This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Methods: The coomassie blue-stained gel slices were cut into smaller pieces followed by destain, reduction, calboxyamidemethylation and in-gel tryptic digestion. The tryptic peptides were then extracted from the gel pieces and profiled by mass spectrometry. Detailed procedures for in-gel digestion and mass spec analysis are shown below. In-gel digestion Coomassie 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 10 mM DTT in 40mM Ambic at 55? C for 1 hr. The DTT solution was exchanged with 55mM 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 40 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 were extracted from the gels 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 LTQ orbitrap mass spectrometry The peptides were resuspended with mobile phase A (0.1% formic acid, FA, in water) and filtered with 0.2 ?m filters prior to sampling. The sample was loaded by off-line onto a nanospray tapered capillary column/emitter self-packed with C18 reverse-phase (RP) resin in a Nitrogen pressure bomb for 5 min at 1000 psi and then separated via a 160 min linear gradient of increasing mobile phase B (80% acetonitrile, ACN, and 0.1% formic acid in water) at a flow rate of ~400 nL/min directly into the mass spectrometer. LC-MS/MS analysis was performed on a LTQ Orbitrap XL mass spectrometer (ThermoFisher) equipped with a nanospray ion source. A full FTMS spectrum at 30,000 resolution was collected at 400?2000 m/z followed by 6 data dependent MS/MS spectra of ITMS in the most intense ion peaks from parent mass list following CID (36 % normalized collision energy). The resulting data was searched against the UniProtKB/Swiss-prot database using the TurboSequest algorithm (Proteome Discoverer 1.0, ThermoFisher) for protein identification. The SEQUEST parameters were set to allow 20.0 ppm of precursor ion mass tolerance and 0.8 Da of fragment ion tolerance with monoisotopic mass. Tryptic peptides were allowed with up to three missed internal cleavage sites and the differential modifications of 57.0215 Da for alkylated cysteine. The resulting peptides found by the sequest search were further filtered based on Xcorr value, 2.0/2.5/3.0 for +1/+2/+3 charged peptide respectively.
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