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. Cell lysis and delipidation The blood samples were spun at 2000 rpm at 4oC for 15 min to remove the plasma. Weighed amounts of wet red blood cells (RBC: Human RBC A, 1.2346 g;Human RBC B, 1.12 g;Turkey RBC, 0.5714 g, all of Guinea pig RBC) were lyzed in a dounce homogenizer (vacuum pulling was done 12 to 15 times) and transferred subsequently with nanopure H2O into conical glass screw-cap tubes. The RBC-H2O solution was added with chloroform and methanol to obtain a final solvent mixture of 2 mL: 4 mL: 1.5 mL, chloroform (C), methanol (M), and water (W), respectively. The tubes were placed on a rocker platform for 2 h, spun at 3000 rpm, 4oC for 15 min. The preceding lipid extraction step was repeated 2 more times and delipidated samples (protein-rich powder) were washed with acetone 2 times. Cleaned cell pellets were dried in a vacuum dessicator for 1 h. Release of N-linked glycans Two 5 to 6 mg of protein-rich powder of each RBC sample were placed in microcentrifuge tubes. The samples were dissolved with 200 ?L protease buffer (0.1 M Tris-HCl pH 8.2 containing 0.01 M CaCl2) and the tubes were placed in a heating block (1000C, 5 min) to denature the protein. After cooling to room temperature, 25 ?L trypsin (2 mg/mL) and 25 ?L chymotrypsin (2 mg/mL) were added to each tube and incubated at 370C for 18 h. The tubes then were heated at 1000C for 5 min to deactivate the enzymes. The digests were passed through a C18 sep pak cartridge to remove contaminants. The glycopeptides were eluted in series with 20% isopropanol in 5% acetic acid, 40% isopropanol in 5% acetic acid, and 100% isopropanol. The eluates were dried under a vacuum centrifuge. The dried digests were redissolved with 25 ?L of H2O and 20 ?L of 0.1 M sodium phosphate buffer (pH 7.5), treated with 7 ?L of PNGase F, and incubated at 370C for 18 h. After the second enzymatic digestion (PNGase F), each of the mixture was passed through a C18 sep pak cartridge to separate the carbohydrate fraction and the peptide-containing fraction. The N-linked glycans were eluted first with 5% acetic acid followed by the elution of O-glycopeptide and peptide fraction in series with 20% isopropanol in 5% acetic acid, 40% isopropanol in 5% acetic acid, and 100% isopropanol. The N-glycan fractions were lyophilized, whereas the O-glycopeptide and peptide fractions were dried under vacuum centrifuge. The N-glycans and O-glycopeptides of same RBC sample were composited into 1 tube. Per-O-methylation of carbohydrates and purification by C18 sep-pak cartridge The N- linked glycans were permethylated for oligosaccharide profiling (Ciucanu and Kerek, 1984). The dried eluates were dissolved in dimethylsulfoxide and then methylated with NaOH and methyl iodide. The reaction was quenched by addition of water and per-O-methylated carbohydrates were extracted with dichloromethane. Per- O-methylated glycans were further cleaned of contaminants. Briefly, the glycans were dissolved in 1:1 methanol:water and loaded into a C18 sep pak cartridge and then washed with nanopure water. Per-O-methyl carbohydrates were eluted with 15% acetonitrile into a screw-cap tube, and with 85% acetonitrile into another screw-cap tube. The glycans eluted with 85% acetonitrile were dried under a stream of nitrogen gas and were dissolved with methanol for analysis by mass spectrometry. N-linked Oligosaccharide Profiling by Matrix-Assisted Laser-Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF MS) MALDI-MS was performed in the positive ion mode using ?-dihyroxybenzoic acid (DHBA, 20 mg/mL in 50% methanol:water) as a matrix. Full mass spectra of all 4 RBC were obtained by using a 4700 Proteomics analyzer (Applied Biosystems).
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