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

被引:59
作者
Bras, Lisandra E. de Castro [1 ,2 ]
Ramirez, Trevi A. [1 ]
DeLeon-Pennell, Kristine Y. [1 ,2 ]
Chiao, Ying Ann [1 ,3 ]
Ma, Yonggang [1 ,2 ]
Dai, Qiuxia [1 ,2 ]
Halade, Ganesh V. [1 ,2 ]
Hakala, Kevin [1 ,4 ]
Weintraub, Susan T. [1 ,4 ]
Lindsey, Merry L. [1 ,2 ,5 ]
机构
[1] San Antonio Cardiovasc Prote Ctr, San Antonio, TX USA
[2] Univ Mississippi, Med Ctr, Dept Physiol & Biophys, Jackson Ctr Heart Res, Jackson, MS 39216 USA
[3] Univ Washington, Dept Pathol, Seattle, WA 98195 USA
[4] Univ Texas Hlth Sci Ctr San Antonio, Dept Biochem, San Antonio, TX USA
[5] GV Sonny Montgomery Vet Affairs Med Ctr, Res Serv, Washington, DC USA
关键词
Extracellular matrix; Enrichment; Decellularization; Heart; Solubility; Matrix metalloproteinases; EXTRACTION PROCEDURES; PROTEOMIC ANALYSIS; MEMBRANE-PROTEINS; COLLAGEN; MATRIX-METALLOPROTEINASE-9; CARTILAGE; SKIN; ANGIOGENESIS; INFARCTION; DIGESTION;
D O I
10.1016/j.jprot.2013.05.004
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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. (C) 2013 Elsevier B.V. All rights reserved.
引用
收藏
页码:43 / 52
页数:10
相关论文
共 33 条
  • [11] Korossis SA, 2005, J HEART VALVE DIS, V14, P408
  • [12] Matrix metalloproteinase-9 gene deletion facilitates angiogenesis after myocardial infarction
    Lindsey, ML
    Escobar, GP
    Dobrucki, LW
    Goshorn, DK
    Bouges, S
    Mingoia, JT
    McClister, DM
    Su, HL
    Gannon, J
    MacGillivray, C
    Lee, RT
    Sinusas, AJ
    Spinale, FG
    [J]. AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY, 2006, 290 (01): : H232 - H239
  • [13] Effect of a cleavage-resistant collagen mutation on left ventricular remodeling
    Lindsey, ML
    Yoshioka, J
    MacGillivray, C
    Muangman, S
    Gannon, J
    Verghese, A
    Aikawa, M
    Libby, P
    Krane, SM
    Lee, RT
    [J]. CIRCULATION RESEARCH, 2003, 93 (03) : 238 - 245
  • [14] Distinct effects of gonadectomy in mate and female mice on collagen fibrillogenesis in the skin
    Markiewicz, Margaret
    Asano, Yoshihide
    Znoyko, Sergey
    Gong, Yong
    Watson, Dennis K.
    Trojanowska, Maria
    [J]. JOURNAL OF DERMATOLOGICAL SCIENCE, 2007, 47 (03) : 217 - 226
  • [15] Effect of ablation or inhibition of stromal matrix metalloproteinase-9 on lung metastasis in a breast cancer model is dependent on genetic background
    Martin, Michelle D.
    Carter, Kathy J.
    Jean-Philippe, Sharon R.
    Chang, Mayland
    Mobashery, Shahriar
    Thiolloy, Sophie
    Lynch, Conor C.
    Matrisian, Lynn M.
    Fingleton, Barbara
    [J]. CANCER RESEARCH, 2008, 68 (15) : 6251 - 6259
  • [16] EXTRACTION OF CARTILAGE PROTEIN-POLYSACCHARIDES WITH INORGANIC SALT SOLUTIONS
    MASON, RM
    MAYES, RW
    [J]. BIOCHEMICAL JOURNAL, 1973, 131 (03) : 535 - 540
  • [17] AGE-RELATED DIFFERENCES IN HUMAN-SKIN COLLAGEN - SOLUBILITY IN SOLVENT, SUSCEPTIBILITY TO PEPSIN DIGESTION, AND THE SPECTRUM OF THE SOLUBILIZED POLYMERIC COLLAGEN MOLECULES
    MIYAHARA, T
    MURAI, A
    TANAKA, T
    SHIOZAWA, S
    KAMEYAMA, M
    [J]. JOURNALS OF GERONTOLOGY, 1982, 37 (06): : 651 - 655
  • [18] Collagens, modifying enzymes and their mutations in humans, flies and worms
    Myllyharju, J
    Kivirikko, KI
    [J]. TRENDS IN GENETICS, 2004, 20 (01) : 33 - 43
  • [19] Interactions between hydrophobic and ionic solutes in aqueous guanidinium chloride and urea solutions: Lessons for protein denaturation mechanism
    O'Brien, Edward P.
    Dima, Ruxandra I.
    Brooks, Bernard
    Thirumalai, D.
    [J]. JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 2007, 129 (23) : 7346 - 7353
  • [20] Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart
    Ott, Harald C.
    Matthiesen, Thomas S.
    Goh, Saik-Kia
    Black, Lauren D.
    Kren, Stefan M.
    Netoff, Theoden I.
    Taylor, Doris A.
    [J]. NATURE MEDICINE, 2008, 14 (02) : 213 - 221