Abstract
Proteomic analysis of extracellular matrix (ECM) and ECM-associated proteins, collectively known as the matrisome, is a challenging task due to the inherent complexity and insolubility of these proteins. Here we present sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH MS) as a tool for the quantitative analysis of matrisomal proteins in both non-enriched and ECM enriched tissue without the need for prior fractionation. Utilising a spectral library containing 201 matrisomal proteins, we compared the performance and reproducibility of SWATH MS over conventional data-dependent analysis mass spectrometry (DDA MS) in unfractionated murine lung and liver. SWATH MS conferred a 15–20% increase in reproducible peptide identification across replicate experiments in both tissue types and identified 54% more matrisomal proteins in the liver versus DDA MS. We further use SWATH MS to evaluate the quantitative changes in matrisome content that accompanies ECM enrichment. Our data shows that ECM enrichment led to a systematic increase in core matrisomal proteins but resulted in significant losses in matrisome-associated proteins including the cathepsins and proteins of the S100 family. Our proof-of-principle study demonstrates the utility of SWATH MS as a versatile tool for in-depth characterisation of the matrisome in unfractionated and non-enriched tissues. SignificanceThe matrisome is a complex network of extracellular matrix (ECM) and ECM-associated proteins that provides scaffolding function to tissues and plays important roles in the regulation of fundamental cellular processes. However, due to its inherent complexity and insolubility, proteomic studies of the matrisome typically require the application of enrichment workflows prior to MS analysis. Such enrichment strategies often lead to losses in soluble matrisome-associated components. In this study, we present sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH MS) as a tool for the quantitative analysis of matrisomal proteins. We show that SWATH MS provides a more reproducible coverage of the matrisome compared to data-dependent analysis (DDA) MS. We also demonstrate that SWATH MS is capable of accurate quantification of matrisomal proteins without prior ECM enrichment and fractionation, which may simplify sample handling workflows and avoid losses in matrisome-associated proteins commonly linked to ECM enrichment.
Highlights
The extracellular matrix (ECM) is a complex network of proteins whose primary scaffolding function confers integrity and elasticity to tissues and organs [1]
Tissue homogenates were subjected to Strong cation exchange (SCX) fractionation in 2 biological replicates and each of the 12 fractions run in dependent analysis (DDA) mode with spiked indexed retention time calibration peptides
While dependent analysis mass spectrometry (DDA mass spectrometry (MS)) has been the foundation of modern proteomics for more than two decades, we show that SWATH MS confers three major advantages in the characterisation of the matrisome
Summary
The extracellular matrix (ECM) is a complex network of proteins whose primary scaffolding function confers integrity and elasticity to tissues and organs [1]. To address this challenge, an effort to integrate proteomic and genomic datasets has led to the generation of a database of ECM and ECM-associated proteins known as the in-silico matrisome [9,10]. An effort to integrate proteomic and genomic datasets has led to the generation of a database of ECM and ECM-associated proteins known as the in-silico matrisome [9,10] This matrisome database is divided into the “core matrisome” which consists of glycoproteins, collagens and proteoglycans subclasses, and the “matrisome-associated proteins” that comprise affiliated proteins, regulators and secreted factors [10]. It should be noted that the matrisome database was designed to be inclusive and contains
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