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Abstract
The use of extracellular matrix (ECM) scaffolds, derived from decellularized tissues for engineered organ generation, holds enormous potential in the field of regenerative medicine. To support organ engineering efforts, we developed a targeted proteomics method to extract and quantify extracellular matrix components from tissues. Our method provides more complete and accurate protein characterization than traditional approaches. This is accomplished through the analysis of both the chaotrope-soluble and -insoluble protein fractions and using recombinantly generated stable isotope labeled peptides for endogenous protein quantification. Using this approach, we have generated 74 peptides, representing 56 proteins to quantify protein in native (nondecellularized) and decellularized lung matrices. We have focused on proteins of the ECM and additional intracellular proteins that are challenging to remove during the decellularization procedure. Results indicate that the acellular lung scaffold is predominantly composed of structural collagens, with the majority of these proteins found in the insoluble ECM, a fraction that is often discarded using widely accepted proteomic methods. The decellularization procedure removes over 98% of intracellular proteins evaluated and retains, to varying degrees, proteoglycans and glycoproteins of the ECM. Accurate characterization of ECM proteins from tissue samples will help advance organ engineering efforts by generating a molecular readout that can be correlated with functional outcome to drive the next generation of engineered organs.
Highlights
A more complete and accurate method for protein characterization would provide a valuable tool for tissue engineering efforts, while shedding light on the possible molecular mechanisms resulting in cell seeding variability and alterations in mechanical properties of engineered lung tissues
Strategies can be employed in an attempt to normalize data [17], there is a distinct advantage to quantification methods using stable isotope labeled (SIL) peptides in this application
Amino acid analysis revealed that this pellet contained protein with a high percentage of glycine and proline compared with the detergent- and chaotrope-soluble fractions. This amino acid profile led us to our first hypothesis; the chaotrope insoluble pellet, which is commonly discarded in proteomic approaches, is primarily composed of fibrillar proteins of the extracellular matrix (ECM)
Summary
This whole organ scaffold can be recellularized using a patient’s own primary or stem-derived cells, eliminating many issues related to graft/host incompatibility This approach was recently used to generate lungs that, when implanted in rat recipients, allowed for gas exchange [4, 5]. Examination of the lung indicated leakage of erythrocytes into the alveolar space, indicating a compromised capillary-endothelial barrier These exciting results highlighted the potential of the method for organ transplantation and the need for improved molecular readouts to guide engineering efforts. Current methods used to characterize the protein composition of native and acellular tissues involve antibody- or dye-based staining, hydroxyproline assays assessing collagen content, or relative quantification of proteins by liquid chromatography tandem mass spectrometry (LCMS/MS) [9, 10]. SIL quantification allows for intra- and intersample comparison of heterogeneous tissues, such as native organs and decellularized scaffolds, with high accuracy and precision
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