Simultaneous proteomic profiling of four different growth states of human fibroblasts, using amine-reactive isobaric tagging reagents and tandem mass spectrometry.
In general, permanent growth arrest due to exhaustive cell replication can be induced prematurely by either stress or overexpression of selected oncogenes. In an attempt to examine key proteins involved in achieving premature senescence, and how they differ from those in serially passaged, replicatively exhausted cells, we used a novel proteomic profiling approach, isobaric tagging for relative and absolute quantitation (iTRAQ), to perform simultaneous four-way comparison of replicatively senescent fibroblasts, oxidatively stressed prematurely senescent fibroblasts, and their young replicating and quiescent counterparts. Two hundred and forty proteins were identified and quantified simultaneously; data analysis reveals: (1) groups of proteins whose expressions are uniformly either up- or down-regulated in all three growth arrest states; (2) signature proteins which may serve as candidate proteomic markers to differentiate the quiescent state from permanent growth arrest by either exhaustive replication or stress induction and (3) that while oxidative stress-induced, prematurely senescent fibroblasts morphologically resemble their replicatively exhausted counterparts, they exhibit different protein expression patterns. Results from simultaneous proteomic profiling were validated by Western blotting for selected proteins: collagen type I, HSP90 and vimentin. In conclusion, this report shows that iTRAQ proteomic profiling is a powerful technique for globally mapping protein signatures for different culture growth states.
Cong
Yu-Sheng
YS
Department of Microbiology and Immunology, University of Louisville School of Medicine, 580 South Preston Street, Louisville, KY 40292, United States.
Fan
Ellen
E
Wang
Eugenia
E
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D002478:Cells, Cultured; D016922:Cellular Senescence; D002845:Chromatography / Q000379:methods; D002853:Chromatography, Liquid; D015536:Down-Regulation; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D015854:Up-Regulation; D001616:beta-Galactosidase / Q000378:metabolism
D010455:Peptides; D011506:Proteins; D001616:beta-Galactosidase; D014357:Trypsin; D010100:Oxygen