An In-solution Ultrasonication-assisted Digestion Method for Improved
Extracellular Matrix Proteome Coverage |
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Authors: | Kirk C Hansen Lauren Kiemele Ori Maller Jenean O'Brien Aarthi Shankar Jaime Fornetti and Pepper Schedin |
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Institution: | From the ‡University of Colorado Cancer Centerx Proteomics and Mass Spectrometry Facility, ;Departments of §Pediatrics and ;**Medicine, ;‖Cancer Biology Program, ;‡‡Reproductive Sciences Program, and ;**Anschutz Medical Campus (AMC) Cancer Research Center, University of Colorado Denver, Aurora, Colorado 80045 |
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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.Extracellular matrix (ECM)1 is a critical component of the tissue microenvironment. ECM plays a pivotal role
in embryonic stem cell development and differentiation (1, 2) as well as many physiological (3) and pathological processes, including cancer
progression (4, 5). Cell regulation by ECM has been studied with high frequency in recent years
(7, 8).
However, our ability to globally characterize ECM composition both in
vitro and in vivo has been severely limited because of several
unique attributes of ECM proteins such as high molecular weight glycans and the presence
of covalent protein cross-links (6, 9, 10).
Traditional proteomics approaches have proven to be ineffective for the identification
of ECM proteins as demonstrated by the fact that collagens, despite being the most
abundant protein in mammals, are significantly underrepresented in tissue-based
proteomics data sets.Ultrasonication has long been used for the digestion of bioorganic materials to allow for
maximal and reproducible extraction and hence the accurate identification of small
molecule and inorganic analytes (11). More
recently, Capelo et al. (12)
have used ultrasonic energy to catalyze tryptic digestion of proteins for subsequent
mass spectrometry-based identification. Here we sought to determine whether this method
could be optimized to prepare ECM samples for mass spectrometry-based analysis. For
method development, we used rat tail collagen as a representative ECM protein for which
current proteomics approaches have proven relatively unsuccessful. Type I collagen is
defined as a right-handed triple helix heterotrimer comprising two identical
α1 chains and one α2 chain that form a fibrillar network (6). The physical properties of the triple helical
structure render the protein resistant to proteasch as trypsin (9). In this work, we focused our efforts on developing a digestion
approach that improves our ability to perform proteomics analysis on a type I collagen
preparation and then used this method to identify the protein composition of EHS murine
chondrosarcoma matrix (10), herein referred to as
Matrigel, and a matrix preparation from rat mammary tissue.In this study, we developed a digestion approach suitable for a two-dimensional liquid
chromatography-tandem mass spectrometry-based analysis of ECM proteins. Our digestion
approach involves three cycles of ultrasonication for rapid initial trypsin digestion
followed by overnight digestion using an acid-labile surfactant. This approach resulted
in significant improvement in collagen peptide identification and the identification of
numerous ECM proteins previously uncharacterized in Matrigel and in mammary tissue. The
application of our ECM-optimized ultrasonic assisted trypsin digestion method is
anticipated to significantly advance the identification of tissue- and disease
state-specific ECM proteins. |
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