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 from5 mg 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 sample was then per-O-trimethylsilylated by treatment with Tri-Sil (Pierce) at 80 C (0.5 mins). [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 0.25mm lD). 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.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR018502-04
Application #
7359067
Study Section
Special Emphasis Panel (ZRG1-BECM (40))
Project Start
2006-07-01
Project End
2007-06-30
Budget Start
2006-07-01
Budget End
2007-06-30
Support Year
4
Fiscal Year
2006
Total Cost
$618
Indirect Cost
Name
University of Georgia
Department
Type
Organized Research Units
DUNS #
004315578
City
Athens
State
GA
Country
United States
Zip Code
30602
Gas-Pascual, Elisabet; Ichikawa, Hiroshi Travis; Sheikh, Mohammed Osman et al. (2018) CRISPR/Cas9 and glycomics tools for Toxoplasma glycobiology. J Biol Chem :
Sheikh, M Osman; Thieker, David; Chalmers, Gordon et al. (2017) O2 sensing-associated glycosylation exposes the F-box-combining site of the Dictyostelium Skp1 subunit in E3 ubiquitin ligases. J Biol Chem 292:18897-18915
Ma, Liang; Chen, Zehua; Huang, Da Wei et al. (2016) Genome analysis of three Pneumocystis species reveals adaptation mechanisms to life exclusively in mammalian hosts. Nat Commun 7:10740
Karumbaiah, Lohitash; Enam, Syed Faaiz; Brown, Ashley C et al. (2015) Chondroitin Sulfate Glycosaminoglycan Hydrogels Create Endogenous Niches for Neural Stem Cells. Bioconjug Chem 26:2336-49
Li, Juan; Murtaugh, Michael P (2015) Functional analysis of porcine reproductive and respiratory syndrome virus N-glycans in infection of permissive cells. Virology 477:82-8
DePaoli-Roach, Anna A; Contreras, Christopher J; Segvich, Dyann M et al. (2015) Glycogen phosphomonoester distribution in mouse models of the progressive myoclonic epilepsy, Lafora disease. J Biol Chem 290:841-50
Dwyer, Chrissa A; Katoh, Toshihiko; Tiemeyer, Michael et al. (2015) Neurons and glia modify receptor protein-tyrosine phosphatase ? (RPTP?)/phosphacan with cell-specific O-mannosyl glycans in the developing brain. J Biol Chem 290:10256-73
Li, Juan; Tao, Shujuan; Orlando, Ron et al. (2015) N-glycosylation profiling of porcine reproductive and respiratory syndrome virus envelope glycoprotein 5. Virology 478:86-98
Panin, Vladislav M; Wells, Lance (2014) Protein O-mannosylation in metazoan organisms. Curr Protoc Protein Sci 75:Unit 12.12.
Ingale, Jidnyasa; Tran, Karen; Kong, Leopold et al. (2014) Hyperglycosylated stable core immunogens designed to present the CD4 binding site are preferentially recognized by broadly neutralizing antibodies. J Virol 88:14002-16

Showing the most recent 10 out of 104 publications