AVS 52nd International Symposium
    Biomaterial Interfaces Wednesday Sessions
       Session BI2-WeA

Paper BI2-WeA9
Identification of Residual ECM Proteins Retained at pNIPAM Surfaces using Time-of-Flight SIMS

Wednesday, November 2, 2005, 4:40 pm, Room 311a

Session: Cell-Surface Interactions
Presenter: H.E. Canavan, University of Washington
Authors: H.E. Canavan, University of Washington
M. Greenfeld, University of Washington
X. Cheng, University of Washington
D.J. Graham, University of Washington
B.D. Ratner, University of Washington
D.G. Castner, University of Washington
Correspondent: Click to Email
Treatment of tissue culture polystyrene (TCPS) with poly(N-isopropyl acrylamide) (pNIPAM) has been developed as a technique for the harvest of intact cell layers. Recently, we demonstrated that although low-temperature liftoff removes the majority of the Extracellular Matrix (ECM) concurrently with the cells, some protein does remain at the pNIPAM surface. However, little is known about the identity of the ECM proteins retained at the pNIPAM surface after cell liftoff. In this work, we characterized the time-of-flight secondary ion mass spectrometry (ToF-SIMS) molecular fragmentation pattern of adsorbed single protein mixtures of important ECM proteins (e.g., laminin, fibronectin, and collagen). We next performed Principal Component Analysis (PCA) to distinguish between the proteins through the identification of unique amino acid fragmentation patterns in the ToF-SIMS positive ion spectra, a technique previously developed in our group. In this way, a model ToF-SIMS projection of the ECM was constructed. We subsequently compared the ToF-SIMS fragmentation pattern of the proteinaceous layer retained on the pNIPAM surface to that of the model ECM. ToF-SIMS fragmentation patterns of bovine serum albumin and serum-containing media controls were compared as positive controls as well. Using the comparison of the model ECM to that of ECM retained on pNIPAM surfaces, we discuss the identity of the proteins retained on the substrate after low-temperature liftoff from pNIPAM surfaces. We then compare our results to those obtained from analysis of the ECM using other surface analytical techniques, including immunoassay, gel electrophoresis, and matrix-assisted laser desorption ionization (MALDI).