Profiling pancreatic cancer-secreted proteome using 15N amino acids and serum-free media.
OBJECTIVE
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.
Xiao
Jing
J
Genomics and Functional Proteomics Laboratories, Osteoporosis Research Center, Creighton University Medical Center, Omaha, NE, USA.
Lee
Wai-Nang Paul
WN
Zhao
Yingchun
Y
Cao
Rui
R
Go
Vay Liang W
VL
Recker
Robert R
RR
Wang
Qi
Q
Xiao
Gary Guishan
GG
D000963:Antimetabolites / Q000494:pharmacology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D053550:Keratin-10 / Q000032:analysis* / Q000378:metabolism; D013058:Mass Spectrometry; D010119:Oxythiamine / Q000494:pharmacology; D010179:Pancreas / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D010190:Pancreatic Neoplasms / Q000378:metabolism / Q000473:pathology; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000032:analysis* / Q000378:metabolism
D000963:Antimetabolites; D016895:Culture Media, Serum-Free; D020543:Proteome; D019715:Tissue Inhibitor of Metalloproteinase-1; D010119:Oxythiamine; D053550:Keratin-10