Dataset Information

Tracking genome-editing and associated molecular perturbations by SWATH mass spectrometry.

ABSTRACT: Advances in gene editing now allow reverse genetics to be applied to a broad range of biological systems. Ultimately, any modification to coding sequences requires confirmation at the protein level, although immunoblotting is often hampered by antibody quality or availability especially in non-model species. Sequential Window Acquisition of All Theoretical Spectra (SWATH), a mass spectrometry (MS) technology with exceptional quantitative reproducibility and accuracy, offers an ideal alternative for protein-based confirmation. Here, using genome edits in mouse, zebrafish and Bicyclus anynana butterflies produced using either homologous recombination or targeted nucleases, we demonstrate absence of the targeted proteins using SWATH, thus confirming successful editing. We show that SWATH is a robust antibody-independent alternative for monitoring gene editing at the protein level and broadly applicable across diverse organisms and targeted genome manipulation techniques. Moreover, SWATH concomitantly defines the global proteome response in the edited organism, which may provide pertinent biological insights.

SUBMITTER: Lin Q

PROVIDER: S-EPMC6811567 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Tracking genome-editing and associated molecular perturbations by SWATH mass spectrometry.

Lin Qifeng Q Low Larry W L LWL Lau Adam A Chua Esther W L EWL Matsuoka Yuji Y Lian Yilong Y Monteiro Antónia A Tate Stephen S Gunaratne Jayantha J Carney Tom J TJ

Scientific reports 20191023 1

Advances in gene editing now allow reverse genetics to be applied to a broad range of biological systems. Ultimately, any modification to coding sequences requires confirmation at the protein level, although immunoblotting is often hampered by antibody quality or availability especially in non-model species. Sequential Window Acquisition of All Theoretical Spectra (SWATH), a mass spectrometry (MS) technology with exceptional quantitative reproducibility and accuracy, offers an ideal alternative ...[more]

PMID: 31645615

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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.

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.

Dataset Information

Tracking genome-editing and associated molecular perturbations by SWATH mass spectrometry.

Publications

Tracking genome-editing and associated molecular perturbations by SWATH mass spectrometry.

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