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: N-Glycosylation Site Mapping With 18O-labeling and LC-MS/MS Forty micrograms of NG was reduced with 25 mM DTT for 1 h at 55 ?C and carboxyamidomethylated with 90mM iodoacetamide in the dark for 45 min. The dried dialyzed sample was resuspended in 50 mM ammonium bicarbonate (NH4HCO3) and digested with 2 ?g of trypsin at 25 ?C for 20 h. Following deactivation of trypsin at 100 ?C for 5 min, the sample was then deglycosylated with 2 ?g of PNGaseF in 36 ?L of 18O Water (H218O) and 2 ?L of 1M NH4HCO3. 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. LC-MS/MS analysis was performed on a LTQ Orbitrap Discoverer mass spectrometer (Thermo Scientific) equipped with a nanospray ion source. The resulting data were searched against the recombinant NG sequence using the TurboSequest algorithm (Proteome Discoverer 1.1, Thermo Scientific). The SEQUEST parameters were set to allow 30.0 ppm of precursor ion mass tolerance and 0.8 Da of fragment ion tolerance with monoisotopic mass. Tryptic peptides were allowed with up to two missed internal cleavage sites, and the differential modifications of 57.02146 Da, 15.9949 Da and 2.98826 Da were allowed for alkylated cysteine, oxidation of methionines and 18O-labeled aspartic acid, respectively.

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