Biochemical characterization of the cell-biomaterial interface by quantitative proteomics

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.

Fig. 1.

Experiment setup and s1‘chematic diagram of machinery used to isolate adherence surface (AS) at cell-biomaterials interface. A, a schematic flow chart of SILAC gel-based proteomics for distinguishing whole cells and AS. B, demonstrates machinery set-up for AS isolation from cells on substrate.

Fig. 2.

Western blottings and immunofluorescence of whole cell and isolated AS for quantifying AS purity. A, shows Immunoblottings on whole cell, crude AS and pure AS fractions against fibronectin, transferrin receptor, cadherin, tubulin,, nucleophosmin and nuclear DNA helicase II. This demonstrates the enrichment and depletion of ECM, membrane, cytoskeleton and nuclear proteins in AS fractions. B, shows immunofluorescence images probing against actin stress fiber, fibronectin and talin, representing cytoskeleton, ECM and focal adhesion, respectively. Whole cell (left) and pure AS (right) can be easily differentiated.

Fig. 3.

Morphology of isolated AS comparing to whole cell visualized with confocol microscopy. Fluorescence confocol micrographs on MDCK whole cells (A) and adherence surface (F). Nucleous, mitochondria and plasma membrane are shown as blue, red and green, respectively. Horizontal sections of (A) are shown in (B–E), and similarly (G–J) are correspond to (F).

Fig. 4.

Morphology of isolated AS comparing to whole cell visualized by AFM and bright field (DIC). Fluorescence image of AS on substratum (A) is compared side by side with its differential interference contrast (DIC) image (B). AFM micrographs (C) shows the contour of whole cells while (D) shows contour of pure AS.

Fig. 5.

Proteomics analysis of AS proteins isolated from cell-biomaterial interface. A, shows SILAC pairs of three peptides in MS spectrums, which belong to plectin isoform 1 isoform 5, polymerase I and transcript release factor and laminin-5 gramma 2, representing “Depleted from AS” (left), “Equally distributed within cell” (Middle) and “Enriched in AS” (right). B, shows normalized H/L ratio of all identified & quantified proteins (204) in descending order, AS enriched proteins (normalized ratio > 0) are in red (24), AS depleted are in black (180). Heat map (C) is complemented to (B), showing proteins subcellular origin, according to GO, at different normalized ratio. D, shows noteworthy proteins and its normalized ratio.