| Popis: |
To assess molecular responses to low dose radiation, a quantitative proteomic approach using 8-plex iTRAQ was employed to identify protein alterations in human skin tissue treated with ionizing radiation. Additionally we investigated how protein modifications complicate reported fold-change for isobaric tagged peptide quantitation, and how multiple forms of identified peptides convolute peptide to protein roll-up. METHODS Sham or irradiated(10 cGy) reconstituted human skin tissues were separated into cytoplasmic, nuclear, chromatin fractions 8h post-exposure. Trypsin digested peptides were labeled with 8-plex iTRAQ reagents and analyzed using online 2D(SCX-RP)LC coupled to an LTQ-Orbitrap Velos, with sequential HCD/CID. SEQUEST was used for peptide identification, in-house developed software was employed for iTRAQ-based peptide quantitation. PRELIMINARY DATA 1129 proteins were identified in one or more subcellular fraction, of these, significant abundance and/or subcellular localization change was observed for 207. Bioinformatics analysis demonstrated that metabolic pathways and regulation of proteolytic processing are key targets of low dose radiation exposure. When rolled-up to the protein level, many of the observed alterations were lower than expected possibly due to effects of post-translational modifications on specific peptides.'As an example, by Western blot, a two-fold increase of vimentin was observed in the nuclear fraction, but the fold-change determined through proteomic analysis was only 0.18(Log2), despite lack of co-fragmentation for these specific peptides. Vimentin is known to be heavily modified, and additionally methionines can be oxidized during sample processing. By removing modified peptides from the analysis, the observed fold-change increased to 0.47(log2). Further results on protein processing and translocation of specific protein tertiary structure, opposed to whole protein changes, will be presented. |
