Dr. Robert N. Eisenman (Fred Hutchinson Cancer Research Center)
Treatment protocol
Serum deprivation for 12 hours.
Extracted molecule
protein
Extraction protocol
as described in J Am Soc Mass Spectrom 14:696-703, 2003.
Label
isotopically-light ICAT reagent
Description
Chromatin-enriched fractions (2.5 mg each), soluble fractions (2.5 mg each), and crude mitochondrial fractions (250 ug each) were prepared as described (1,2), dissolved in the ICAT labeling buffer (0.05% SDS, 6M urea, 200 mM Tris [pH 8.3], and 5 mM EDTA), reduced with 5 mM Tris(2-carboxyethyl) phosphine (TCEP) for 30 min at 37 ºC, and labeled (Rat1/vector cell lysate: isotopically-light ICAT reagent; Rat1/Myc cell lysate: isotopically-heavy ICAT reagent). The two labeled fractions were combined, proteolyzed to peptides with trypsin, and fractionated by cation-exchange chromatography. ICAT reagent-labeled peptides were purified using the biotin tag present in the reagent and analyzed by microcapillary high performance liquid chromatography-tandem mass spectrometry (µLC-MS/MS) as described (1,3-5). MS/MS spectra were searched against the NCBI rat/mouse/human protein database. Peptide/protein identification was validated by Peptide/ProteinProphet software tools (6,7). Peptide and protein abundance ratios were calculated using ASAPRatio software tool (8). 1. Shiio, Y., Eisenman, R. N., Yi, E. C., Donohoe, S., Goodlett, D. R., and Aebersold, R. (2003) J Am Soc Mass Spectrom 14(7), 696-703 2. Juin, P., Hueber, A. O., Littlewood, T., and Evan, G. (1999) Genes Dev 13(11), 1367-1381 3. Gygi, S. P., Rist, B., Gerber, S. A., Turecek, F., Gelb, M. H., and Aebersold, R. (1999) Nat Biotechnol 17(10), 994-999 4. Han, D. K., Eng, J., Zhou, H., and Aebersold, R. (2001) Nat Biotechnol 19(10), 946-951 5. Shiio, Y., Donohoe, S., Yi, E. C., Goodlett, D. R., Aebersold, R., and Eisenman, R. N. (2002) Embo J 21(19), 5088-5096 6. Keller, A., Nesvizhskii, A. I., Kolker, E., and Aebersold, R. (2002) Anal Chem 74(20), 5383-5392 7. Nesvizhskii, A. I., Keller, A., Kolker, E., and Aebersold, R. (2003) Anal Chem 75(17), 4646-4658 8. Li, X. J., Zhang, H., Ranish, J. A., and Aebersold, R. (2003) Anal Chem 75(23), 6648-6657
Data processing
Supplementary data files contain peptides with PeptideProphet probability score greater than/equal to 0.5 (6,7). Peptide and protein abundance ratios were calculated using ASAPRatio software tool (8). Abundance ratios of 0, 9999, and -1 denote that acceptable peptide peak(s) were not detected for one or both of the ICAT-labeled peptide(s).
