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. Lipid Extraction One milliliter of each sample was transferred to test tubes. Four milliliters of chloroform:methanol (1:2, v/v) was added to the samples. The lipids were extracted overnight at 4?C. Samples were centrifuged at 3000 rpm for 15 min and lipids were decanted leaving the completely clear supernatant. A chloroform:methanol:water (4:8:3, v/v/v) solvent mixture was added to the sample again. The samples were reextracted, centrifuged, and supernatant removed. Acetone precipitation Cold acetone was added to the samples, which were then centrifuged at 4 oC for 15 min and supernatant was removed. Cold acetone was added to the sample again and re-centrifuged. The samples were dried down under a nitrogen stream. Release of N-linked glycans from glycopeptide The dried samples were dissolved in ammonium bicarbonate buffer (50 mM, pH 8.4), and denatured immediately by heating at 100 oC for 5min prior to trypsin and chymotrypsin digestion at 37 oC for 20 hours. After trypsin digestion, the samples were heated at 100 oC for 5 min to deactivate the enzyme. The samples were centrifuged at 4 oC for 15 min. The supernatant was colleted into a clean tube. Nanopure water was added and the samples were centrifuged again and supernatant was collected. The samples were dried using a SpeedVac. Two milligrams of each sample 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 from the peptides. The N-linked glycan fraction of the samples were eluted with 5% acetic acid and then lyophilized. 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 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 the spectra of 50 laser shots.
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