This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.18O-labeling of N-linked glycosylation sites One hundred micro liters of the sample (TEP1r) was placed in microcentrifuge tube and added 100 L of 50 mM ammonium bicarbonate buffer (pH 8.4) and followed immediately by reduction with 25 mM dithiothreitol (45 min at 50oC) and carboxyamidomethylation with 90 mM iodoacetamide (45 min at room temperature in the dark). The sample was digested with sequencing grade trypsin (Promega, Madison, WI) overnight incubation at 37 C. After trypsin digestion, the sample was added 1 % formic acid to deactivate the enzyme and then was passed through a C18 reversed phase cartridge. The sample was dried down in a speed vac and added with ammonium bicarbonate buffer (50mM, pH 8.4) and then dried down in a speed vac. Sodium phosphate (100mM, pH 7.5) was added to the dried sample. The sample then was dried to completeness and rehydrated in 19 L of H218O. PNGase F was added and the mixture was incubated for 20 hours at 37 C and dried in the Speed Vac. The sample was digested with sequencing grade trypsin for 5 hours at 37 C. The sample was filtered by use of a NanoSep centrifugation concentrator (Pall Inc.) and analyzed by liquid chromatography and mass spectrometry (LC-MS). Liquid chromatography and mass spectrometric analysis The peptides were loaded onto a capillary column packed with C18 and washed with 0.1 % formic acid (mobile phase A). Subsequently, the peptide was eluted over 160 min with a flow of 200 nl/min using a linear gradient of 5 ~ 95 % of mobile phase B (80 % acetonitrile + 0.1 % formic acid). The eluted peptides were analyzed by nanoelectrospray ionization mass spectrometry (NSI-MS) using a LTQ-MS (Thermo Finnigan).
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