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

Abstract

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.

• Authors: Jing Xiao, Wai-Nang Paul Lee, Yingchun Zhao, Rui Cao, Vay Liang W Go, Robert R Recker, Qi Wang, Gary Guishan Xiao
• Journal: Pancreas (Pancreas) Vol. 39 Issue 1 Pg. e17-23 (Jan 2010) ISSN: 1536-4828 [Electronic] United States
• PMID: 19904223 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

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

Topics

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