An in-solution ultrasonication-assisted digestion method for improved extracellular matrix proteome coverage

Kirk C. Hansen, Lauren Kiemele, Ori Maller, Jenean O'Brien, Aarthi Shankar, Jaime Fornetti, Pepper Schedin

Research output: Contribution to journalArticle

60 Citations (Scopus)

Abstract

Epithelial cell behavior is coordinated by the composition of the surrounding extracellular matrix (ECM); thus ECM protein identification is critical for understanding normal biology and disease states. Proteomic analyses of ECM proteins have been hindered by the insoluble and digestion-resistant nature of ECM. Here we explore the utility of combining rapid ultrasonication- and surfactant-assisted digestion for the detailed proteomics analysis of ECM samples. When compared with traditional overnight digestion, this optimized method dramatically improved the sequence coverage for collagen I, revealed the presence of hundreds of previously unidentified proteins in Matrigel, and identified a protein profile for ECM isolated from rat mammary glands that was substantially different from that found in Matrigel. In a three-dimensional culture assay to investigate epithelial cell-ECM interactions, mammary epithelial cells were found to undergo extensive branching morphogenesis when plated with mammary gland-derived matrix in comparison with Matrigel. Cumulatively these data highlight the tissue-specific nature of ECM composition and function and underscore the need for optimized techniques, such as those described here, for the proteomics characterization of ECM samples.

Original languageEnglish (US)
Pages (from-to)1648-1657
Number of pages10
JournalMolecular and Cellular Proteomics
Volume8
Issue number7
DOIs
StatePublished - 2009
Externally publishedYes

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Proteome
Extracellular Matrix
Digestion
Extracellular Matrix Proteins
Proteomics
Epithelial Cells
Human Mammary Glands
Morphogenesis
Surface-Active Agents
Breast
Collagen
Chemical analysis
Rats
Assays
Proteins
Tissue
matrigel

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Analytical Chemistry

Cite this

An in-solution ultrasonication-assisted digestion method for improved extracellular matrix proteome coverage. / Hansen, Kirk C.; Kiemele, Lauren; Maller, Ori; O'Brien, Jenean; Shankar, Aarthi; Fornetti, Jaime; Schedin, Pepper.

In: Molecular and Cellular Proteomics, Vol. 8, No. 7, 2009, p. 1648-1657.

Research output: Contribution to journalArticle

Hansen, Kirk C. ; Kiemele, Lauren ; Maller, Ori ; O'Brien, Jenean ; Shankar, Aarthi ; Fornetti, Jaime ; Schedin, Pepper. / An in-solution ultrasonication-assisted digestion method for improved extracellular matrix proteome coverage. In: Molecular and Cellular Proteomics. 2009 ; Vol. 8, No. 7. pp. 1648-1657.
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