Divide-and-Conquer Matrisome Protein (DC-MaP) Strategy: An MS-Friendly Approach to Proteomic Matrisome Characterization

Abstract

Currently, the extracellular matrix (ECM) is considered a pivotal complex meshwork of macromolecules playing a plethora of biomolecular functions in health and disease beyond its commonly known mechanical role. Only by unraveling its composition can we leverage related tissue engineering and pharmacological efforts. Nevertheless, its unbiased proteomic identification still encounters some limitations mainly due to partial ECM enrichment by precipitation, sequential fractionation using unfriendly-mass spectrometry (MS) detergents, and resuspension with harsh reagents that need to be entirely removed prior to analysis. These methods can be technically challenging and labor-intensive, which affects the reproducibility of ECM identification and induces protein loss. Here, we present a simple new method applicable to tissue fragments of 10 mg and more. The technique has been validated on human ovarian tissue and involves a standardized procedure for sample processing with an MS-compatible detergent and combined centrifugation. This two-step protocol eliminates the need for laborious sample clarification and divides our samples into 2 fractions, soluble and insoluble, successively enriched with matrisome-associated (ECM-interacting) and core matrisome (structural ECM) proteins.

In-Depth Analysis of the Pancreatic Extracellular Matrix during Development for Next-Generation Tissue Engineering

“…The label-free quantification, which consists of measuring precursor ions’ intensities, is routinely used for quantification and comparison of proteins in biological tissue. Here, we adapted a proteomic workflow [ 16 ], developed to allow identification and quantification of ECM proteins, to small E14.5 and P1 pancreatic tissues. Briefly, we collected and lysed 5 mg (wet weight) of pancreatic tissue with the help of steel beads in a tissue homogenizer ( Figure 1 A, Step 1).…”

“…To accommodate the small size of the embryonic pancreas, we had to adapt and scale down available proteomics processes. We utilized a label-free protocol developed by Ouni et al [ 16 ], but with a reduced amount of initial biological material (only 5 mg). We also introduced an extra fractionation step before mass spectrometry analysis to increase the number of detected proteins.…”

“…The mechanical properties of biological tissues are crucial for their function and development. In vitro studies have shown that substrate elasticity influences lineage determination, with a rigid matrix favoring mesenchymal stem cell differentiation towards osteoblasts and a soft matrix favoring neuronal fate regardless of serum conditions [ 16 ]. Remodeling of the matrisome content between E14.5 and P1 suggests potential changes in the mechanical properties of the ECM, which could impact cell adhesion, migration, and differentiation.…”

MatrisomeDB 2.0: 2023 updates to the ECM-protein knowledge database

“…Upon completion of the curation process, we added 772 raw files from the datasets of 25 studies (Table 1 and Supplementary Table S1 ). This significantly expands the content of the database which includes new healthy tissues, such as the ECM of umbilical arteries ( 35 ) and the aorta ( 36 ), the stomach ( 37 ), the skin, including healthy skin from different anatomical regions and patients of different ages, but also scars and keloids ( 38–40 ), the Fallopian tubes ( 41 ) and ovaries ( 42 ), different musculoskeletal structures ( 43–48 ), and different regions of the brain ( 49 , 50 ). It also includes two cancer types not included before: pancreatic ductal adenocarcinoma ( 51 ) and gastric antrum adenocarcinoma ( 37 ), and data on the ECM of metastases arising from mammary tumors growing in different distant organs ( 52 ) and from metastases of ovarian tumors growing in the omentum treated or not with chemotherapy ( 53 ).…”

Ten Years of Extracellular Matrix Proteomics: Accomplishments, Challenges, and Future Perspectives

“…The Amorim lab has proposed a simpler two-step fractionation approach using commercially available MS-compatible reagents and separating tissue lysates into two pools of protein: detergent soluble and detergent insoluble ( 89 ). Similarly, the Ge lab has developed a photocleavable detergent called azo and applied it in a two-step fractionation approach to characterize the ECM composition of murine mammary tumors ( 90 ).…”