STRUCTRUAL ANALYSIS OF N-LINKED OLIGOSACCHARIDES IN MEN2234 GLYCOPROTEIN

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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. Neutral and amino sugars were released from a portion of the sample through hydrolysis with 2 N trifluoroacetic acid at 100¿C for 4 h. After hydrolysis the samples were dried using a centrifugal vacuum evaporator, then resuspended in 100 ¿l water, and then sonicated on ice. For analysis, 10 ¿l were injected. Blanks (water) were injected and run between the standards and the sample (data not shown) to prevent carry-over. The calibration standards were hydrolyzed in parallel with the samples, and then injected at three levels (0.5, 1.0 and 2.0 nmol). The quantitation was based on a linear interpolation from these standards. Sialic acids were released from a portion of the sample by mild acid hydrolysis using 2M acetic acid at 80¿C for 3 h. The samples were dried as above, and resuspended in 100 ¿l water. For analysis, 10 ¿l were injected. Blanks (water) were injected and run between the standards and the sample (data not shown) to prevent carry-over. The calibration standards were hydrolyzed in parallel with your samples, and then injected at three levels (0.25, 0.75 and 1.5 nmol). The quantitation was based on a linear interpolation from these standards. Monosaccharides were analyzed using a Dionex DX500 system equipped with a GP40 gradient pump, an ED40 electrochemical detector and a Thermo-Separations AS3500 autosampler containing a stainless steel needle. Analyses were performed using a Dionex CarboPac PA20 (3 x 150 mm) analytical column with an Amino Trap column. The gradient programs used eluents A, water; B, 100 mM NaOH for the neutral and amino sugars; C, 100 mM NaOH; and D, 1 M sodium acetate in 100 mM NaOH for the sialic acids. Injections were made every 50 minutes for the neutral and amino sugar determinations and every 60 minutes for the sialic acid determinations. All methods were based on protocols described by Hardy and Townsend (Hardy, M. R., and Townsend, R. R., High-pH anion-exchange chromatography of glycoprotein-derived carbohydrates, 1994, Methods Enzymol. 230: 208-225). Instrument control and data processing were accomplished using Dionex PeakNet software, version 5.01. N-linked oligosaccharide profiling by MALDI-MS N-linked oligosaccaharides were released from 5mg of the sample (client weight) using the enzyme digestion protocol supplier by the manufacturer (New England Biolabs, Beverly,MA). The sample was dried and resuspended in 200¿l d H2O and 20¿l of 10X denaturing buffer (5% SDS and 10%¿-mercaptoethanol). The sample was then denatured by heating for 5 minutes at 100¿ C. After cooling, 20¿l of 10X buffer and 10% NP-40 were added. The sample was mixed, and 4¿l of enzyme (30U) were added. The sample was incubated overnight at 37¿C. The N-linked oligosaccharides were collected by passing the sample through a C18 Sep-Pak equilibrated with 5% acetic acid. The N-linked fraction was dried and passed through an ion exchange resin in order to remove sodium ions. The carbohydrate fraction was dried and permethylated by the method 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/water and analyzed by MALDI-TOF Mass Spectrometry. Exoglycosidase analysis N-linked oligosaccharides were released from a 5 mg portion of the sample as described above. The sample was then treated with 1,3-galactosidase, 1,6-galactosidase, and hexosaminidase. A portion of the sample was taken for permethylation and analysis after each enzymatic digest. A separate portion of the released N-linked glycans after ¿1,3- galactosidase, ¿ 1,6-galactosidase were further digested with ¿1-(2,3,4) fucosidase and subsequently permethylated prior to MALDI-MS analysis. Each fraction was dried and permethylated by the method 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/water and analyzed by MALDI-TOF Mass Spectrometry. N-linked oligosaccharide profiling by LCQ-MS/MS The permethylated N-linked oligosaccharides were analyzed by electrospray mass spectrometry in order to verify the structures that were identified by MALDI-TOF MS. Structures and referenced spectra are discussed in the results section. N-linked oligosaccharide profiling by HPAEC N-linked oligosaccaharides were released from 200¿g of the sample (client weight) as well as from fetuin (Sigma, from fetal calf serum, 400 ¿g) and ribonuclease B (RNase B, Sigma Type XII-B from bovine pancreas, 800¿g) 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 sample and the standards were dried and resuspended in 100¿l d H2O and 10¿l of 10X denaturing buffer (5% SDS and 10%¿-mercaptoethanol). The sample and standards were then denatured by heating for 5 minutes at 100¿ C. After cooling, 10¿l of 10X buffer and 10% NP-40 were added. The samples were mixed, and 4¿l of enzyme (30U) were added. Samples were incubated overnight at 37¿C. The oligosaccharides were isolated after treatment with 15¿l of 1% acetic acid (2h, room temperature) to convert the resultant glycosylamine to a single reducing form (Hardy, M. R., and Townsend, R. R., High-pH anion-exchange chromatography of glycoprotein-derived carbohydrates, 1994, Methods Enzymol. 230: 208-225).