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Analyzing trapped protein complexes by Virotrap and SFINX

Nature Protocols volume 12, pages 881–898 (2017)Cite this article

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Abstract

The analysis of protein interaction networks is one of the key challenges in the study of biology. It connects genotypes to phenotypes, and disruption of such networks is associated with many pathologies. Virtually all the approaches to the study of protein complexes require cell lysis, a dramatic step that obliterates cellular integrity and profoundly affects protein interactions. This protocol starts with Virotrap, a novel approach that avoids the need for cell homogenization by fusing the protein of interest to the HIV-1 Gag protein, trapping protein complexes in virus-like particles. By using the straightforward filtering index (SFINX), which is a powerful and intuitive online tool (http://sfinx.ugent.be) that enables contaminant removal from candidate lists resulting from mass-spectrometry-based analysis, we provide a complete workflow for researchers interested in mammalian protein complexes. Given direct access to mass spectrometers, researchers can process up to 24 samples in 7 d.

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Figure 1: General overview of the protocol.
Figure 2: Light microscopy images of HEKT293T cell density at different time points in the Virotrap protocol.
Figure 3: Fluorescence microscopy image of HEK293T cells transfected with pMET7-GAG-EGFP.
Figure 4: Chromatogram of eluted peptides.

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Acknowledgements

K.T. is a PhD student with the Agency for Innovation by Science and Technology (IWT). K.G. acknowledges support from the Fund for Scientific Research-Flanders (FWO-Vlaanderen, grant G011312N) and the Ghent University Concerted Research Actions (grant BOF14/GOA/013). J.T. was supported by grants from IUAP P6/36, the GROUP-ID MRP-UGent and the Fund for Scientific Research-Flanders (grants G.0747.10N and G.0864.10), and is a recipient of a European Research Council (ERC) Advanced Grant (Cytokine Receptor Signaling Revisited, 340941). S.E. was supported by a Methusalem grant to J.T.

Author information

Author notes

  1. Kevin Titeca and Emmy Van Quickelberghe: These authors contributed equally to this work.

Authors and Affiliations

  1. VIB-UGent Center for Medical Biotechnology, Ghent, Belgium

    Kevin Titeca, Emmy Van Quickelberghe, Noortje Samyn, Delphine De Sutter, Annick Verhee, Kris Gevaert, Jan Tavernier & Sven Eyckerman

  2. Department of Biochemistry, Ghent University, Ghent, Belgium

    Kevin Titeca, Emmy Van Quickelberghe, Noortje Samyn, Delphine De Sutter, Annick Verhee, Kris Gevaert, Jan Tavernier & Sven Eyckerman

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Contributions

The Virotrap protocol was developed by S.E., E.V.Q. and K.T. with excellent assistance from N.S., A.V. and D.D.S., and support and advice from K.G. and J.T. SFINX was developed by K.T. under the guidance of J.T. and S.E. The manuscript was written by K.T., E.V.Q. and S.E., with corrections from K.G. and J.T.

Corresponding author

Correspondence to Sven Eyckerman.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Home page of the SFINX web site interface.

(a) Circular SFINX button. (b) Tabs to navigate through the website.

Supplementary Figure 2 Analysis page of the SFINX web site interface before data input.

(a) Left panel with input possibilities. (b) Right panel with short explanation. (c) Check box for the use of example files. (d) Button to browse computer and upload personal “Basic data” file. (e) Button to browse computer and upload personal “Bait Identification” file. (f) Features to specify the use of alternative input formats for the “Basic data” file. (g) Features to specify the use of alternative input formats for the “Bait Identities” file.

Supplementary Figure 3 Filtered interactions tab of the analysis page of the SFINX web site interface after data input.

(a) Circular SFINX button. (b) Tabs to navigate through the website. (a) Results display. (b) Slider bar to determine the level of strictness. (c) Alternative result tabs in the analysis page. (d) Tabular display of the detailed results of the SFINX filtering. (e) Additional information about the quality of the data and results, and buttons for downloading of the filtered interactions. (f) Toggle for the amount of displayed detailed interactions. (g) General search bar for all the detailed results. (h) Search bars specific for each column of the detailed results table.

Supplementary Figure 4 Distribution tab of the analysis page of the SFINX web site interface after data input.

(a) Distribution display. (b) Indicator of the cut-off position.

Supplementary Figure 5 Bait data tab of the analysis page of the SFINX web site interface after data input.

(a) Tabular display of the original “Bait Identities” file as observed by SFINX.

Supplementary Figure 6 Original data tab of the analysis page of the SFINX web site interface after data input.

(a) Limited tabular display of the original “Basic data” file as observed by SFINX. (b) Check box for the visualization of the complete “Basic data” file as observed by SFINX.

Supplementary Figure 7 Network tab of the analysis page of the SFINX web site interface after data input.

(a) Interactive network visualization of the filtered interactions.

Supplementary information

Supplementary Figures and Tables

Supplementary Figures 1–7. (PDF 966 kb)

Supplementary Data 1

Example file for 'Basic data' input (semicolon-separated). (CSV 263 kb)

Supplementary Data 2

Example file for 'Bait identities' input. (TXT 0 kb)

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Titeca, K., Van Quickelberghe, E., Samyn, N. et al. Analyzing trapped protein complexes by Virotrap and SFINX. Nat Protoc 12, 881–898 (2017). https://doi.org/10.1038/nprot.2017.014

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