An in-solution ultrasonication-assisted digestion method for improved extracellular matrix proteome coverage.
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.
University of Colorado Cancer Center Proteomics and Mass Spectrometry Facility, Department of Pediatrics, University of Colorado Denver, Aurora, Colorado 80045, USA. kirk.hansen@UCDenver.edu
Kirk C
KC
Hansen
Lauren
L
Kiemele
Ori
O
Maller
Jenean
J
O'Brien
Aarthi
A
Shankar
Jaime
J
Fornetti
Pepper
P
Schedin
D000818:Animals; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000737:chemistry* / Q000378:metabolism; D005260:Female; D008321:Mammary Glands, Animal / Q000737:chemistry / Q000166:cytology; D008969:Molecular Sequence Data; D020543:Proteome / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012996:Solutions / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods; D014465:Ultrasonics
D016326:Extracellular Matrix Proteins; D020543:Proteome; D012996:Solutions