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

Pancreas. 2010 Jan;39(1):e17-23. doi: 10.1097/MPA.0b013e3181bc44dd.


Objectives: A new method of determining protein turnover by labeling protein with N 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% N 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 N 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 N 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antimetabolites / pharmacology
  • Blotting, Western
  • Cell Line, Tumor
  • Culture Media, Serum-Free / pharmacology
  • Electrophoresis, Gel, Two-Dimensional
  • Enzyme-Linked Immunosorbent Assay
  • Humans
  • Keratin-10 / analysis*
  • Keratin-10 / metabolism
  • Mass Spectrometry
  • Oxythiamine / pharmacology
  • Pancreas / drug effects
  • Pancreas / metabolism
  • Pancreas / pathology
  • Pancreatic Neoplasms / metabolism
  • Pancreatic Neoplasms / pathology
  • Proteome / analysis*
  • Proteome / metabolism
  • Proteomics / methods*
  • Tissue Inhibitor of Metalloproteinase-1 / analysis*
  • Tissue Inhibitor of Metalloproteinase-1 / metabolism


  • Antimetabolites
  • Culture Media, Serum-Free
  • Proteome
  • Tissue Inhibitor of Metalloproteinase-1
  • Oxythiamine
  • Keratin-10