Matrisome and Extracellular Matrix (ECM) Proteomics Service

Sequential Chemical Extraction · Insoluble Matrix Solubilization · Fibrosis & Tumor Desmoplasia

Conventional whole-tissue workflows can under-represent highly crosslinked, insoluble, or heavily glycosylated extracellular matrix proteins. Creative Proteomics provides a specialized Matrisome & ECM Proteomics Service using fit-for-purpose sequential extraction, optional enzymatic deglycosylation, and DIA mass spectrometry to quantify core matrisome components and matrix-associated regulators.

  • 4-step sequential extraction: Solubilizes crosslinked fibrillar collagens without pellet protein loss
  • In Silico Matrisome annotation: Quantifies Core Matrisome (collagens, glycoproteins, proteoglycans) and Matrisome-Associated factors
  • Deglycosylation pre-treatment: PNGase F and Chondroitinase ABC can improve peptide recovery from selected proteoglycan-rich samples
  • Translational disease applications: Fibrotic liver (MASH), lung (IPF), desmoplastic tumor stroma (PDAC, TNBC), and decellularized scaffolds

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What Is Matrisome and ECM Proteomics?

Matrisome and extracellular matrix (ECM) proteomics applies specialized sample preparation and LC-MS/MS to quantitatively characterize structural ECM proteins and matrix-associated regulators. It is designed for fibrotic tissue, tumor stroma, and decellularized scaffolds, where crosslinked or glycosylated matrix proteins can be under-represented by conventional workflows.

Our DIA quantitative proteomics workflow integrates sample-appropriate fractionation, solubilization, and, where appropriate, deglycosylation to improve recovery of collagens, glycoproteins, proteoglycans, and ECM regulators. Results are delivered as relative-abundance and fraction-specific matrisome profiles to support mechanism-focused studies in fibrosis, oncology, and regenerative medicine.

Content Guide

  • Matrisome Classification
  • Sequential Extraction SOP
  • Disease Applications
  • Workflow
  • Platform Selection
  • Sample Submission
  • Deliverables

Matrisome Classification: Core Matrisome vs. Matrisome-Associated Regulators

Proteins are mapped directly against the In Silico Matrisome Project framework:

1. Collagens (Core Matrisome)

All 44 human collagen chains: fibrillar types (COL1A1/A2, COL3A1, COL5A1), basement membrane networks (COL4A1–A6), and microfibrillar collagens (COL6A1–A3).

2. ECM Glycoproteins (Core Matrisome)

Multi-domain adhesive proteins: Fibronectin (FN1), Laminins (LAMA/B/C), Tenascins (TNC, TNXB), Fibrillins (FBN1/2), Periostin (POSTN), and Vitronectin (VTN).

3. Proteoglycans (Core Matrisome)

Compressive and hydrating matrix components: Aggrecan (ACAN), Versican (VCAN), Decorin (DCN), Biglycan (BGN), Lumican (LUM), and Perlecan (HSPG2).

4. ECM Regulators & Growth Factors

Crosslinkers (LOX, LOXL1–4, TGM2), proteases (MMP1/2/9/14, ADAMTS), inhibitors (TIMP1–4), and sequestered ligands (TGF-β, BMPs, VEGFs).

Pre-Analytical Protocol: 4-Step Sequential Chemical Extraction

To enrich insoluble matrix components while reducing cellular protein masking, we use a sequential fractionation workflow that is selected during project feasibility review:

Matrisome sequential extraction protocol
  • Fraction 1 (Cytosolic Depletion): Hypotonic buffer (0.5 M NaCl + 0.1% Triton X-100) washes away soluble intracellular proteins while preserving the intact matrix scaffold.
  • Fraction 2 (Membrane Clearance): Mild 0.1% SDS incubation clears organellar debris and cellular chaperones, yielding an enriched decellularized matrix pellet.
  • Fraction 3 (Chaotropic Extraction): 4M Guanidine-HCl + reducing agents solubilizes non-covalently crosslinked glycoproteins and matrisome-associated factors.
  • Fraction 4 (Core Pellet Solubilization & Deglycosylation): The insoluble crosslinked pellet is treated with PNGase F and Chondroitinase ABC to strip GAG chains, followed by high-temperature S-Trap enzymatic digestion into tryptic peptides.

Translational Disease Applications: Fibrosis, Desmoplasia, and Biomaterials

Application Model Systems Analytical Goal Key Molecular Readouts
Fibrotic Disease Profiling MASH/NASH liver, IPF lung, renal interstitial fibrosis. Quantify collagen accumulation and relative abundance patterns of crosslinking regulators. COL1A1/COL3A1 ratios, LOXL2, Periostin, Tenascin-C, Fibronectin ED-A/B.
Tumor Stroma & Desmoplasia Pancreatic ductal adenocarcinoma (PDAC), TNBC, CAF co-cultures. Characterize ECM features associated with T-cell exclusion and reduced therapeutic penetration. Versican (VCAN), basement membrane remodelers (COL4/COL6), MMP/TIMP ratios.
Decellularized Scaffolds (dECM) Tissue engineering matrices, hydrogels, cardiac patches. Verify cellular antigen clearance while confirming matrix protein retention. Relative depletion of cellular marker proteins alongside retention profiles for structural laminins and collagens.
Anti-Fibrotic Drug Screening Fibrotic tissues treated with LOX inhibitors, TGF-β blockers, Pirfenidone. Measure treatment-associated changes in matrix deposition and crosslinking-regulator abundance. Dose-associated changes in LOX/TGM2 abundance and ECM remodeling markers.

Step-by-Step Matrisome Proteomics Workflow

1
Study Scoping & Tissue Dissection

Define tissue cohorts (healthy vs. fibrotic, stroma vs. tumor core) and anti-fibrotic treatment arms.

2
4-Step Sequential Extraction

Execute hypotonic cellular depletion, 0.1% SDS wash, 4M Guanidine-HCl extraction, and core pellet recovery.

3
Deglycosylation & S-Trap Digestion

Apply selected deglycosylation steps when compatible with the sample type, followed by S-Trap trypsin/Lys-C digestion.

4
LC-MS/MS Acquisition

High-depth single-shot DIA on Orbitrap Astral or 4D-DIA (dia-PASeF on timsTOF Pro 2) with pooled QC monitoring.

5
Matrisome Project Annotation

Automated classification into Core Matrisome (Collagens, Glycoproteins, Proteoglycans) vs. Associated factors.

6
Statistical & Pathway Reporting

Matrix subcategory breakdown, crosslinking network mapping, GSEA fibrosis scoring, and PRM validation shortlists.

Study Design
Cohort & tissue definition
Fractionation
4-step chemical extraction
Deglycosylation
PNGase F & S-Trap digestion
DIA / 4D-MS
timsTOF / Orbitrap with pooled QC
QC & Annotation
Matrisome Project classification
Matrisome Report
Composition & fibrosis score
  • Insoluble matrix recovery: 4-step chemical extraction captures crosslinked collagens without pellet loss
  • Matrisome Project classification: direct mapping of Core Matrisome and Matrisome-Associated factors
  • Enzymatic deglycosylation: PNGase F and Chondroitinase ABC pre-treatment ensures deep proteoglycan coverage
  • Decellularization validation: quantitative confirmation of cellular antigen removal for biomaterial scaffolds
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Matrisome Proteomics Analytical Platform Decision Guide

Study Goal Strategy Primary Platform Deliverable Scope
Bulk Matrisome Discovery
(Fibrotic Liver / Lung / Stroma)		 Discovery DIA Proteomics Orbitrap Astral / Exploris 480 / timsTOF Pro 2 Fit-for-purpose DIA depth and quantitative precision for qualified tissue cohorts; expected coverage and CV are confirmed during feasibility review.
Microscale & Needle Biopsies
(Core Biopsies / LCM Stroma)		 High-Sensitivity 4D-DIA timsTOF Pro 2 (dia-PASeF) / timsTOF Ultra TIMS ion mobility supports limited tissue input and microdissected stroma projects; expected coverage depends on tissue region, extraction yield, and matrix composition.
Decellularized Scaffolds (dECM)
(Hydrogels / Biomaterials)		 dECM Quality & Purity DIA Orbitrap Exploris 480 / timsTOF Pro 2 Profiles structural matrisome retention and relative depletion of cellular protein markers; pair with DNA and histology controls when qualifying decellularization.
Mechanosensing Signaling
(Integrin-FAK-YAP Kinetics)		 4D Phosphoproteomics timsTOF Pro 2 / Orbitrap Exploris 480 Provides high-coverage phosphoproteomics for integrin-FAK-YAP pathway investigation; achievable phosphosite coverage depends on input and enrichment performance.
Targeted Fibrosis Biomarkers
(LOX, MMPs, Collagen Turnover)		 Targeted PRM Panels Orbitrap PRM / Triple Quadrupole Absolute quantification can be configured for selected matrisome biomarkers when suitable internal standards and assay performance criteria are established.

Sample Submission Requirements and Guidelines

Sample Type Recommended Input Minimum Input Handling & Shipping
Fresh-Frozen Fibrotic Tissue 30–50 mg wet weight 10 mg wet weight Dissect rapidly; rinse excess blood in cold saline; snap-freeze in liquid N2. Ship on dry ice (-80°C).
Tumor Stroma / Margin Tissue 30–50 mg wet weight 10 mg wet weight Dissect tumor vs. margin; snap-freeze immediately in liquid N2. Ship on dry ice.
Decellularized Scaffolds (dECM) 10–20 mg dry / wet weight 5 mg wet weight Rinse thoroughly with sterile deionized water to remove detergents; snap-freeze. Ship on dry ice.
3D Organoid / Hydrogel Cultures 50–100 organoids in dome 20 organoids Recover domes with cell recovery solution or freeze intact dome in liquid N2. Ship on dry ice.
Pre-Extracted Fractions 20–50 μL at 1–2 mg/mL 10 μg total protein Document extraction buffer composition (Guanidine/Urea). Ship on dry ice.

Deliverables and Decision-Ready Outputs

Quantitative matrisome matrices, Matrisome Project classifications, and fibrosis scorecards

PCA clustering of healthy, fibrotic, and anti-fibrotic treated tissue matrisomes

Sample-level PCA evaluates whether matrisome composition clusters strictly by disease stage (early vs. late fibrosis).

Heatmap of Core Matrisome and Matrisome-Associated factors across cohorts

Hierarchical clustering resolves distinct matrix subcategories (collagens, proteoglycans, glycoproteins) across groups.

Volcano plot of differential matrisome protein abundance in fibrosis

Differential volcano plots prioritize statistically significant matrix crosslinking enzymes (LOX/LOXL2), MMPs, and collagen chains.

Matrisome classification pie chart and integrin signaling network

Matrisome division breakdown and integrin-FAK mechanotransduction networks map biomechanical remodeling pathways.

Discuss Your Project

Classified Matrisome Intensity Tables

  • Normalized protein- and peptide-level matrices annotated by Matrisome Project divisions (Core vs. Associated).

Quality & Decellularization Summary

  • Extraction efficiency metrics, digestion completeness, pooled QC CV distributions, and intracellular protein clearance reports.

Core-to-Associated Ratio Metrics

  • Quantitative ratios of fibrillar collagens (COL1/COL3), crosslinking-regulator abundance patterns, and fraction-specific matrix remodeling profiles.

Pathway & Mechanotransduction Networks

  • GSEA fibrosis pathway enrichment, Integrin-FAK-YAP/TAZ mechanotransduction signaling, and pre-metastatic niche scorecards.

Comprehensive Final Report

  • Publication-ready figures, auditable methods documentation, and candidate shortlists for targeted PRM validation.

Matrisome & ECM Proteomics Frequently Asked Questions

Why do standard cell lysis buffers (RIPA, 1% SDS) fail to solubilize ECM proteins?
Core matrisome proteins—particularly fibrillar and basement membrane collagens—can form covalently crosslinked and highly glycosylated networks. Mild lysis conditions may therefore under-recover parts of the insoluble matrix fraction. Our sequential extraction workflow uses chaotropic solubilization, reducing conditions, and selected deglycosylation steps to improve recovery of matrix proteins; the final protocol is matched to the sample type and study objective.
How do you classify identified proteins into the Matrisome database?
We map all identified and quantified protein accessions directly against the curated In Silico Matrisome Project database (Naba et al.). Identified proteins are systematically categorized into: (1) Core Matrisome, comprising 44 collagen alpha chains, ECM Glycoproteins (~200 proteins, e.g., FN1, Laminins, Tenascins), and Proteoglycans (~35 proteins, e.g., ACAN, VCAN, DCN); and (2) Matrisome-Associated, comprising ECM Regulators (LOX/LOXL, MMPs, TIMPs, ADAMTS), ECM-Affiliated proteins (Annexins, Galectins), and Secreted Factors (TGF-β, BMPs, Wnts).
Why is enzymatic deglycosylation (PNGase F & Chondroitinase ABC) required prior to mass spectrometry?
Proteoglycans such as Versican, Aggrecan, Decorin, and Lumican can carry glycosaminoglycan chains and glycans that reduce trypsin accessibility and complicate mass spectrometry analysis. PNGase F removes N-glycans, while Chondroitinase ABC digests chondroitin and dermatan sulfate chains. These steps can improve peptide recovery for suitable samples, but they do not establish complete digestion or coverage of every glycan and GAG class.
Can matrisome proteomics evaluate decellularized ECM (dECM) scaffolds for tissue engineering?
Yes. DIA proteomics can compare residual cellular protein markers with structural matrisome proteins in decellularized ECM scaffolds, supporting assessment of relative cellular-component removal and matrix retention. These protein-level profiles should be interpreted alongside established decellularization controls such as residual dsDNA measurement and histologic assessment; proteomics alone does not qualify a scaffold as decellularized.
How does matrisome proteomics profile tumor desmoplasia and immune exclusion in cancer?
In desmoplastic tumors such as pancreatic ductal adenocarcinoma and breast cancer, cancer-associated fibroblasts can produce collagen-rich stroma associated with altered immune-cell access and therapeutic penetration. We quantify ECM signatures including Periostin, Tenascin-C, collagen ratios, and LOXL2 to evaluate treatment-associated matrix changes. These data support mechanism hypotheses and prioritization; they do not alone establish restored anti-tumor immunity.
What is the minimum tissue input required for matrisome sequential extraction?
For standard bulk fibrotic tissue, we recommend submitting 30–50 mg wet weight tissue; 10 mg can be considered when the tissue type and extraction yield are suitable. Clinical needle cores and microdissected stroma sections can also be assessed through a feasibility review. Coverage from limited material is sample-dependent and should be defined against the study objective before analysis.
How do you evaluate collagen crosslinking and turnover in fibrosis drug screening?
We quantify structural collagen chains and crosslinking-regulator abundance, including LOX, LOXL1–4, TGM2, and P4HA1/2. Differential extraction can compare operationally defined soluble and insoluble matrix fractions and identify treatment-associated shifts in matrix composition. Direct measurement of collagen crosslinks, synthesis rates, or turnover requires dedicated crosslink assays or stable-isotope experimental designs.
What sample submission format and project metadata are required?
Tissue samples should be submitted as flash-frozen pieces (≥30–50 mg, rinsed briefly in ice-cold saline to remove excess blood) shipped on dry ice (-80°C). For decellularized biomaterial scaffolds, submit 10–20 mg dry/wet weight rinsed thoroughly with sterile deionized water. Please provide project metadata including: (1) tissue type and species (human/mouse/rat/pig), (2) disease model (MASH, IPF, tumor stroma, dECM scaffold), (3) control conditions (healthy/sham), and (4) requested analytical layers (matrisome DIA, 4D-phosphoproteomics, PRM validation).
* For Research Use Only. Not for use in the treatment or diagnosis of disease.

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