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. Glycopeptide analysis by LC-MS/MS Fifty micrograms of S178-73A was reduced with 25 mM DTT for 1 h at 55 ?C and carboxyamidomethylated with 90 mM iodoacetamide in the dark for 45 min. The dried dialyzed sample was resuspended in 50 mM ammonium bicarbonate (NH4HCO3) and digested with 2.5 ?g of trypsin, chymotrypsin or chymotrypsin/Glu-C (DE) at 37 ?C for 20 h. The digested glypeptides were resuspended with 200 ?L of mobile phase A (0.1% formic acid in water). The glypeptides were 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 10 min at 1,000 psi (~5 uL load) and then separated via a 160 min linear gradient of increasing mobile phase B (80% acetonitrile in 0.1% formic acid) at a flow rate of~500 nL/min directly into the mass spectrometer (LTQ Orbitrap Discoverer, Thermo Scientific). For improving the detection of glycopeptides with Asn36, chymotryptic digest was also separated and analyzed under low pH condition (pH ~1.7, 3% formic acid) according to the method of Hyunwoo et. al. A full mass spectrum was collected first with 30,000 resolution followed by MS/MS spectra using CID at 35%-50% collision energy of the sixth most intense peaks. The spectra of the glycopeptides were searched and analyzed manually.
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