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  • Cell Cycle Compound Library br Phosphorylated proteins were

    2018-11-07


    Phosphorylated proteins were enriched from the total protein extract using the Al(OH)3-MOAC method as previously described with modifications [6]. Approximately 8mg of total protein extract was added to 2g of Al(OH)3. After centrifugation (6000g for 10min), the matrix with retained proteins was washed four times with the wash buffer (similar to IB except for the amino Cell Cycle Compound Library concentration was 0.15M). The bound putative phosphoproteins were then eluted with 20mL of elution buffer (0.3M potassium pyrophosphate, 8M urea, pH 9). After centrifugation (10,000g for 5min), the supernatant was removed from the matrix and recentrifuged to avoid matrix carryover. The eluted proteins were amended with 2% w/v sodium deoxycholate and 100% w/v trichloroacetic acid (TCA) and placed at 4°C overnight. After centrifugation (18,000g for 15min), precipitated proteins were successively collected and washed with 25% w/v TCA. The pellets were then resuspended in 10mM Tris–HCl (pH 7.5) in 80% v/v acetone and washed with 90% v/v acetone after centrifugation. After air drying, the phosphoproteins were dissolved in resolubilization/rehydration buffer (RB) (7M urea, 2M thiourea, 2% v/v NP-40, 65mM DTT, and 1% IPG buffer).
    Gel electrophoresis and staining of gels Two-dimensional gel electrophoresis (2-DE) was performed as described previously [7]. Briefly, gels were loaded with 150μg of proteins in 350μL RB buffer. Isoelectric focusing (IEF) was performed in a stepwise manner: at 100V for 0.5h, 500V for 0.5h, 1000V for 2h, 8000V gradients for 4h and 8000V for total of 60,000Vh. SDS-PAGE in the second dimension was then conducted with 12% gels. For phosphoproteins detection, 2-D gels were stained with a modified protocol using Pro-Q Diamond fluorescent gel stain [8]; total proteins were stained with the improved Coomassie brilliant blue G-250 (CBB) staining method [9]. Briefly gels were treated with fixation solution (50% methanol, 10% acetic acid) (2×30min), washed with deionized water (3×10min), stained with 3-fold diluted Pro-Q DPS in deionized water (2h), destained with destaining solution (20% ACN, 50mM sodium acetate pH 4.0) (4×30min) to remove gel-bound nonspecific Pro-Q DPS, and washed again with deionized water (2×5min). The gel was then scanned with a Typhoon Trio Variable Mode Imager (GE Healthcare, Uppsala Sweden) with a 532nm laser excitation and a 580nm bandpass emission filter. Spot identification, background elimination, point matching, and differential analysis of the protein spots were completed using PDQuest software (Version 8.0, Bio-Rad, Hercules, CA, USA). The proteins spots showing ≥2-fold changes in all three biological replicas were considered as differentially regulated putative phosphoproteins.
    MALDI-TOF/TOF MS analysis and database searching Selected protein spots were excised from the CBB-stained gels, and then destained with 100mM NH4HCO3 in 30% acetonitrile (ACN). After removing the destaining buffer, the gel pieces were lyophilized and rehydrated in 30μL of 50mM NH4HCO3 containing 50ng trypsin. After overnight digestion at 37°C, the peptides were extracted three times with 0.1% TFA in 60% ACN. Extracts were pooled together and lyophilized. A protein-free gel piece was treated as above and used as a control to identify autoproteolysis products derived from trypsin. MS and MS/MS spectra were obtained using the ABI 4800 Proteomics Analyzer MALDI-TOF/TOF (Applied Biosystems, Foster City, CA). Peptide mass maps were acquired in positive ion reflector mode with 1000 laser shots per spectrum. Monoisotopic peak masses were automatically determined within the mass range 800–4000Da with a signal to noise ratio minimum set to10 and a local noise window width of m/z 250. After excluding common trypsin autolysis peaks and matrix ion signals, up to five most intense ions with a minimum signal to noise ratio higher than 50 were selected as candidates for MS/MS acquisition. In MS/MS-positive ion mode, spectra were averaged, collision energy was set at 2kV, and default calibration was selected. Monoisotopic peak masses were automatically determined with a signal to noise ratio minimum set to 5 and a local noise window width of m/z 250. The MS together with MS/MS spectra were searched against the UniprotKB/SwissProt database (v.2013.01.24) using the software GPS Explorer version 3.6 (Applied Biosystems) and MASCOT version 2.2 (Matrix Science, London, UK) with the following parameter settings: trypsin cleavage, one missed cleavage allowed, carbamidomethylation set as fixed modification, peptide mass tolerance set to 100ppm, fragment tolerance set to ±0.3Da, and minimum ion score confidence interval for MS/MS data set to 95%.