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. In-gel digestion Coomassie blue-stained gel slices were cut into smaller pieces (~1 mm3) and destained alternately with 40mM Ammonium bicarbonate (AmBic) and 100% acetonitrile until the color turned clear. Destained gel was reswelled in 10 mM DTT in 40mM Ambic at 55? C for 1 hr. The DTT solution was exchanged with 55mM Iodoacetamide (IAM) and incubated in the dark for 45 min. Incubation was followed by washing alternately with 40mM AmBic and 100% acetonitrile twice. Dehydrated gel was reswelled with trypsin solution (trypsin in 40 mM Ambic) on ice for 45 min initially, and protein digestion was carried out at 37? C overnight. The supernatant was transferred into another tube. Peptides and the glycopeptides were extracted from the gel in series with 20% acetonitrile in 5% formic acid, 50% acetonitrile in 5% formic acid and then 80% acetonitrile in 5% formic acid. The sample solutions were dried and combined into one tube. Glycan preparation Extracted tryptic digest was passed through a C18 sep-pak cartridge and washed with 5% acetic acid to remove contaminants (salts, SDS, etc.). Peptides and glycopeptides were eluted in series with 20% iso-propanol in 5% acetic acid, 40% iso-propanol in 5% acetic acid and 100% iso-propanol and dried in a speed vacuum concentrator. The dried samples were combined and then reconstituted with 50 mM sodium phosphate buffer (pH 7.5) and heated at 100? C for 5 min to inactivate trypsin. The tryptic digest was incubated with PNGase F at 37? C overnight to release N-glycans. After digestion, the sample was passed through a C18 sep-pak cartridge and the carbohydrate fraction was eluted with 5% acetic acid and dried by lyophilization. Released N-linked oligosaccharides were permethylated based on the method of Anumula and Taylor (Anumula and Taylor, 1992) and profiled by mass spectrometry. Matrix-assisted laser-desorption ionization 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 (LTQ) 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 MS analysis was performed in the positive ion mode. For total ion mapping, automated MS/MS analysis (at 35 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.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Biotechnology Resource Grants (P41)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-CB-L (40))
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Georgia
Organized Research Units
United States
Zip Code
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
Gasimli, Leyla; Hickey, Anne Marie; Yang, Bo et al. (2014) Changes in glycosaminoglycan structure on differentiation of human embryonic stem cells towards mesoderm and endoderm lineages. Biochim Biophys Acta 1840:1993-2003
Panin, Vladislav M; Wells, Lance (2014) Protein O-mannosylation in metazoan organisms. Curr Protoc Protein Sci 75:Unit 12.12.

Showing the most recent 10 out of 104 publications