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. 2018 Oct 30:189:11-22.
doi: 10.1016/j.jprot.2018.02.026. Epub 2018 Mar 1.

SWATH mass spectrometry as a tool for quantitative profiling of the matrisome

Lukas Krasny  1 , Philip Bland  2 , Naoko Kogata  2 , Patty Wai  3 , Beatrice A Howard  2 , Rachael C Natrajan  3 , Paul H Huang  4
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SWATH mass spectrometry as a tool for quantitative profiling of the matrisome (VSports)

V体育安卓版 - Lukas Krasny et al. J Proteomics. .

"V体育官网入口" 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 VSports手机版. 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. SIGNIFICANCE: The 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. .

Keywords: DIA MS; Extracellular matrix; Mass spectrometry; Matrisome; Proteomics; SWATH MS. V体育安卓版.

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Fig. 1
Fig. 1
Schematic of the experimental workflow employed in this study. Key steps that were undertaken for the generation of the spectral library (blue) and sample processing, data acquisition and analysis (orange) in this study are highlighted. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2
Fig. 2
Comparative analysis of the performance of SWATH MS versus DDA MS. Venn diagrams depicting number and overlap of matrisomal proteins detected by DDA and SWATH MS in A) liver and B) lung tissue across 2 biological replicates and 3 technical replicates. Proteins uniquely identified by each method are highlighted. Venn diagrams depicting the number and overlap of unique matrisome peptides identified in C) liver and D) lung tissue across 3 technical replicates (R1-R3) in DDA and SWATH MS. Only peptides with 99% confidence of identification or higher were compared.
Fig. 3
Fig. 3
Comparative analysis of the liver and lung matrisome by SWATH MS. A) Venn diagram depicting the number and overlap of unique matrisomal proteins quantified by SWATH MS in liver and lung tissue across 6 biological replicates. B) A heatmap of the 90 common matrisomal proteins that were quantified in both liver and lung tissue (n = 6). Samples were subjected to two-way hierarchical clustering based on Euclidian distance. Relative quantification of C) Collagen IV and D) Galectin-9 in liver and lung tissue across the 6 biological replicates. Mean Log2 signal peak area from 3 technical replicates of each sample is shown with the standard deviation.
Fig. 4
Fig. 4
Effect of ECM enrichment on the matrisomal content in liver and lung as measured by SWATH MS. Venn diagram depicting the number and overlap of unique matrisomal proteins in ECM enriched versus non-enriched samples in A) liver and B) lung tissue across 6 biological replicates. Heatmaps of the common matrisomal proteins that were quantified in both ECM enriched and non-enriched samples in C) liver and D) lung tissue (n = 6). Samples were subjected to two way hierarchical clustering based on Euclidian distance. Core matrisomal proteins (black text) and matrisome associated proteins (orange text) are highlighted to illustrate gains in core matrisome proteins and losses in matrisome-associated proteins as a result of ECM enrichment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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