per-O-methylated carbohydrates were extracted with dichloromethane. The organic phase was dried under the nitrogen stream and then dissolved with methanol. After permethylated glycans were further purified by a C18 and then dried under the nitrogen stream. Matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI)MALDI-MS was performed in the positive ion mode using -dihyroxybenzoic acid (DHBA, 20 mg/mL in 50% methanol: water) as the matrix. Full mass spectrum of sample was obtained initially using a MALDI TOF Mass Spectrometer (Applied Biosystems).ElectroSpray Ionization Linear Ion Trap mass Spectrometry (ESI-LTQ/MSn)Mass analysis of oligosaccharides was performed by direct infusion of permethylated glycans dissolved in 1 mM NaOH in 50% methanol (5 pmol/ L) into an LCQ-MS (Thermo Finnigan) instrument using a nanoelectrospray source at syringe flow rate of 0.40 L/min. The capillary temperature was set to 210oC and MS analysis was performed in positive ion mode. The collision energy was set at 28% for fragmentation in MS/MS.Composition analysis by HPAECThe dried N-glycan fraction of the sample 1317042 was dissolved in 1 mL nanopure H2O. About 600 L were pipetted into a screw-cap and lyophilized for neutral and amino sugars and analyzed by High pH-Anion-Exchange Chromatography (HPAEC). Hydrolysis of each of the sample was performed with 2 N trifluoroacetic acid (TFA) at 100 C for 4 h. After hydrolysis, the samples were lyophilized and resuspended in nanopure water, sonicated in cold water and transferred to an injection vial. For analysis, the autosampler was set to deliver 10 L of sample or standard solution per injection. Blanks (water) were injected between standards and the samples to prevent carry-over contamination. A mix of sugar standards with a known number of moles was hydrolyzed at the same time as the sample. Three concentrations of standards (0.5, 1.0, and 2.0 nmoles per injection) were prepared and injected to establish a calibration equation. The number of moles of each sugar was quantified by linear interpolation from the calibration equation.Linkage analysis by Exoglycosidase digestions -Glucosidase from Bacillus stearothermophilus (Sigma-Aldrich): a portion of the N-glycan fraction was dissolved in the 100 mM potassium phosphate buffer (pH 6.8) and incubated at 37oC with -Glucosidase from Bacillus stearothermophilus (Sigma-Aldrich).
Showing the most recent 10 out of 104 publications
