Phosphoproteome profiling of human skin fibroblast cells in response to low- and high-dose irradiation

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

Research output: Contribution to journalArticle

99 Scopus citations

Abstract

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

Original languageEnglish (US)
Pages (from-to)1252-1260
Number of pages9
JournalJournal of Proteome Research
Volume5
Issue number5
DOIs
StatePublished - May 1 2006

Keywords

  • High-dose irradiation
  • Low-dose irradiation
  • Mass spectrometry
  • Normal human skin fibroblast phosphoproteome
  • Phosphoproteome profiling

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

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    Yang, F., Stenoien, D. L., Strittmatter, E. F., Wang, J., Ding, L., Lipton, M. S., Monroe, M. E., Nicora, C. D., Gristenko, M. A., Tang, K., Fang, R., Adkins, J. N., Camp, D. G., Chen, D. J., & Smith, R. D. (2006). Phosphoproteome profiling of human skin fibroblast cells in response to low- and high-dose irradiation. Journal of Proteome Research, 5(5), 1252-1260. https://doi.org/10.1021/pr060028v