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 glycansThe dried samples were dissolved in Trypsin buffer (0.1M Tris-HCl, pH 8.2 containing 0.01M CaCl2). The samples then were denatured by heating for 5 minutes at 100 C. After cooling, the samples were digested with the trypsin (37oC, overnight). After tryptic digestion, the samples were heated at 100 C for 5 minutes to de-activate the trypsin. After cooling to room temperature, the N-glycans were treated with PNGase F (New England BioLabs) to release the N-glycans. After digestion, the samples were passed through a C18 reversed phase cartridge. The carbohydrate fraction (N-linked glycans) was first eluted with 5% acetic acid and then the O-linked glycopeptides and peptides were eluted in series with 20% iso-propanol in 5% acetic acid, 40% iso-propanol in 5% acetic acid and 100% iso-propanol each into a microcentrifuge tube. The carbohydrate fraction was dried by lyophilization, whereas the other propanol fractions were dried in a speed vacuum concentrator and then combined into one tube.Release of O-linked glycansO-linked carbohydrate fractions were cleaved from the glycoprotein by -elimination procedures. Briefly, 500 L of 50 mM Sodiumhydroxide (NaOH) containing 19 mg of sodium borohydride were added to the samples and incubated overnight at 450C. The incubated samples then were 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 samples were cleaned of borate with methanol:acetic acid (9:1) under a stream of nitrogen gas. Then the samples were passed through a C18 reversed phase cartridge to purify the O-glycans. The carbohydrate fractions (O-linked glycans) were first eluted with 5% acetic acid and then the peptides were eluted with 100% iso-propanol. The carbohydrate fractions were dried by lyophilization, whereas the propanol fractions were dried under a stream of nitrogen gas.Preparation of the per-O-methylated carbohydrates, cleaning up by C18The carbohydrate fraction 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. Per- O-methylated glycans were further cleaned of contaminants. Briefly, the glycans were loaded into a C18 sep pak cartridge and then washed with nanopure water and 15% acetonitrile. The glycans then were eluted with 85% acetonitrile. Purified glycans were dried under a stream of nitrogen gas and were dissolved with methanol prior to analysis by mass spectrometry.Matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI/TOF-MS)MALDI/TOF-MS was performed in the reflector positive ion mode using -dihyroxybenzoic acid (DHBA, 20mg/mL solution in 50%methanol: water) as a matrix. All spectra were obtained by using a 4700 Proteomics analyzer (Applied Biosystems).NanoSpray ionization-Linear Ion Trap Mass Spectrometry (NSI-LTQ/MSn)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). 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 windows that overlapped the preceeding window by 2 mass units.
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