Screening for membrane proteins differentially expressed between the lung adenocarcinoma cell lines with high- and low-metastatic potential using iTRAQ technology / 肿瘤

ABSTRACT

Objective: To screen for the membrane proteins differentially expressed between the lung adenocarcinoma cell lines with high- and low- metastatic potential, and to explore the potential targets for biomarkers and biological therapy. Methods: The membrane proteins were extracted from lung adenocarcinoma SPC-A-1 and SPC-A-1 sci cells which were generated from the same parental cell type and had low- and high- metastatic potential, respectively. The membrane proteins were labeled and the peptides were separated and analyzed by iTRAQ (isobaric tags for relative and absolute quantitation) technology combined with Nano-LC-MS/MS (nano liquid chromatography-tandem mass spectrometry). The identification and quantitation of the proteins were analyzed by Proteinpilot 4.0 solfware. The membrane proteins differentially expressed were analyzed and verified by GO (Gene Ontology) terms and real-time fluorescence quantitative-PCR in combination with Western blotting, respectively. Results: The identified numbers of proteins with FDRs (false discovery rates) < 1% were 1 413, 1 374, 1 297, and 1 351 in the experiment which were repeated four times by Nano LC-MS/MS, and the rate of labelling was above 95%. Among the 27 proteins up-regulated in total four experiments, 20 proteins were identified as membrane protein. Among the 32 proteins down-regulated in total four experiments, 25 proteins were identified as membrane protein. The GO analysis demonstrated the major molecular functions of the proteins differentially expressed including cytoskeletal protein binding, identical protein binding and enzyme binding as well as the catabolic process and cellular localization in biological processes. The expression levels of ITGA3 (integrin alpha-3), MYH9 (myosin, heavy chain 9), PLEC1 (plectin 1), HADHA (3-hydroxyacyl- CoA dehydrogenase), HK1 (hexokinase-1), KTN1 (kinectin 1), ESYT1 (extended synaptotagmin-like protein 1), ALDH18A1 (aldehyde dehydrogenase 18 family, member A1), ATP5A1 (ATP synthase alpha-subunit), LMNB2 (lamin-B2), CAV1 (caveolin-1) and CK-19 (keratin, type I cytoskeletal 19) mRNAs in SPC-A-1 sci cells were higher than those in the SPC-A-1 cells. The expression levels of CLTC (clathrin, heavy chain), HK1, LMNB2 and CK-19 in SPC-A-1 sci cells were also higher than those in SPC-A-1 cells. These results were consistent with those from quantitative mass spectrometry. Conclusion: A high-throughput screen for metastasis-related membrane proteins can be performed by a combined use of iTRAQ and Nano LCMS/ MS and may provide the potential metastasis-related biomarkers and therapeutic targets in diagnosis, prognostic prediction and treatment for patients with lung adenocarcinoma. Copyright © 2013 by TUMOR.