Project PXD002855

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Summary

Title

A novel inducible retroviral expression system for StrepHA-tandem affinity purification mass-spectrometry-based proteomics

Description

Tandem-affinity purification mass spectrometry using the streptavidin-hemagglutinin (SH)-tag has been successfully employed to map signaling networks in several large-scale studies. However its application has so far been restricted to the small number of Flp/FRT-recombination competent cell lines. We present pRSHIC, a novel retroviral, doxycycline-inducible Tet-On vector system suitable for expression of SH-tagged target proteins in a wide range of human and mouse cell systems. The additional feature of concomitant reporter fluorophore expression makes pRSHIC a valuable tool for a diverse set of phenotypic analyses beyond TAP-MS experiments. The dataset demonstrates the application of pRSHIC for TAP-MS analysis of two showcase bait proteins involved in cancer cell proliferation as well as cell death induction and identified novel high- confidence interacting proteins with possible pharmacological intervention potential.

Sample Processing Protocol

Cells were seeded, induced with doxycycline, lysed and subjected to TAP-LC-MSMS analysis. In brief, bait proteins were sequentially tandem-affinity purified via StrepTactin sepharose and HA-agarose beads. Samples were eluted with formic acid, subjected to tryptic digestion and analyzed on a hybrid linear trap quadrupole (LTQ) Orbitrap Velos mass spectrometer coupled to an Agilent 1200 HPLC nanoflow system.

Data Processing Protocol

The RAW files were processed using ProteoWizard software (v2.1.2708) to extract the MS1 and MS2 spectra. Mass lists were re-calibrated using an initial database search using Mascot (www.matrixscience.com, version 2.3.02) with broad mass tolerance as well as conservative score threshold. Mass tolerance of ± 10 ppm and ± 0.6 Da was used for precursor and fragment ions, respectively. Other parameters include, fully-tryptic peptides with maximum of 1 missed cleavage; carbamidomethyl cysteine as fixed modification; and methionine oxidation variable modification. In addition, Mascot peptide ion score of 30 or above and a minimum of 3 unique peptides per protein were required. Re-calibrated mass lists were searched using Mascot and Phenyx (GeneBio, SA, version 2.5.14) database search algorithms with mass tolerances of ± 4 ppm and ± 0.3 Da at precursor and fragment ions, respectively. Fixed and variable modifications were the same as in the initial search. Searches were performed against the human UniProtKB/SwissProt database (www.uniprot.org release 2013.01) including all protein isoforms. In-house Perl-based programs were used to merge identifications from Mascot and Phenyx as described in PubMed ID 20940065. Finally, using a reverse database search with same parameters, a false discovery rate of <1% and <0.1% for protein and peptide identifications, respectively.

Contact

Richard Kumaran Kandasamy, CeMM Center for Molecular Medicine
Giulio Superti-Furga, Scientific Director, CeMM - Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria; Professor for Medical Systems Biology, Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria (lab head) ( lab head )

Submission Date

16/12/2015

Publication Date

05/01/2016

Publication

    Bigenzahn JW, Fauster A, Rebsamen M, Kandasamy RK, Scorzoni S, Vladimer GI, Mueller AC, Gstaiger M, Zuber J, Bennett KL, Superti-Furga G. An inducible retroviral expression system for tandem affinity purification mass-spectrometry-based proteomics identifies MLKL as an HSP90 client. Mol Cell Proteomics. 2015 Dec 29. pii: mcp.O115.055350 PubMed: 26714523

Assay

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Showing 1 - 10 of 16 results
# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 60609 no assay title provided (mzIdentML) 105 1713 163 13764 645
2 60608 no assay title provided (mzIdentML) 85 1655 149 13276 651
3 60607 no assay title provided (mzIdentML) 108 1787 152 14482 666
4 60599 no assay title provided (mzIdentML) 100 1778 143 14337 670
5 60606 no assay title provided (mzIdentML) 134 2083 262 15373 1080
6 60605 no assay title provided (mzIdentML) 140 2064 259 14976 1059
7 60604 no assay title provided (mzIdentML) 85 1691 156 13865 877
8 60613 no assay title provided (mzIdentML) 89 1587 157 13589 823
9 60614 no assay title provided (mzIdentML) 62 921 100 8106 519
10 60611 no assay title provided (mzIdentML) 68 917 107 7703 503