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.Release of N-linked glycans The dried cell pellets 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 tryptic and chymotryptic digests were spun at 3000 rpm, 40C for 15 min and the supernatants were transferred into another tube. The pellets were added subsequently with 200 L of H2O, vortexed and spun as described previously and the supernatants were collected into their respective sample tubes and lyophilized. The dried digests were dissolved with 200 L of 5% acetic acid and passed through 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 stream of N2, redissolved with 25 L of H2O and 20 L of 0.1 M sodium phosphate buffer (pH 7.5), treated with 5 L of PNGase F, and incubated at 370C for 18 h. After the second enzymatic digestion (PNGase F), 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 with 100% isopropanol. Both fractions were dried either by lyophilization (N-glycan) or under a stream of nitrogen gas (O-glycopeptide and peptide).Release of O-linked glycans by -elimination The O-linked glycans were cleaved from the glycopeptide by -elimination procedures. Briefly, 250 L of 50 mM NaOH were added to each of the samples 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 samples, vortexed, and incubated overnight at 450C. The incubated samples then were neutralized with 10% acetic acid, desalted by passing through a packed column of Dowex resins and then were lyophilized. Dried samples were cleaned of borate with methanol:acetic acid (9:1) under a stream of nitrogen gas. The samples were passed through C18 reversed phase cartridge to collect the cleaved O-glycans (eluted with 5% acetic acid). The O-glycan fraction was dried under a stream of nitrogen.Per-O-methylation of carbohydrates and purification by C18 sep-pak cartridgeThe N-and O-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 methanol:water (1:1)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.Oligosaccharide Profiling by Matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF)Profiling of N-linked and O-linked glycans was performed initially using MALDI/TOF-MS. The machine used was a 4700 Proteomics analyzer (Applied Biosystems), which was set in the reflector positive ion mode. Permethylated glycans were crystallized on a MALDI plate with -dihydroxybenzoic acid (DHBA, 20 mg/mL solution in 50% methanol:water) as a matrix.Oligosaccharide Profiling by NanoSpray ionization-Linear Ion Trap Mass Spectrometry (LTQ)The N-linked oligosaccharides detected by MALDI-TOF MS was confirmed by ESI-LTQ mass spectrometry. Mass spectrometric analysis was performed following the method developed at the Complex Carbohydrates Research Center (Aoki K, Perlman M, Lim JM, Cantu R, Wells L, Tiemeyer M. J Biol Chem. 2007 Mar 23;282(12):9127-42. Epub 2007 Jan 29). Mass analysis was determined by using NSI-LTQ/MSn. Briefly, permethylated glycans were dissolved in 1mM NaOH in 50% methanol and infused directly into the instrument (LTQ,Thermo Finnigan) at a constant flow rate of 0.4 L/min. The capillary temperature was set at 210o C and MS analysis was performed in the positive ion mode. The collision energy was set at 28 for MS/MS fragmentation. For total ion mapping, automated MS/MS analysis (at 28 collision energy), m/z range from 500 to 2000 was scanned in successive 2.8 mass unit window that overlapped the preceeding window by 2 mass units.
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