Differential protein expression profiling by iTRAQ-Two-dimensional LC-MS/MS of human bladder cancer EJ138 cells transfected with the metastasis suppressor KiSS-1 gene

Ruppen, Isabel, Grau, Laura, Orenes-Pinero, Esteban, Ashman, Keith, Gil, Marta, Algaba, Ferran, Bellmunt, Joaquin and Sanchez-Carbayo, Marta (2010) Differential protein expression profiling by iTRAQ-Two-dimensional LC-MS/MS of human bladder cancer EJ138 cells transfected with the metastasis suppressor KiSS-1 gene. Molecular and Cellular Proteomics, 9 10: 2276-2291. doi:10.1074/mcp.M900255-MCP200


Author Ruppen, Isabel
Grau, Laura
Orenes-Pinero, Esteban
Ashman, Keith
Gil, Marta
Algaba, Ferran
Bellmunt, Joaquin
Sanchez-Carbayo, Marta
Title Differential protein expression profiling by iTRAQ-Two-dimensional LC-MS/MS of human bladder cancer EJ138 cells transfected with the metastasis suppressor KiSS-1 gene
Formatted title
Differential protein expression profiling by iTRAQ-Two-dimensional LC-MS/MS of human bladder cancer EJ138 cells transfected with the metastasis suppressor KiSS-1 gene
Journal name Molecular and Cellular Proteomics   Check publisher's open access policy
ISSN 1535-9476
1535-9484
Publication date 2010-10-01
Sub-type Article (original research)
DOI 10.1074/mcp.M900255-MCP200
Open Access Status DOI
Volume 9
Issue 10
Start page 2276
End page 2291
Total pages 16
Place of publication Bethesda, MD, United States
Publisher American Society for Biochemistry and Molecular Biology
Language eng
Formatted abstract
KiSS-1 is a metastasis suppressor gene reported to be involved in the progression of several solid neoplasias. The loss of KiSS-1 gene expression has been shown to be inversely correlated with increasing tumor stage, distant metastases, and poor overall survival in bladder tumors. To identify the molecular pathways associated with the metastasis suppressor role of KiSS-1 in bladder cancer, we carried out a proteomics analysis of bladder cancer cells (EJ138) transiently transfected with a vector encompassing the full-length KiSS-1 gene using an iTRAQ (isobaric tags for relative and absolute quantitation) approach. Protein extracts collected after 24- and 48-h transfection were fractionated and cleaved with trypsin, and the resulting peptides were labeled with iTRAQ reagents. The labeled peptides were separated by strong cation exchange and reversed phase LC and analyzed by MALDI-TOF/TOF MS. Three software packages were utilized for data analysis: ProteinPilot for identification and quantification of differentially expressed proteins, Protein Center for gene ontology analysis, and Ingenuity Pathways Analysis to provide insight into biological networks. Comparative analysis among transfected, mock, and empty vector-exposed cells identified 1529 proteins with high confidence (>99%) showing high correlation rates among replicates (70%). The involvement of the identified proteins in biological networks served to characterize molecular pathways associated with KiSS-1 expression and to select critical candidates for verification analyses by Western blot using independent transfected replicates. As part of complementary clinical validation strategies, immunohistochemical analyses of proteins regulated by KiSS-1, such as Filamin A, were performed on bladder tumors spotted onto tissue microarrays (n = 280). In summary, our study not only served to uncover molecular mechanisms associated with the metastasis suppressor role of KiSS-1 in bladder cancer but also to reveal the biomarker role of Filamin A in bladder cancer progression and clinical outcome.
Keyword Tandem mass-spectrometry
Multidimensional liquid-chromatography
Coupled receptor GPR54
Breast cancer
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: UQ Centre for Clinical Research Publications
 
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