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