This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Methods: B-elimination followed by Michael addition (BEMAD) for O-Glycosylation site mapping One hundred micrograms of CBH2b-cDNA and CBH2b-YO were reduced with 5 mM DTT for 1 h at 55 ?C and carboxyamidomethylated with 15 mM iodoacetamide in the dark for 45 min. The dried dialyzed samples were resuspended in 50 mM ammonium bicarbonate (NH4HCO3) and digested with 5 ?g of trypsin at 37 ?C for 20 h. Following deactivation of protease at 100 ?C for 5 min, the samples were digested with 5 ?g of Glu-C at 25 ?C for 20 h and then dried down in a Speed Vac. Dried peptides were then B-eliminated and subjected to Michael addition with DTT via resuspension in 1% triethylamine, 0.1% NaOH, and 10 mM DTT. The reaction was incubated at 42 ?C for 3 h, and the reaction was quenched with 1% trifluoroacetic acid. The labeled peptides were clean up by reverse phase C18 columns, eluted in 0.1 % formic acid, 80 % acetonitrile, and dried in a Speed Vac. Protein identification by LC-MS/MS LC-MS/MS analysis was performed on a LTQ Orbitrap Discoverer mass spectrometer (Thermo Scientific) equipped with a nanospray ion source. The labeled peptides were resuspended with 200 ?L of mobile phase A (0.1% formic acid in water). The sample was then loaded onto a nanospray tapered capillary column/emitter (360x75x15 ?m, PicoFrit, New Objective, Woburn, MA) self-packed with C18 reverse-phase resin (10.5 cm, Waters, Milford, MA) in a Nitrogen pressure bomb for 5 min at 1,000 psi (~5 uL load) and then separated via a 160 min linear gradient of increasing mobile phase B at a flow rate of~500 nL/min directly into the mass spectrometer. The resulting data were searched against the recombinant CBH2b-cDNA or CBH2b-YO sequence using the TurboSequest algorithm (Proteome Discoverer 1.1, Thermo Scientific). The SEQUEST parameters were set to allow 2 Da of precursor ion mass tolerance and 0.8 Da of fragment ion tolerance with monoisotopic mass. Digested peptides were allowed with up to two missed internal cleavage sites, and the differential modifications of 57.02146 Da, 15.9949 Da, and 136.002 Da were allowed for alkylated cysteine, oxidation of methionines, and DTT-labeled serine or theonine, respectively.
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