Evaluation and Refinement of Sample Preparation Methods for Extracellular Matrix Proteome Coverage

Abstract

The extracellular matrix is a key component of tissues, yet it is underrepresented in proteomic datasets. Identification and evaluation of proteins in the extracellular matrix (ECM) has proved challenging due to the insolubility of many ECM proteins in traditional protein extraction buffers. Here we separate the decellularization and ECM extraction steps of several prominent methods for evaluation under real-world conditions. The results are used to optimize a two-fraction ECM extraction method. Approximately one dozen additional parameters are tested, and recommendations for analysis based on overall ECM coverage or specific ECM classes are given. Compared with a standard in-solution digest, the optimized method yielded a fourfold improvement in unique ECM peptide identifications.

Extracellular Matrix-Oriented Proteomic Analysis of Periodontal Ligament Under Mechanical Stress

“…This may lead to the decreased proportion of collagen in the analytical sample from the distal side of PDL. To overcome technical limitations attributable to the insolubility of the ECM, several ECM-enrichment protocols including decellularization, deglycosylation, and chemical digestion have been developed ( de Castro Bras et al, 2013 ; McCabe et al, 2021 ). Although these methods were shown to effectively enrich the core matrisome proteins, they also present a significant loss in the number of detected matrisome-associated proteins ( Krasny and Huang, 2021 ).…”

Comparison of extracellular matrix enrichment protocols for the improved characterization of the skin matrisome by mass spectrometry

“…2). A recent study actually suggested that this method leads to high losses of matrisome components in the various extraction buffers when applied to a whole mouse powder [37].…”

“…It should also be noted here that a possible optimization applicable to all methods, especially if accurate quantification of proteoglycans and glycoproteins is desired, would be to combine several deglycosylation enzymes (in addition to the classical PNGaseF) to remove glycosaminoglycan (GAG) chains and other O-glycosylations, as described by others [32]. However, the systematic integration of a GAG removal step in ECM-centered analyses must be validated first as a recent study [37] suggested that it may decrease the number of PSMs for collagens and glycoproteins.…”

Tissue-Specific Decellularized Extracellular Matrix Bioinks for Musculoskeletal Tissue Regeneration and Modeling Using 3D Bioprinting Technology

“…Recently, proteomic analysis has been highlighted because of its complementary strategy, providing both compositional and quantitative data. It also allows an in-depth study of enriched proteins such as growth factors and cytokines in dECM [68]. In addition, as well as the development of analysis tools, the process of decellularization should be also optimized in order to qualify the dECM components.…”