Extraction of cartilage protein–polysaccharides with inorganic salt solutions

Mason, Roger M.; Mayes, R. W.

doi:10.1042/bj1310535

Cited by 43 publications

(18 citation statements)

References 21 publications

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“…1). The results with cartilage corresponded to published data Mason & Mayes, 1973). With the possible exceptions of 8M-urea and 4M-MgCI2, the amount of hexuronate extracted from disc appeared to be independent of the extractant used.…”

Section: Resultssupporting

confidence: 79%

The proteoglycans of the human intervertebral disc

Emes

Pearce

1975

Biochemical Journal

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The methods of were used to compare the proteoglycans of human intervertebral disc with those of bovine nasal cartilage. In contrast with cartilage, most of the hexuronate of disc could be extracted at low shear with water or dilute salt solutions. Extracts of disc with 4M-guanidinium chloride were centrifuged in 0.4M-guanidinium chloride in a CsCl gradient. Analytical ultracentrifugation of the hexuronatecontaining heavy component revealed two fractions, both more polydisperse than those of cartilage. Also the more rapidly sedimenting component was a much smaller fraction of the total. After prior extraction with 0.4M-guanidinium chloride, 4M-guanidinium chloride extracts of disc were found, by ultracentrifugal analysis, to be enriched in components resembling the proteoglycan monomer and aggregating factors of cartilage.

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Section: Resultssupporting

confidence: 79%

The proteoglycans of the human intervertebral disc

Emes

Pearce

1975

Biochemical Journal

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“…) in other organs and that approach was adapted for use with liver tissue here . The NaCl extraction displaces polyionic interactions between proteins, thereby solubilizing loosely bound proteins . Following NaCl extraction, the remaining liver tissue was decellularized using 1% sodium dodecyl sulfate (SDS).…”

Section: Resultsmentioning

confidence: 99%

The hepatic “matrisome” responds dynamically to injury: Characterization of transitional changes to the extracellular matrix in mice

et al. 2016

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The extracellular matrix (ECM) consists of diverse components that work bidirectionally with surrounding cells to create a responsive microenvironment. In some contexts (e.g., hepatic fibrosis), changes to the ECM are well recognized and understood. However, it is becoming increasingly accepted that the hepatic ECM proteome (i.e., matrisome) responds dynamically to stress well before fibrosis. The term “transitional tissue remodeling” describes qualitative and quantitative ECM changes in response to injury that do not alter the overall architecture of the organ; these changes in ECM may contribute to early disease initiation and/or progression. The nature and magnitude of these changes to the ECM in liver injury are poorly understood. The goals of this work were to validate analysis of the ECM proteome and compare the impact of 6 weeks of ethanol diet and/or acute lipopolysaccharide (LPS). Liver sections were processed in a series of increasingly rigorous extraction buffers to separate proteins by solubility. Extracted proteins were identified using liquid chromatography/tandem mass spectrometry (LC-MS/MS). Both ethanol and LPS dramatically increased the number of matrisome proteins ~25%. The enhancement of LPS-induced liver damage by ethanol preexposure was associated with unique protein changes. Conclusion An extraction method to enrich the hepatic ECM was characterized. The results demonstrate that the hepatic matrisome responds dynamically to both acute (LPS) and chronic (ethanol) stresses, long before more-dramatic fibrotic changes to the liver occur. The changes to the mastrisome may contribute, at least in part, to the pathological responses to these stresses. It is also interesting that several ECM proteins responded similarly to both stresses, suggesting a common mechanism in both models. Nevertheless, there were responses that were unique to the individual and combined exposures.

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“…LV samples are initially incubated in a neutral low salt, non-denaturing buffer (0.5 M NaCl, 10 mM Tris base pH 7.5 and 1× PI) for extraction of soluble proteins, including newly synthesized ECM proteins and degradation products (Step 1). The salts in the buffer interrupt ionic interactions between proteins, enabling the removal of non-covalently bound extracellular proteins [22]. The remaining tissue is then decellularized by treatment with SDS (Step 2).…”

Section: Resultsmentioning

confidence: 99%

Texas 3-Step decellularization protocol: Looking at the cardiac extracellular matrix

Brás

Ramirez²,

DeLeon‐Pennell

et al. 2013

Journal of Proteomics

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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 resolubilization. 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.

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Extraction of cartilage protein–polysaccharides with inorganic salt solutions

Cited by 43 publications

References 21 publications

The proteoglycans of the human intervertebral disc

The proteoglycans of the human intervertebral disc

The hepatic “matrisome” responds dynamically to injury: Characterization of transitional changes to the extracellular matrix in mice

Texas 3-Step decellularization protocol: Looking at the cardiac extracellular matrix

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