Profiling pancreatic cancer-secreted proteome using 15N amino acids and serum-free media

Jing Xiao, Wai Nang Paul Lee, Yingchun Zhao, Rui Cao, Vay Liang W. Go, Robert R. Recker, Qi Wang, Gary Guishan Xiao

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Abstract

Objectives: A new method of determining protein turnover by labeling protein with 15N amino acids was used in conjunction with serum-free cell culture to profile secreted proteins that are released by MIA PaCa-2 pancreatic cancer cells in culture. Methods: MIA PaCa-2 cells were first cultured in Dulbecco modified Eagle medium (Gibco by Invitrogen, Carlsbad, Calif) with 10% fetal bovine serum, then in serum-free modified Eagle medium with or without 50% 15N algal amino acid mixture. The effect of oxythiamine chloride on secreteome was studied. Secreteome from cell culture media was analyzed by 2-dimensional (2D) gel electrophoresis. Differentially expressed proteins were detected and identified. Protein turnover rates were calculated according to the newly established method. Western blot and enzyme-linked immunosorbent assay were used to validate identified proteins. Results: Among the 14 differentially expressed proteins after oxythiamine treatment, tissue inhibitor of metalloproteases-1 and cytokeratin-10 were identified as 2 newly synthesized secreted proteins caused by substantial 15N incorporation. The inhibition of tissue inhibitor of metalloproteases-1 expression in MIA PaCa-2 cells by oxythiamine treatment was first demonstrated by 2D gel electrophoresis and further validated by Western blotting and enzyme-linked immunosorbent assay analyses. Conclusions: Our method of labeling protein with 15N amino acids in conjunction with serum-free cell culture allows the identification of actively secreted proteins from pancreatic cancer cells and is a useful method for serum biomarker discovery.

Original languageEnglish (US)
Pages (from-to)e17-e23
JournalPancreas
Volume39
Issue number1
DOIs
StatePublished - Jan 1 2010

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All Science Journal Classification (ASJC) codes

  • Internal Medicine
  • Endocrinology, Diabetes and Metabolism
  • Hepatology
  • Endocrinology

Cite this

Xiao, J., Lee, W. N. P., Zhao, Y., Cao, R., Go, V. L. W., Recker, R. R., Wang, Q., & Xiao, G. G. (2010). Profiling pancreatic cancer-secreted proteome using 15N amino acids and serum-free media. Pancreas, 39(1), e17-e23. https://doi.org/10.1097/MPA.0b013e3181bc44dd