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

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

Author information

1
Genomics and Functional Proteomics Laboratories, Osteoporosis Research Center, Creighton University Medical Center, Omaha, NE, USA.

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.

PMID:
19904223
PMCID:
PMC2835986
DOI:
10.1097/MPA.0b013e3181bc44dd
[Indexed for MEDLINE]
Free PMC Article

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