SWATH mass spectrometry as a tool for quantitative profiling of the matrisome.
SWATH mass spectrometry as a tool for quantitative profiling of the matrisome.
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DOI：
10.1016/j.jprot.2018.02.026
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发表时间：
2018-10-30
影响因子：
3.3
通讯作者：
Huang PH
中科院分区：
文献类型：
--
作者：
Krasny L;Bland P;Kogata N;Wai P;Howard BA;Natrajan RC;Huang PH
细胞外基质（ECM）和ECM相关蛋白（统称为基质体）的蛋白质组学分析是一项具有挑战性的任务，这是由于这些蛋白质固有的复杂性和不溶性。在这里，我们提出了连续窗口采集的所有理论碎片离子光谱质谱（SWATH MS）作为一种工具，用于定量分析matrisomal蛋白质在非富集和ECM富集组织，而不需要事先分馏。利用光谱库包含201 matrisomal蛋白质，我们比较了性能和重现性的SWATH MS在传统的数据依赖性分析质谱（DDA MS）在普通小鼠肺和肝脏。SWATH MS在两种组织类型的重复实验中赋予了15-20%的可重复肽鉴定增加，并且与DDA MS相比，在肝脏中鉴定了54%以上的基质体蛋白。我们进一步使用SWATH MS来评估伴随ECM富集的基质体含量的定量变化。我们的数据表明，ECM富集导致核心matrisomal蛋白的系统性增加，但导致基质体相关蛋白，包括组织蛋白酶和S100家族蛋白的显着损失。我们的原理验证研究证明了SWATH MS作为深入表征未分级和非富集组织中基质体的通用工具的实用性。基质体是细胞外基质（ECM）和ECM相关蛋白的复杂网络，其为组织提供支架功能并且在基本细胞过程的调节中起重要作用。然而，由于其固有的复杂性和不溶性，基质体的蛋白质组学研究通常需要在MS分析之前应用富集工作流程。这种富集策略通常导致可溶性基质体相关组分的损失。在这项研究中，我们提出了连续窗口采集的所有理论碎片离子光谱质谱（SWATH MS）作为一种工具，用于定量分析基质体蛋白质。我们表明，SWATH MS提供了一个更可重复的覆盖率的基质体相比，数据依赖性分析（DDA）MS。我们还表明，SWATH MS能够准确定量的基质体蛋白，而无需事先ECM富集和分馏，这可能会简化样品处理工作流程，并避免损失的基质体相关蛋白通常与ECM富集。SWATH MS能够定量分析非富集和ECM富集组织中的基质体。SWATH MS提供了一个更可重复的覆盖率相比，DDA MS的基质体。定量分析SWATH MS揭示，肺有一个更高的基质体蛋白质含量比肝脏。比较分析发现，ECM富集导致基质体相关蛋白的显著损失。
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. 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. SWATH MS is capable of quantitative analysis of the matrisome in both non-enriched and ECM enriched tissues. SWATH MS provides a more reproducible coverage of the matrisome compared to DDA MS. Quantitative analysis by SWATH MS reveals that the lung has a higher matrisomal protein content versus the liver. Comparative analysis finds that ECM enrichment leads to significant losses in matrisome-associated proteins.
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影响因子：
4.4
作者：
Barrett, Alexander S.;Wither, Matthew J.;Hansen, Kirk C.
通讯作者：
Hansen, Kirk C.
DOI：
10.1074/mcp.m110.001693
发表时间：
2010-09
期刊：
Molecular & cellular proteomics : MCP
影响因子：
--
作者：
Didangelos A;Yin X;Mandal K;Baumert M;Jahangiri M;Mayr M
通讯作者：
Mayr M
DOI：
10.1111/j.2517-6161.1995.tb02031.x
发表时间：
1995-01-01
影响因子：
5.8
作者：
BENJAMINI, Y;HOCHBERG, Y
通讯作者：
HOCHBERG, Y
DOI：
10.1074/mcp.ra117.000314
发表时间：
2017-12
期刊：
Molecular & cellular proteomics : MCP
影响因子：
--
作者：
Bruderer R;Bernhardt OM;Gandhi T;Xuan Y;Sondermann J;Schmidt M;Gomez-Varela D;Reiter L
通讯作者：
Reiter L
影响因子：
4.4
作者：
Muntel J;Xuan Y;Berger ST;Reiter L;Bachur R;Kentsis A;Steen H
通讯作者：
Steen H