Phosphoproteome Profiling of Human Skin Fibroblast Cells in Response to Low- and High-Dose Irradiation

• DOI: 10.1021/pr060028v
• Publication Date: 2006-05-05T04:41:07Z
• Authors: Feng Yang; David L. Stenoien; Eric F. Strittmatter; Junhua Wang; Lianghao Ding; Mary S. Lipton; Matthew E. Monroe; Carrie D. Nicora; Marina A. Gristenko; Keqi Tang; Ruihua Fang; Joshua N. Adkins; David G. Camp; David J. Chen; Richard D. Smith
• Keywords: Proteomics; 0301 basic medicine; Spectrometry, Mass, Electrospray Ionization; Amino Acid Motifs; Molecular Sequence Data; Dose-Response Relationship, Radiation; Fibroblasts; Protein Serine-Threonine Kinases; Phosphoproteins; Chromatography, Affinity; Cyclin-Dependent Kinases; Mass Spectrometry; 3-Phosphoinositide-Dependent Protein Kinases; Oncogene Protein v-akt; 03 medical and health sciences; Protein Biosynthesis; Humans; Amino Acid Sequence; Phosphorylation; Cells, Cultured; Chromatography, High Pressure Liquid; Signal Transduction

A hallmark of the response to high-dose radiation is up-regulation and phosphorylation proteins involved in cell cycle checkpoint control, DNA damage signaling, repair, apoptosis. Exposure cells low doses has well documented biological effects, but underlying regulatory mechanisms are still poorly understood. The objective this study provide an initial profile normal human skin fibroblast (HSF) phosphoproteome explore potential differences between low- irradiation responses at protein level. Several techniques including Trizol extraction proteins, methylation tryptic peptides, enrichment phosphopeptides with immobilized metal affinity chromatography (IMAC), nanoflow reversed-phase HPLC (nano-LC)/electrospray ionization, tandem mass spectrometry were combined for analysis HSF phosphoproteome. Among 494 unique phosphopeptides, 232 singly phosphorylated, while 262 peptides had multiple sites indicating overall effectiveness IMAC technique enrich both multiply phosphorylated peptides. We observed approximately 1.9-fold 3.6-fold increases number identified low-dose samples respectively, suggesting levels stimulate signaling pathways. 6-fold increase cyclin dependent kinase (cdk) motifs was after dose irradiation, stimulated 3-phosphoinositide-dependent kinase-1 (PDK1) AKT/RSK 8.5- 5.5-fold, respectively. High- resulted increased pathways as apoptosis control phosphoproteins broadly distributed among processes.