Comparison of native extracellular matrix with adsorbed protein films using secondary ion mass spectrometry.
In the past decade, the temperature-responsive behavior of poly(N-isopropyl acrylamide) (pNIPAM) has come to be recognized as a convenient method for the nondestructive harvest of confluent cell layers. Recently, we have utilized this nondestructive cell harvest method as a means to ascertain the nature of the extracellular matrix (ECM) secreted from cells. In this work, we compare the ECM obtained after cell liftoff to individual ECM proteins adsorbed directly onto RF-plasma-deposited pNIPAM (ppNIPAM). Using X-ray photoelectron spectroscopy, we find that the composition of ppNIPAM post-cell liftoff surfaces is consistent with those of the ppNIPAM post-protein adsorption surface, both of which differ from control surfaces. Using principal component analysis of positive-ion time-of-flight secondary ion mass spectrometry (ToF-SIMS) data, we show that the major ECM proteins examined can effectively be identified from their amino acid compositions. By comparing the positive-ion ToF-SIMS data from each of the ppNIPAM post-protein adsorption surfaces to that of ppNIPAM post-cell liftoff, we find that ppNIPAM post-cell liftoff surfaces are distinctly separate from fibronectin (FN). This result is consistent with our previous observation using immunoassay that FN is clearly associated with the cell sheet after low-temperature liftoff from ppNIPAM.
Canavan
Heather E
HE
National ESCA and Surface Analysis Center for Biomedical Problems, University of Washington Engineered Biomaterials, and Departments of Bioengineering and Chemical Engineering, University of Washington, Seattle, Washington 98195, USA. canavan@unm.edu
Graham
Daniel J
DJ
Cheng
Xuanhong
X
Ratner
Buddy D
BD
Castner
David G
DG
D000180:Acrylic Resins / Q000737:chemistry*; D000327:Adsorption; D000818:Animals; D001011:Aorta / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D005109:Extracellular Matrix / Q000737:chemistry*; D016326:Extracellular Matrix Proteins / Q000737:chemistry*; D006801:Humans; D011485:Protein Binding; D018629:Spectrometry, Mass, Secondary Ion
D000180:Acrylic Resins; D016326:Extracellular Matrix Proteins; C052970:poly-N-isopropylacrylamide