Project PXD000685

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Summary

Title

Virotrap: Abducting Protein Complexes from Mammalian Cells

Description

Cell lysis is an inevitable step in classical affinity purification - mass spectrometry (AP-MS) strategies to map intracellular protein interactions. Cell homogenization implies a drastic change of the protein complex environment which strongly impedes comprehensive analysis. Complementary lysis conditions, in situ crosslinking strategies and proximal labeling techniques have been recently developed to address part of this problem. We here demonstrate the use of a viral particle sorting approach as an alternative lysis-free method. By fusing a bait protein to the HIV-1 GAG protein, we show that interaction partners of the bait protein become trapped within virus-like particles (VLPs) that bud from mammalian cells. This so-called Virotrap approach obviates the need for cell homogenization and preserves the protein complexes during purification. Using an efficient VLP enrichment protocol, Virotrap allows the detection of known binary interactions as well as MS-based identification of novel protein partners. In addition, we show the identification of stimulus-dependent interactions and demonstrate trapping of protein partners for small molecules. Virotrap constitutes an elegant complementary approach to the current arsenal of MS-based methods to study protein complexes.

Sample Processing Protocol

For mass spectrometry, 107 HEK293T cells were seeded in 3-4 75 cm2 bottles and transfected the next day with a total of 15 g DNA per bottle using polyethyleneimine (PEI) reagent. The following DNA/PEI quantities were used: GAG-bait 7.5 g; mock vector 5.4 g; 2.1 g of a 1/2 pMD2.G - pcDNA3-FLAG-VSV-G mix vs. 37.5 l PEI. The cellular supernatant was harvested after 32 h and centrifuged for 3 min at 450xg to remove cellular debris. For simvastatin, tamoxifen and reversin experiments, the bivalent methotrexate-polyethylene glycol-small molecules 20, 36 were added after transfection to the producing cells at a concentration of 1 M. Producer cells transfected with the A20 bait, VSV-G capture proteins and 1.15 g TNFR plasmid were treated with 300 IU/ml of human TNF alpha during production to monitor dynamic A20 complexes. The cleared supernatant was then filtered using 0.45 m filters (Millipore). A total of 100 l MyOne™ Streptavidin T1 beads pre-loaded with 10 l ANTI-FLAG® BioM2-Biotin antibody was used to bind the tagged particles. Particles were allowed to bind for 2 h by end-over-end rotation. The total supernatant was processed in 3 consecutive binding steps. Bead-particle complexes were washed once with washing buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl) and were then eluted with FLAG peptide (30 min at 37°C; 200 g/ml in washing buffer) and lysed by addition of SDS to a final concentration of 0.1%. After 5 min, SDS was removed using HiPPR Detergent Removal Spin Columns (Pierce, Thermo Scientific) followed by boiling and overnight digestion with 0.5 g sequence-grade trypsin (Promega). After acidification (addition of 1 l of 10% trifluoroacetic acid), the peptides were separated by nano-LC and directly analyzed with a Q Exactive instrument (Thermo Scientific) operating in MS/MS mode as described before

Data Processing Protocol

Searches were performed using the MASCOT algorithm (Version 2.4.1. Matrix Science) at 99% confidence against human and bovine SWISSPROT accessions (Release 2013_02) complemented with HIV-1, EGFP, VSV-G and FLAG-VSV-G protein sequences. FDR rates were obtained by searches against the reversed version of the complete search database and by retaining only the PSMs with the highest score in standard or reverse search. FDR was then calculated by dividing the number of PSMs against the reversed database by the number of PSMs against both databases. Identifications are reported by unique GENE NAME in the tables. Raw data files, search settings, MGFs and identification lists were submitted to PRIDE using Proteome Exchange. Protein spectral count files and peptide spectral count files are provided as Excel files.

Contact

Sven Eyckerman, VIB Center for Medical Biotechnology
Sven Eyckerman, VIB Medical Biotechnology Center, VIB, A. Baertsoenkaai 3 B-9000 Ghent, Belgium ( lab head )

Submission Date

08/02/2016

Publication Date

27/04/2016

Cell Type

epithelial cell

Disease

disease free

Instrument

Q Exactive

Software

Unknown

Experiment Type

Shotgun proteomics

Assay count

15

Publication

Publication pending

Assay

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Page size 10
Showing 1 - 10 of 15 results
# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 61672 Virotrap: Abducting Protein Complexes from Mammalian Cells 917 11504 3465 79828 11504
2 61673 Virotrap: Abducting Protein Complexes from Mammalian Cells 1157 10664 5252 40027 10664
3 61661 Virotrap: Abducting Protein Complexes from Mammalian Cells 1157 10664 5252 40027 10664
4 61670 Virotrap: Abducting Protein Complexes from Mammalian Cells 1662 18567 7316 78387 18567
5 61662 Virotrap: Abducting Protein Complexes from Mammalian Cells 1157 14032 4551 60290 14032
6 61671 Virotrap: Abducting Protein Complexes from Mammalian Cells 1400 14459 5885 68852 14459
7 61667 Virotrap: Abducting Protein Complexes from Mammalian Cells 1248 11636 4939 62024 11636
8 61668 Virotrap: Abducting Protein Complexes from Mammalian Cells 727 9581 3001 28801 9581
9 61669 Virotrap: Abducting Protein Complexes from Mammalian Cells 1656 13466 6955 43256 13466
10 61663 Virotrap: Abducting Protein Complexes from Mammalian Cells 1215 13185 4974 55451 13185