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. Glycosyl composition analysis was performed by combined gas chromatography/mass spectrometry (GC/MS) of the per-O-trimethylsilyl (TMS) derivatives of the monosaccharide methyl glycosides produced from the sample by acidic methanolysis. Methyl glycosides were first prepared from 300 l of the dry sample provided by the client by methanolysis in 1 M HCl in methanol at 80 C (18-22 hours), followed by re-N-acetylation with pyridine and acetic anhydride in methanol (for detection of amino sugars). The samples were then per-O-trimethylsilylated by treatment with Tri-Sil (Pierce) at 80 C (0.5 hours). [These procedures were carried out as previously described in Merkle and Poppe (1994) Methods Enzymol. 230: 1-15; York, et al. (1985) Methods Enzymol. 118:3-40.] GC/MS analysis of the TMS methyl glycosides was performed on an HP 5890 GC interfaced to a 5970 MSD, using a Supelco DB-1 fused silica capillary column (30m x 0.25 mm ID). Alditol acetates were prepared from 300 g of the dry sample. The sample was hydrolyzed using 2 M trifluoroacetic acid (2 h in sealed tube at 121 C), reduced with NaBD4, and acetylated using acetic anhydride/TFA. The resulting alditol acetates were analyzed on a Hewlett Packard 5890 GC interfaced to a 5970 MSD (mass selective detector, electron impact ionization mode); separation was performed on a 30 m Supelco 2330 bonded phase fused silica capillary column. 1 mg of recombinant erythropoietin (REPO) and 500 g of alpha- and beta- erythropoietin standards were subjected to PNGase F treatment, and the released glycans were subjected to 2-aminopyridine (PA) labeling. For the PNGase F treatment, protein aliquots were dried at reduced pressure on a SpeedVac concentrator. Dried protein fractions were dissolved in a buffer that contained 50mM Tris pH 7.5, 50mM beta-mercaptoethanol and 1.0 % SDS; then protein was denatured by heating at 100 C for 10 minutes. The sample was cooled on ice for 5 minutes and then diluted to reach a final concentration of 0.1 SDS and added Nonidet P-40 to a final concentration of 0.5%. After addition of PNGase F, the sample was incubated at 37 C for 16 hours. The released glycans were purified from the protein and SDS by solid phase extraction (SPE) on a Oasis HLB 30 mg cartridge (Waters). Before use the SPE cartridge was a washed with 2ml of methanol, and then equilibrated with of 5% acetic acid/H2O. The digest was loaded onto the C18 cartridge and the glycans were eluted in 2ml of 5% acetic acid/ H2O. The eluted glycan fraction was dried overnight in a Speed Vac Concentrator. The eluted glycans were subjected to PA labeling. PA labeling of glycans was performed by reductive amination (Hase 1994).The coupling reagent was prepared by adding 33 lt of acetic acid to 100 mg of pure 2-aminopyridine crystals. This mixture was heated to 95 C and 20 lt were added to the dry glycans. The coupling reaction was incubated at 90 C during 1 hour. After this first incubation, 50 lt of the reducing reagent (200 mg of borane-dimethylamine complex dissolved in 50 lt of H2O and 80 lt of acetic acid) were added and the reduction was carried at 80 C for 1 hour. To stop the reaction, 50 lt of triethyl:methanol (1:3) and 40 lt of toluene were added and the resulting mixture was evaporated under a stream of N2 at 37 C for 30 minutes. The labeled glycans were then added with 50 lt of toluene:methanol (2:1) and evaporated 20 minutes under a stream of N2. This last step was repeated 5 times. To remove the excess of reagents the mixture was injected to a 1X50 cm Sephadex G-15 column and eluted with 10 mM NH4HCO3. The labeled glycans that eluted in the Vo of this column were pooled, dried under reduced pressure and subjected to HPLC analysis. To separate the PA- labeled glycans according to their content of terminal sialic acid residues,these glycans were injected to an Anion exchange TSKgel DEAE-5PW 7.5X75 mm HPLC column (Tosoh) (Nakagawa, Kawamura et al. 1995). PA-oligosaccharides were detected by a fluorescence monitor at an excitation wavelength of 320 nm and an emission wavelength of 400 nm. Prior to injection, this column was previously equilibrated with Solvent A (10% acetonitrile in water adjusted to pH 9.5 with triethylamine) at a flow rate of 1 ml/min and a temperature of 30 C. After injection, neutral glycans were recovered by eluting the column with 100 % of solvent A, then the mono- di- and tri-sialyl glycans were eluted with a 45 minute linear gradient to reach 100% of solvent B (3% acetic acid in water, adjusted to pH 7.3 with TEA, mixed with Acetonitrile 90:10). Finally tetra-sialylated PA glycans were eluted during 10 minutes with 100% of Solvent B. To separate PA-labelled glycans according to their size, they were injected to a 4.6X250 mm TSK-amide 80 column (Tosoh). Prior to injection, column was equilibrated with 100% of solvent C (80% acetonitrile, 3, acetic acid adjusted to pH 7.3 with triethylamine) at a flow rate of 1 ml/min and a temperature of 35 C. After injection, column was eluted during 2 minutes with 100% of solvent C and then with a 60 minute linear gradient to 100% of solvent D (an aqueous solution with 3% acetic acid adjusted to pH 7.3 with triethylamine). PA-labeled dextran oligomers (4 to 21 glucose units) were used as standards in separate calibration runs. N-linked oligosaccharides were released from the samples by treatment with peptide N-glycosidase F (PNGase F, N-glycanase) using the enzyme digestion protocol supplier by the manufacturer (New England Biolabs, Beverly, MA). The samples were resuspended in 400 l d H20 and 40 l of 10X denaturing buffer (5% SDS and 10% beta-mercaptoethanol.).. The sample and standards were then denatured by heating for 5 minutes at 100 C. After cooling, 40 l of 10X buffer and 40% NP-40 were added. The samples were mixed, and 4 l of enzyme (30U) were added. Samples were incubated overnight at 37 C. The digested samples were acidified and applied to a C18 classic SEP-PAK. This was eluted with 5 ml of 5% acetic acid to collect the N-linked sugars. The sample fractions were dried and permethylated by the methods of Ciukanu and Kerek (1984) Carbohydr. Res. 131:209-217 (treatment with sodium hydroxide and methyl iodide in dry DMSO). After permethylation, the samples were extracted three times with methylene chloride/water in order to remove any impurities. The resulting permethylated samples were resuspended in methanol and analyzed by MALDI-TOF. MALDI-TOF-MS was performed with a Voyager mass spectrometer operated in the positive ion mode. The mass spectrometer was calibrated with a mixture of maltooligosaccharides. Methanolic solutions of samples were diluted 1:1 with 2,5-dihydroxybenzoic acid (DHB) and a portion (1.0 l) was applied to the sample plate of the MS. Samples were desorbed from the sample plate with a nitrogen laser having a guide wire reading of 0.05% and a laser intensity of approximately 2100. For Glycosyl linkage analysis, the N-linked sample was permethylated, depolymerized, reduced, and acetylated and the resultant partially methylated alditol acetates (PMAAs) analyzed by gas chromatography-mass spectrometry (GC-MS) as described by York et al (1985) Methods Enzymol. 118:3-40. Initially, an aliquot of the sample was permethylated by the method of Ciukanu and Kerek (1984) Carbohydr. Res. 131:209-217 (treatment with sodium hydroxide and methyl iodide in dry DMSO). The permethylation was repeated twice in order to aid complete methylation of the polymer. Following sample workup, the permethylated material was hydrolyzed using 2 M trifluoroacetic acid (2 h in sealed tube at 121 C), reduced with NaBD4 and acetylated using acetic anhydride at 100 C for 1 hour. The resulting PMAAs were analyzed on an HP 5890 GC interfaced to a 5970 MSD, using a Supelco DB-1 fused silica capillary column (30m x 0.25 mm lD). The sample was dissolved in 100 ml of 1M sodium borohydride in 0.05M sodium hydroxide (with sonication) and incubated 18 hours at 45 C. The reaction was stopped with 10 ml of glacial acetic. Sodium salts were removed by using cationic resin (Dowex). The resulting solution was dried using centrifugal evaporation. The sample was resuspended in 0.5 ml of a 0.1M acetic acid solution in methanol and dried under a stream of nitrogen. This was repeated a total of five times in order to remove the borates.
Showing the most recent 10 out of 104 publications
