Dataset Information

Precise Temporal Profiling of Signaling Complexes in Primary Cells Using SWATH Mass Spectrometry.

ABSTRACT: Spatiotemporal organization of protein interactions in cell signaling is a fundamental process that drives cellular functions. Given differential protein expression across tissues and developmental stages, the architecture and dynamics of signaling interaction proteomes is, likely, highly context dependent. However, current interaction information has been almost exclusively obtained from transformed cells. In this study, we applied an advanced and robust workflow combining mouse genetics and affinity purification (AP)-SWATH mass spectrometry to profile the dynamics of 53 high-confidence protein interactions in primary T cells, using the scaffold protein GRB2 as a model. The workflow also provided a sufficient level of robustness to pinpoint differential interaction dynamics between two similar, but functionally distinct, primary T cell populations. Altogether, we demonstrated that precise and reproducible quantitative measurements of protein interaction dynamics can be achieved in primary cells isolated from mammalian tissues, allowing resolution of the tissue-specific context of cell-signaling events.

SUBMITTER: Caron E

PROVIDER: S-EPMC5382234 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Precise Temporal Profiling of Signaling Complexes in Primary Cells Using SWATH Mass Spectrometry.

Caron Etienne E Roncagalli Romain R Hase Takeshi T Wolski Witold E WE Choi Meena M Menoita Marisa G MG Durand Stephane S García-Blesa Antonio A Fierro-Monti Ivo I Sajic Tatjana T Heusel Moritz M Weiss Tobias T Malissen Marie M Schlapbach Ralph R Collins Ben C BC Ghosh Samik S Kitano Hiroaki H Aebersold Ruedi R Malissen Bernard B Gstaiger Matthias M

Cell reports 20170301 13

Spatiotemporal organization of protein interactions in cell signaling is a fundamental process that drives cellular functions. Given differential protein expression across tissues and developmental stages, the architecture and dynamics of signaling interaction proteomes is, likely, highly context dependent. However, current interaction information has been almost exclusively obtained from transformed cells. In this study, we applied an advanced and robust workflow combining mouse genetics and af ...[more]

PMID: 28355572

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Project description: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. 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.

Project description:The lack of early diagnostic maneuvers and effective pharmacotherapy for osteoarthritis (OA) is predominantly attributed to current limited understanding of its pathogenesis. In the present study, the alteration of synovial fluid (SF) proteome in OA were analyzed using SWATH‑mass spectrometry (SWATH‑MS) to further elucidate the pathogenesis of OA. SF samples were collected from 10 OA and 10 rheumatoid arthritis (RA) patients undergoing arthroplasty and 10 patients undergoing arthroscopy for traumatic arthritis (meniscus injury without cartilage lesion). According to the Kellgren‑Lawrence (KL) radiographic grading criteria, all the OA and RA patients were classified as KL grade 4. SWATH‑MS was applied to identify differentially expressed proteins specifically regulated in OA. Differentially expressed proteins identified by SWATH‑MS were subjected to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation. Proteins of interest were quantified by enzyme‑linked immunosorbent assay (ELISA) following identification. With the use of SWATH‑MS, 131 proteins were identified to be differentially expressed in OA, of which 93 corresponded to upregulation and 38 to downregulation. Complement C1r was the most significantly upregulated protein in OA. Twenty‑eight out of the 131 proteins were specifically regulated in OA, of which 17 were upregulated and 11 were downregulated. Dickkopf‑related protein 2 (DKK2) was one of the proteins specifically upregulated in OA. Furthermore, KEGG pathway annotation indicated that differentially expressed proteins in OA were enriched in complement and coagulation cascades. ELISA indicated that OA severity was positively correlated with the levels of complement C1r (r=0.549; P<0.001) and DKK2 (r=0.79; P<0.001) in the SF. The results indicate that complement and coagulation cascades are involved in the pathogenesis of OA. Differentially expressed proteins, including complement C1r and DKK2 may be used as potential biomarkers or drug targets, which may facilitate with intervention of OA.

Dataset Information

Precise Temporal Profiling of Signaling Complexes in Primary Cells Using SWATH Mass Spectrometry.

Publications

Precise Temporal Profiling of Signaling Complexes in Primary Cells Using SWATH Mass Spectrometry.

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