Biochemical Characterization of the Cell-Biomaterial Interface by Quantitative Proteomics

W.Y Tong,Y.M Liang,V Tam,H.K Yip,Y.T Kao,K.M.C Cheung,K.W.K Yeung,Y.W Lam

doi:10.1074/mcp.m110.001966

Abstract

Surface topography and texture of cell culture substrata can affect the differentiation and growth of adherent cells. The biochemical basis of the transduction of the physical and mechanical signals to cellular responses is not well understood. The lack of a systematic characterization of cell-biomaterial interaction is the major bottleneck. This study demonstrated the use of a novel subcellular fractionation method combined with quantitative MS-based proteomics to enable the robust and high-throughput analysis of proteins at the adherence interface of Madin-Darby canine kidney cells. This method revealed the enrichment of extracellular matrix proteins and membrane and stress fibers proteins at the adherence surface, whereas it shows depletion of extracellular matrix belonging to the cytoplasmic, nucleus, and lateral and apical membranes. The asymmetric distribution of proteins between apical and adherence sides was also profiled. Apart from classical proteins with clear involvement in cell-material interactions, proteins previously not known to be involved in cell attachment were also discovered.

Highlights

Surface topography and texture of cell culture substrata can affect the differentiation and growth of adherent cells
With this simple model system, ingenious experiments have shown that physical forces applied through the extracellular matrix (ECM)11 can induce changes in cell adhesion molecules and stress-induced ion channels, which lead to changes in the cytoskeleton and gene expressions [11,12,13]
1 The abbreviations used are: ECM, extracellular matrix; AS, adherence surface; MDCK, Madin-Darby canine kidney; iTRAQ, isobaric tag for relative and absolute quantitation; SILAC, stable isotope labeling with amino acids in cell culture; Dulbecco’s modified eagle medium (DMEM), Dulbecco’s modified Eagle’s medium; R6, L-13c6-arginine; k4, L-4,4,5-D4-lysine; R0, Larginine; k0, L-lysine; NDH II, nuclear DNA helicase II; IF, immunofluorescence; AFM, atomic force microscopy

Summary

Introduction

Surface topography and texture of cell culture substrata can affect the differentiation and growth of adherent cells. Given the complexity of this process, it is clear that the understanding of this phenomenon cannot be achieved merely by studying individual biological parts in isolation It is necessary, to systematically characterize the biochemical factors that mediate the interactions between cells and materials to yield insights into intracellular signaling processes that are responsible for such cellular responses. To systematically characterize the biochemical factors that mediate the interactions between cells and materials to yield insights into intracellular signaling processes that are responsible for such cellular responses Toward this goal, we seek to investigate the biochemical basis of how different biomaterials may impose changes in the composition of the AS of adherent cells. Xu et al [20] investigated proteome differences of human osteoblasts on various nano-sized hydroxyapatite powders with different shapes and chemical compositions using iTRAQ-based twodimensional LC-MS/MS

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Conclusion