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February 2005

Identifying shed and secreted proteins by mass spectrometry after exposure of CHO cells to low-dose radiation

David L. Springer and John H. Miller, Principal Investigators

In work sponsored by the U.S. Department of Energy's Low-Dose Radiation Program, scientists at Pacific Northwest National Laboratory (PNNL) are unraveling effects that produce biological responses in cells not directly hit by radiation—called bystander cells. Their findings will appear in Proteomics and have been submitted to Mutation Research.


The hypothesis being addressed is that radiation-induced bystander effects (RIBE) are caused by release of cell surface proteins generated by changes in either regulated proteolysis or secretory processes in response to low doses of radiation. Shed proteins were previously discovered in media taken from human mammary epithelial cells after treatment with phorbol ester, a known shedding inducer (Ahram et al. in press). However, after radiation exposure similar changes were not observed, and subsequent assays showed no bystander activity (Springer et al. submitted).

Subsequently, the research team conducted a radiation study using Chinese hamster ovary (CHO) cells because the bystander response is better characterized in these cells and large numbers of cells can be grown thus providing enough material to rigorously analyze for shed and secreted proteins. Samples were evaluated by both tandem ion trap and FTICR mass spectrometry in the Environmental Molecular Sciences Laboratory at PNNL. Mass spectrometry outputs were analyzed using a recently developed discriminant analysis procedure developed in EMSL (Strittmatter et al. 2004), which provides a confidence score based on SEQUEST parameters, the tryptic nature of the peptide, mass, and retention time.

Current findings

Figure 1. Possible network linking down-regulated proteins by signaling downstream of the activin A receptor in the TGF-? superfamily. Full Image

Using this approach the PNNL group identified approximately 500 proteins in the media, about 10% of which contain one or more transmembrane domains; hence, they likely shed (Ahram et al., in press). Because of the increased sensitivity of the FTICR and the improvements in informatics, the researchers observed several changes that appear to be attributable to radiation. These include 1) down-regulation of activin A receptor (a member of the TGF-β receptor superfamily), stromal interaction molecule (STIM) 2, vitronectin (an adhesive protein in plasma and serum), heat shock protein (HSP) 70 (a protein that plays roles in the folding, transport, synthesis, and quality control of other proteins), and fibronectin (an extracellular adhesion molecule involved in cellular processes); and 2) up-regulation of metalloproteinase carboxypeptidase and alpha-1-microglobulin (Table 1).

Figure 1 shows a possible link between the down-regulated proteins based on known pathways in eukaryotic cells. The upper part of the pathway illustrates the well-established mechanism by which receptors of activin and TGF-β; activate receptor-phosphorylated SMADs (R-SMADs) causing transport into the nucleus to assemble transcription factors through binding to Co-SMADs (Massague 2000). Through linkages with the metalloproteinase 13 and the Creb-binding protein, transcription factors p53 and NF-Β are activated, which leads to links to HSP70, fibronectin, and vitronectin. By placing the proteins with modified abundance in the context of known regulatory pathways, hypotheses can be formulated for modeling and new experiments.

The radiation-induced down-regulation of activin A receptor may be particularly relevant to RIBE because TGF-β has been implicated in bystander effects by other studies (Iyer and Lehnart 2000). Receptors on vesicles in the extracellular medium tie up ligands that initiate signaling pathways if they bind to receptors on the cell surface. Decreased activin A receptor in the medium could, therefore, imply a shift toward more TGF-β available to interact with cell surface receptors. Work continues to investigate this hypothesis.


  • Ahram M, JN Adkins, DL Auberry, DS Wunschel DS, and DL Springer. 2005. "A Proteomic Approach to Characterize Shed Proteins." Proteomics 5(1):123-131. DOI:10.1002/pmic.200400912.
  • Ahram M and DL Springer. 2004. "Large-Scale Proteomic Analysis of Membrane Proteins." Expert Opinions in Proteomics 1(3):293-302. DOI:10.1586/14789450.1.3.293.
  • Ahram M, EF Strittmatter, ME Monroe, JN Adkins, J Hunter, JH Miller, and DL Springer. 2005. "Identification of Shed Proteins from CHO cells: Application of Statistical Confidence using Human and Mouse Protein Databases." Proteomics 5(7):1815-1826. DOI:10.1002/pmic.200401072.
  • Iyer R and BE Lehnert. 2000. "Factors Underlying the Cell Growth-Related Bystander Responses to α Particles." Cancer Research 60(5):1290-1298. DOI:10.1586/14789450.1.3.293. 
  • Massague J. 2000. "How Cells Read TGF-β Signals." Nature Reviews 1(3):169-178. DOI:10.1038/35043051.
  • Strittmatter EF, LJ Kangas, K Petritis, HM Mottaz, GA Anderson, Y Shen, JM Jacobs, DG Camp, and RD Smith. 2004. "Application of Peptide Retention Time Information in a Discriminant Function for Peptide Identification by Tandem Mass Spectrometry." Journal of Proteome Research 3(4):760-769. DOI:10.1021/pr049965y.

Table 1

The proteins listed showed either up- or down-regulation following low-dose radiation exposure of CHO cells as determined by FTICR mass spectrometry.

Reference NumberProtein Name
gi25070247 Activin A receptor, type 1B
gi41349446 Stromal interaction molecule 2
gi4502067 Alpha-1-microglobulin/bikunin precursor
gi4503011 Carboxypeptidase N, polypeptide 1, 50 kDa
gi16933542 Fibronectin 1 isoform 1 preprotein
gi5123454 Heat shock 70kDa protein 1A
gi18201911 Vitronectin precursor

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