Detection of tyrosine‑phosphorylated proteins in hepatocellular carcinoma tissues using a combination of GST‑Nck1‑SH2 
pull‑down and two‑dimensional electrophoresis

Qiu,Fanghua; Huang,Dehong; Xiao,Hongguang; Qiu,Fangying; Lu,Liming; Nie,Jing

doi:10.3892/mmr.2013.1324

Detection of tyrosine‑phosphorylated proteins in hepatocellular carcinoma tissues using a combination of GST‑Nck1‑SH2 pull‑down and two‑dimensional electrophoresis

Authors:
- Fanghua Qiu
- Dehong Huang
- Hongguang Xiao
- Fangying Qiu
- Liming Lu
- Jing Nie
View Affiliations

Affiliations: Department of Clinical Laboratory, Guangzhou Hospital of Traditional Chinese Medicine, Guangzhou, Guangdong, P.R. China, Department of Neurology, Guangzhou Hospital of Traditional Chinese Medicine, Guangzhou, Guangdong, P.R. China, Department of Clinical Laboratory, The First Affiliated Hospital of Guangzhou Medical College, Guangzhou, Guangdong, P.R. China, Department of Emergency, The Fourth Affiliated Hospital of Guangzhou Medical College, Guangzhou, Guangdong, P.R. China, Division of Nephrology, Nanfang Hospital, Southern Medical University, Research Institute of Nephrology Guangdong Province, Key Laboratory for Organ Failure Research, Ministry of Education, Guangzhou, Guangdong, P.R. China
Published online on: February 19, 2013 https://doi.org/10.3892/mmr.2013.1324
Pages: 1209-1214

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Tyrosine‑phosphorylated proteins govern a host of cell functions, such as growth, division, adhesion and motility. We previously identified a group of Nck Src homology 2 (SH2) domain‑binding proteins by combining the GST‑Nck1‑SH2 pull‑down method with two‑dimensional electrophoresis (2‑DE) in hepatocellular carcinoma (HCC) tissues. In the present study, different methods and conditions for key procedures of GST‑Nck1‑SH2 pull‑down and 2‑DE were investigated and optimized. High‑resolution results were obtained using the following conditions: a total amount of 100 µl GST‑Nck1‑SH2 fusion proteins/10 mg liver proteins to execute the pull‑down procedure; 7 M urea and 2 M thiourea as lysis buffer; ultrafiltration depletion of interferential materials. Moreover, we performed a negative control experiment using GST‑4T3 during the pull‑down procedure, and further demonstrated that the proteins obtained using the aforementioned method interacted with Nck in a tyrosine phosphorylation‑dependent manner. The optimized method offers a rapid, efficient alternative for the high‑quantity screening of tyrosine‑phosphorylated protein expression and solubility, which in turn facilitates future studies on tyrosine‑phosphorylated proteins.

View Figures

View References

1	Barr AJ: Protein tyrosine phosphatases as drug targets: strategies and challenges of inhibitor development. Future Med Chem. 2:1563–1576. 2010. View Article : Google Scholar : PubMed/NCBI
2	Soderblom EJ, Philipp M, Thompson JW, Caron MG and Moseley MA: Quantitative label-free phosphoproteomics strategy for multifaceted experimental designs. Anal Chem. 83:3758–3764. 2011. View Article : Google Scholar : PubMed/NCBI
3	Morishita A, Gong J, Nomura T, Yoshida H, Izuishi K, Suzuki Y, Kushida Y, Haba R, D’Armiento J and Masaki T: The use of protein array to identify targetable receptor tyrosine kinases for treatment of human colon cancer. Int J Oncol. 37:829–835. 2010. View Article : Google Scholar : PubMed/NCBI
4	Alcolea MP, Kleiner O and Cutillas PR: Increased confidence in large-scale phosphoproteomics data by complementary mass spectrometric techniques and matching of phosphopeptide data sets. J Proteome Res. 8:3808–3815. 2009. View Article : Google Scholar : PubMed/NCBI
5	Kuban-Jankowska A, Gorska M, Debicki A, Popowska U, Knap N and Wozniak M: Protein tyrosine phosphatases - endogenous markers of oxidative stress. Postepy Biochem. 56:269–273. 2010.(In Polish).
6	Machida K, Mayer BJ and Nollau P: Profiling the global tyrosine phosphorylation state. Mol Cell Proteomics. 2:215–233. 2003.
7	Blagoev B, Kratchmarova I, Ong SE, Nielsen M, Foster LJ and Mann M: A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling. Nat Biotechnol. 21:315–318. 2003. View Article : Google Scholar : PubMed/NCBI
8	Bladt F, Aippersbach E, Gelkop S, Strasser GA, Nash P, Tafuri A, Gertler FB and Pawson T: The murine Nck SH2/SH3 adaptors are important for the development of mesoderm-derived embryonic structures and for regulating the cellular actin network. Mol Cell Biol. 23:4586–4597. 2003. View Article : Google Scholar : PubMed/NCBI
9	Dierck K, Machida K, Voigt A, Thimm J, Horstmann M, Fiedler W, Mayer BJ and Nollau P: Quantitative multiplexed profiling of cellular signaling networks using phosphotyrosine-specific DNA-tagged SH2 domains. Nat Methods. 3:737–744. 2006. View Article : Google Scholar : PubMed/NCBI
10	Shen SL, Qiu FH, Dayarathna TK, Wu J, Kuang M, Li SS, Peng BG and Nie J: Identification of Dermcidin as a novel binding protein of Nck1 and characterization of its role in promoting cell migration. Biochim Biophys Acta. 1812:703–710. 2011. View Article : Google Scholar : PubMed/NCBI
11	Machida K, Thompson CM, Dierck K, et al: High-throughput phosphotyrosine profiling using SH2 domains. Mol Cell. 26:899–915. 2007. View Article : Google Scholar : PubMed/NCBI
12	Blume-Jensen P and Hunter T: Oncogenic kinase signalling. Nature. 411:355–365. 2001. View Article : Google Scholar : PubMed/NCBI
13	Machida K, Eschrich S, Li J, Bai Y, Koomen J, Mayer BJ and Haura EB: Characterizing tyrosine phosphorylation signaling in lung cancer using SH2 profiling. PLoS One. 5:e134702010. View Article : Google Scholar : PubMed/NCBI
14	Kudo M and Okanoue T; Japan Society of Hepatology. Management of hepatocellular carcinoma in Japan: consensus-based clinical practice manual proposed by the Japan Society of Hepatology. Oncology. 72(Suppl 1): 2–15. 2007. View Article : Google Scholar : PubMed/NCBI
15	Kikuchi T and Carbone DP: Proteomics analysis in lung cancer: challenges and opportunities. Respirology. 12:22–28. 2007. View Article : Google Scholar : PubMed/NCBI
16	Wu Y, Zhou J, Zhang X, Zheng X, Jiang X, Shi L, Yin W and Wang J: Optimized sample preparation for two-dimensional gel electrophoresis of soluble proteins from chicken bursa of Fabricius. Proteome Sci. 7:382009. View Article : Google Scholar : PubMed/NCBI
17	Liu B, Qiu FH, Voss C, Xu Y, Zhao MZ, Wu YX, Nie J and Wang ZL: Evaluation of three high abundance protein depletion kits for umbilical cord serum proteomics. Proteome Sci. 9:242011. View Article : Google Scholar : PubMed/NCBI