An In-solution Ultrasonication-assisted Digestion Method for Improved Extracellular Matrix Proteome Coverage

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. Molecular & Cellular Proteomics 8:1648 -1657, 2009. 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 crosslinks (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 ␣1 chains and one ␣2 chain that form a fibrillar network (6). The physical properties of...

Quantification of Extracellular Matrix Proteins from a Rat Lung Scaffold to Provide a Molecular Readout for Tissue Engineering

“…Currently accepted and widely used digestion methods require proteins to be solubilized for bottom-up proteomic analysis (22). Recent papers have reported characterization of the ECM fraction from tissues through the use of strong chaotropes (11,21,(23)(24)(25)(26)(27) or cellular fractionation followed by strong detergent (10,28,29). However, in our experience, these protocols invariably yield various sizes of an insoluble protein-containing pellet when applied to a variety of tissue samples (heart, lung, and mammary gland).…”

“…Maintaining structural rigidity and native microarchitecture through the decellularization process makes an acellular organ a good candidate to serve as a tissue scaffold (19,20). These same characteristics are a central reason why the ECM is challenging to characterize using common bottom-up proteomics approaches (21). Currently accepted and widely used digestion methods require proteins to be solubilized for bottom-up proteomic analysis (22).…”

Comparative secretome analysis of human follicular dermal papilla cells and fibroblasts using shotgun proteomics

“…Table 1 shows a complete list of identified ECM proteins with PHS≥2, based on gene ontology and emPAI-mol%. Most of the ECM proteins identified were skeletal proteins, such as serpin, galectin, collagen, and secreted protein acidic and rich in cysteine (SPARC), which were previously found to be the ECM components of hair follicular fibroblasts (12). There were 28 DPC-specific ECM proteins including transporters (ECM1, A2M, APOA1), enzymes (LOX, PON2, DNASE1L1), and peptidases (C3, C1R, C1S, LTF, HTRA1, MMP2).…”

Detection of Differentially Expressed Basal Cell Proteins by Mass Spectrometry

“…The other general approach to isolate components of the ECM involves using detergents to lyse cells on the surfaces to which they are attached and collect the remaining cell debris for analysis (15). Although progress has been made with respect to the creation of "mass spectrometry-friendly" detergents (16), the use of chemicals for the purpose of protein solubilization is generally not ideal. To overcome these problems, we adapted a fast, simple method of isolating extracellular, transmembrane, and associated proteins (from here on collectively referred to as "basal cell proteins") from cells attached to a solid substrate.…”