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

Research output: Contribution to journalArticle

7 Citations (Scopus)

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
JournalPancreas
Volume39
Issue number1
DOIs
StatePublished - Jan 2010

Fingerprint

Serum-Free Culture Media
Proteome
Pancreatic Neoplasms
Amino Acids
Oxythiamine
Proteins
Cell Culture Techniques
Serum
Eagles
Metalloproteases
Electrophoresis
Gels
Enzyme-Linked Immunosorbent Assay
Far-Western Blotting
Keratins
Culture Media
Chlorides
Biomarkers
Western Blotting

All Science Journal Classification (ASJC) codes

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

Cite this

Profiling pancreatic cancer-secreted proteome using 15N amino acids and serum-free media. / Xiao, Jing; Lee, Wai Nang Paul; Zhao, Yingchun; Cao, Rui; Go, Vay Liang W; Recker, Robert R.; Wang, Qi; Xiao, Gary Guishan.

In: Pancreas, Vol. 39, No. 1, 01.2010.

Research output: Contribution to journalArticle

Xiao, Jing ; Lee, Wai Nang Paul ; Zhao, Yingchun ; Cao, Rui ; Go, Vay Liang W ; Recker, Robert R. ; Wang, Qi ; Xiao, Gary Guishan. / Profiling pancreatic cancer-secreted proteome using 15N amino acids and serum-free media. In: Pancreas. 2010 ; Vol. 39, No. 1.
@article{298f47b20f0c46ee9f78d72e5f591a31,
title = "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 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.",
author = "Jing Xiao and Lee, {Wai Nang Paul} and Yingchun Zhao and Rui Cao and Go, {Vay Liang W} and Recker, {Robert R.} and Qi Wang and Xiao, {Gary Guishan}",
year = "2010",
month = "1",
doi = "10.1097/MPA.0b013e3181bc44dd",
language = "English",
volume = "39",
journal = "Pancreas",
issn = "0885-3177",
publisher = "Lippincott Williams and Wilkins",
number = "1",

}

TY - JOUR

T1 - Profiling pancreatic cancer-secreted proteome using 15N amino acids and serum-free media

AU - Xiao, Jing

AU - Lee, Wai Nang Paul

AU - Zhao, Yingchun

AU - Cao, Rui

AU - Go, Vay Liang W

AU - Recker, Robert R.

AU - Wang, Qi

AU - Xiao, Gary Guishan

PY - 2010/1

Y1 - 2010/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=74049102336&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=74049102336&partnerID=8YFLogxK

U2 - 10.1097/MPA.0b013e3181bc44dd

DO - 10.1097/MPA.0b013e3181bc44dd

M3 - Article

VL - 39

JO - Pancreas

JF - Pancreas

SN - 0885-3177

IS - 1

ER -