Research

Proteome-level display by 2-dimensional chromatography of extracellular matrix-dependent modulation of the phenotype of bladder cancer cells

Robert E Hurst^1,2,3 , Kimberly D Kyker¹ , Mikhail G Dozmorov¹ , Nobuaki Takemori² , Anil Singh² , Hiroyuki Matsumoto² , Ricardo Saban⁴ , Edna Betgovargez⁵ and Michael H Simonian⁵

¹  Department of Urology, Oklahoma University Health Sciences Centre, Oklahoma City, OK 73104, USA

²  Department of Biochemistry and Molecular Biology, Oklahoma University Health Sciences Centre, Oklahoma City, OK 73104, USA

³  Department of Occupational and Environmental Health, Oklahoma University Health Sciences Centre, Oklahoma City, OK 73104, USA

⁴  Department of Physiology, Oklahoma University Health Sciences Centre, Oklahoma City, OK 73104, USA

⁵  Biomedical Research Division, Beckman Coulter Inc. 4300 N. Harbor Blvd., Fullerton, CA 92834, USA

author email corresponding author email

Proteome Science 2006, 4:13doi:10.1186/1477-5956-4-13

Published:	2 June 2006

Abstract

Background

The extracellular matrix can have a profound effect upon the phenotype of cancer cells. Previous work has shown that growth of bladder cancer cells on a matrix derived from normal basement membrane suppresses many malignant features that are displayed when the cells are grown on a matrix that has been modified by malignant tumors. This work was undertaken to investigate proteome-level changes as determined by a new commercially available proteome display involving 2-dimensional chromatography for bladder cancer cells grown on different extracellular matrix preparations that modulate the expression of the malignant phenotype.

Results

Depending on the matrix, between 1300 and 2000 distinct peaks were detected by two-dimensional chromatographic fractionation of 2.1 – 4.4 mg of total cellular protein. The fractions eluting from the reversed-phase fractionation were suitable for mass spectrometric identification following only lyophilization and trypsin digestion and achieved approximately 10-fold higher sensitivity than was obtained with gel-based separations. Abundant proteins that were unique to cells grown on one of the matrices were identified by mass spectrometry. Following concentration, peaks of 0.03 AU provided unambiguous identification of protein components when 10% of the sample was analyzed, whereas peaks of 0.05 AU was approximately the lower limit of detection when the entire sample was separated on a gel and in-gel digestion was used. Although some fractions were homogeneous, others were not, and up to 3 proteins per fraction were identified. Strong evidence for post-translational modification of the unique proteins was noted. All 13 of the unique proteins from cells grown on Matrigel were related to MYC pathway.

Conclusion

The system provides a viable alternative to 2-dimensional gel electrophoresis for proteomic display of biological systems. The findings suggest the importance of MYC to the malignant phenotype of bladder cancer cells.