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. 2017 Aug 4;16(8):3083-3091.
doi: 10.1021/acs.jproteome.7b00191. Epub 2017 Jul 19.

Characterization of the Extracellular Matrix of Normal and Diseased Tissues Using Proteomics

Alexandra Naba  1 , Oliver M T Pearce  2 , Amanda Del Rosario  3 , Duanduan Ma  4 , Huiming Ding  4 , Vinothini Rajeeve  2 , Pedro R Cutillas  2 , Frances R Balkwill  2 , Richard O Hynes  1   5
Affiliations

Characterization of the Extracellular Matrix of Normal and Diseased Tissues Using Proteomics

Alexandra Naba et al. J Proteome Res. .

Abstract

The extracellular matrix (ECM) is a complex meshwork of insoluble fibrillar proteins and signaling factors interacting together to provide architectural and instructional cues to the surrounding cells. Alterations in ECM organization or composition and excessive ECM deposition have been observed in diseases such as fibrosis, cardiovascular diseases, and cancer. We provide here optimized protocols to solubilize ECM proteins from normal or tumor tissues, digest the proteins into peptides, analyze ECM peptides by mass spectrometry, and interpret the mass spectrometric data. In addition, we present here two novel R-script-based web tools allowing rapid annotation and relative quantification of ECM proteins, peptides, and intensity/abundance in mass spectrometric data output files. We illustrate this protocol with ECMs obtained from two pairs of tissues, which differ in ECM content and cellularity: triple-negative breast cancer and adjacent mammary tissue, and omental metastasis from high-grade serous ovarian cancer and normal omentum. The complete proteomics data set generated in this study has been deposited to the public repository ProteomeXchange with the data set identifier: PXD005554.

Keywords: collagens; extracellular matrix; hydroxylation; mass-spectrometry-based proteomics; matrisome; microenvironment.

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Figures

Figure 1.
Figure 1.
Experimental pipeline. (A) Masson’s trichrome staining (blue) highlight fibrillar collagen content of the four tissues profiled in this study. (B) Experimental pipeline. A description of the samples analyzed in this study is presented in Supplementary Table S1A.
Figure 2.
Figure 2.
Comparison of matrisome and non-matrisome proteins identified in urea-soluble versus crude ECM samples. Bar charts represent the distribution of matrisome (blue) versus non-matrisome (gray) proteins in terms of peptide abundance (upper panels), number of spectra (middle panels), and number of proteins (lower panels) in urea-soluble or crude ECM extracts from normal omental tissue and omental metastases from ovarian tumors (A) and normal breast tissues and triple-negative breast tumors (B). Two independent samples were compared for each tissue and methodology. Related to Supplementary Table S1B,C.
Figure 3.
Figure 3.
Peptide fractionation increases the number of ECM and ECM-associated proteins identified by mass spectrometry in both urea-soluble and crude ECM samples. (A) Bar charts represent the number of matrisome proteins identified proteins in urea-soluble or ECM samples from normal omentum (left) or omental metastasis from ovarian cancer (right) fractionated or not by basic-reverse phase liquid chromatography prior to LC−MS/MS. (Bar charts represent the number of identified spectra corresponding to core matrisome (B) or matrisome-associated (C) proteins in urea-soluble or ECM samples from normal omentum (left) or omental metastasis from ovarian cancer (right) fractionated or not by basic-reverse phase liquid chromatography prior to LC−MS/MS. Related to Supplementary Table S1C,D.
Figure 4.
Figure 4.
Importance of allowing hydroxylation of prolines when searching databases to identify and quantify ECM proteins. Bar charts represent the number of spectra (A), peptide abundance (B), and number of proteins (C) for matrisome (left) and non-matrisome (right) proteins identified by searching the data allowing or not for prolines or lysines hydroxylations as dynamic modifications. Post-fractionation data to conduct this comparative analysis were acquired on urea-soluble or crude ECM extracts from omental metastasis from high-grade-serous ovarian cancer. Related to Supplementary Table S1E,F.
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