Quantification of decellularized human myocardial matrix: A comparison of six patients

Abstract

Purpose The purpose of this study was to characterize and quantitatively analyze human cardiac extracellular matrix (ECM) isolated from six different cadaveric donor hearts. Experimental Design ECM was isolated by decellularization of six human cadaveric donor hearts and characterized by quantifying sulfated glycosaminoglycan content (sGAG) and via polyacrylamide gel electrophoresis (PAGE). The protein content was then quantified using ECM-targeted Quantitative conCATamers (QconCAT) by Liquid Chromatography - Selected Reaction Monitoring (LC-SRM) analysis using 83 stable isotope labeled (SIL) peptides representing 48 different proteins. Non-targeted global analysis was also implemented using liquid chromatography tandem mass spectrometry (LC-MS/MS). Results The sGAG content, PAGE, and QconCAT proteomics analysis showed significant variation between each of the six patient samples. The quantitative proteomics indicated that the majority of the protein content was composed of various fibrillar collagen components. Also, quantification of difficult to remove cellular proteins represented less than 1% of total protein content, which is very low for a decellularized biomaterial. Global proteomics identified over 200 distinct proteins present in the human cardiac ECM. Conclusions and Clinical Relevance In conclusion, quantification and characterization of human myocardial ECM showed significant patient-to-patient variability between the six investigated patients. This is an important outcome for the development of allogeneic derived biomaterials and for increasing our understanding of human myocardial ECM composition.

Comparative proteomic assessment of matrisome enrichment methodologies

“…This list of common matrisomal components will be useful for the development of selected reaction monitoring (SRM) approaches for quantitative profiling of the matrisome in multiple tissue types, where the identity of the proteins and peptides of interest are required a priori for assay development [40]. A recent study describes the development of an SRM method based on QconCat technology, which utilises 83 stable isotope-labelled peptides representing 48 different ECM proteins to measure decellularised human hearts [14]. Of our 29 common matrisomal proteins, 60% are found in the QconCat library, indicating that this quantitative SRM method can be readily extended to quantitative matrisomal measurements in lung, mammary gland and liver.…”

Hydroxylamine Chemical Digestion for Insoluble Extracellular Matrix Characterization

“…Samples were analyzed by LC-SRM and LC-MS/MS as described 17 . Equal volumes from each post-digestion sample were combined and injected every third run and used to monitor technical reproducibility.…”

Controlling stem cell behavior with decellularized extracellular matrix scaffolds

“…Additionally, the majority of studies that included stiffness in their design were limited to 2D cultures, as opposed to 3D cultures, which are arguably more physiologically relevant. The combination of more quantitative analysis of the components of the ECM [86, 87] along with high throughput cell-biomaterial interaction screening [88, 89], which can simultaneously examine multiple biochemical cues and mechanical properties, could enable more mechanistic future studies.…”

“…It is therefore important to continue working towards improved processing, but to also keep the processing consistent when comparing results. Furthermore, more quantitative analysis of the ECM, such as QconCat based mass spectrometry [86, 87], is needed to more rigorously assess differences in the ECM and efficiency of decellularization after processing.…”