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: 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 5 mM DTT in 40mM Ambic at 55? C for 1 hr. The DTT solution was exchanged with 15 mM 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 140 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. Protein identification by LC-MS/MS The 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 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. 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 target portein 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. Digested peptides were allowed with up to two missed internal cleavage sites, and the differential modifications of 57.02146 Da and 15.9949 Da were allowed for alkylated cysteine and oxidation of methionines, respectively. 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. Nanospray Ionization-Linear Ion Trap Mass Spectrometry Mass analysis by NSI-MS was performed on a LTQ Orbitrap Discoverer mass spectrometer (Thermo Scientific) equipped with a nanospray ion source. Briefly, permethylated glycans were dissolved in 1 mM NaOH in 50% methanol and infused directly into a linear ion trap mass spectrometer using a nanospray source at a syringe flow rate of 0.5 ?l/min. The capillary temperature was set to 210 ?C, and MS analysis was performed in positive ion mode. Fragmentation by CID in MS/MS modes 50% collision energy was applied. The nomenclature of Domon and Costello (Domon and Costello, 1988) was used to guide the depiction of fragmentation derived from MS/MS spectra.
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