In the present study, we examined the response of confluent, primary human fibroblasts and cells of a
melanoma (YUSAC2) cell line to ionizing radiation mediated through post-translational
protein phosphorylation. Since the purpose of our study was to identify novel radiation-induced
phosphoproteins in the DNA damage stress response of
melanoma cells, we were primarily interested in changes in
protein phosphoserine expression at early times after irradiation. Our rationale was that by examining the overall
protein phosphorylation profile (the phosphoproteome) in irradiated cells, we might discover novel radiation-induced
phosphoproteins that distinguish fibroblasts from
melanoma cells. Cell
proteins were separated by gel electrophoresis and
phosphoproteins were identified by Western blot analysis using nonspecific anti-phosphoamino
acid antibodies. This approach was not pursued previously since adequate
antibodies for examining global
protein phosphoserine expression were unavailable. While some radiation-induced
phosphoprotein changes in high-abundance
proteins were identified, in general the sensitivity of this approach was not sufficient to detect changes in low-abundance, regulatory
proteins. Characterization of these
phosphoproteins will require greater enrichment of low-abundance
proteins.