Texas 3-step decellularization protocol: looking at the cardiac extracellular matrix

Abstract

The extracellular matrix (ECM) is a critical tissue component, providing structural support as well as important regulatory signaling cues to govern cellular growth, metabolism, and differentiation. The study of ECM proteins, however, is hampered by the low solubility of ECM components in common solubilizing reagents. ECM proteins are often not detected during proteomics analyses using unbiased approaches due to solubility issues and relatively low abundance compared to highly abundant cytoplasmic and mitochondrial proteins. Decellularization has become a common technique for ECM protein-enrichment and is frequently used in engineering studies. Solubilizing the ECM after decellularization for further proteomic examination has not been previously explored in depth. In this study, we describe testing of a series of protocols that enabled us to develop a novel optimized strategy for the enrichment and solubilization of ECM components. Following tissue decellularization, we use acid extraction and enzymatic deglycosylation to facilitate re-solubilization. The end result is the generation of three fractions for each sample: soluble components, cellular components, and an insoluble ECM fraction. These fractions, developed in mass spectrometry-compatible buffers, are amenable to proteomics analysis. The developed protocol allows identification (by mass spectrometry) and quantification (by mass spectrometry or immunoblotting) of ECM components in tissue samples.

Biological significance: The study of extracellular matrix (ECM) proteins in pathological and non-pathological conditions is often hampered by the low solubility of ECM components in common solubilizing reagents. Additionally, ECM proteins are often not detected during global proteomic analyses due to their relatively low abundance compared to highly abundant cytoplasmic and mitochondrial proteins. In this manuscript we describe testing of a series of protocols that enabled us to develop a final novel optimized strategy for the enrichment and solubilization of ECM components. The end result is the generation of three fractions for each sample: soluble components, cellular components, and an insoluble ECM fraction. By analysis of each independent fraction, differences in protein levels can be detected that in normal conditions would be masked. These fractions are amenable to mass spectrometry analysis to identify and quantify ECM components in tissue samples. The manuscript places a strong emphasis on the immediate practical relevance of the method, particularly when using mass spectrometry approaches; additionally, the optimized method was validated and compared to other methodologies described in the literature.

Fig. 1

Description of the evolution of strategies developed for the enrichment of cardiac ECM proteins to facilitate analysis by mass spectrometry.

Fig. 2

a. Image of decellularized and untreated mouse left ventricle (LV); b. proteins in decellularized LV samples from young (4 to 8 month old) wild type mice (n = 10 biological replicas) were visualized in a 1-D SDS-PAGE gel (strategy 4). Lane 1 is the molecular weight (MW) marker. Poor sample solubility caused insoluble proteins to stay trapped in the wells (boxed region), resulting in decreased electrophoretic mobility; c. immunoblots (5 μg total protein) of fibronectin, MnSOD, and GAPDH in soluble (lysis buffer) and insoluble (Protein Extraction Reagent 4) young LV protein extracts. Decellularization buffer # 1 (1% SDS): lanes 1 and 4. Decellularization buffer # 2 (1% SDS, 20 mM NH₄OH): lanes 2 and 5. Untreated LV (lanes 3 and 6) and cellular extract (lane 7) were used as controls. Lane descriptions apply to Fig. 2c.

Fig. 3

Two treatments were used to increase sample solubility and enhance protein resolution by electrophoresis: protein reduction/alkylation and filtration of high molecular weight proteins. The Coomassie Blue stained gel shows proteins in decellularized LV samples before and after treatments. Lane 1: molecular weight (MW) marker; lane 2: control untreated sample; lane 3: LV sample after reduction/ alkylation; lane 4: >300 kDa protein fraction of a decellularized LV sample after membrane filtration; lane 5: <300 kDa protein fraction of decellularized LV sample (same sample as in lane 4). Reduction and alkylation of the decellularized LV increased sample solubility but proteins were smeared, suggesting over-denaturation. Filtration to remove ultra-high MW proteins did prevent proteins from remaining in the well (<300 kDa, lane 5), but as observed in lane 4 the filtration device also removed proteins with molecular weights below 300 kDa (boxed area).

Fig. 4

Coomassie Blue stained gel of decellularized LV extracts after acid extraction in conjunction with pepsin digestion. Pepsin treatment improved protein resolution on the gel. The same LV extract was treated with increasing quantities of pepsin for 30 min or 1 h. MW, molecular weight marker.

Fig. 5

Protein fractions resulting from the Texas 3-Step protocol. a. Coomassie Blue stained gel (1 μg total protein per lane). No proteins were observed trapped in the wells, which shows an improved protein resolution compared to the previous tested strategies (boxed area). Immunoblots: b. collagen type I; c. fibronectin; and d) MnSOD; and d. fibronectin, densitometry analysis was normalized to total protein, *p < 0.05 versus respective control. By using a differential solubility-based, protein fractionation strategy, we unmasked protein differences (arrows) that otherwise would not be noticeable by a 1-fraction method. Step 1: soluble proteins; Step 2: cellular proteins; Step 3: insoluble proteins. MW: molecular weight; Ctr: LV control tissue from unoperated mice (n = 4); LVI: LV infarcted tissue 5 days post-MI (n = 4).