MassIVE MSV000083364

Complete Public PXD012516

MudPIT analyses of the proteins co-purified with components of the LSD1 (KDM1A) transcriptional repressor complex and the non-canonical BAF complex, immuno-precipitated from MKL-1 and WaGa MCC cell lines

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Description

Protein Complexes Purification: Immunoprecipitations were performed with antibodies to LSD1 (2139; Cell signaling technology), INSM1 Mouse (SC-271408, Santa Cruz Biotechnology), RCOR2 (23969-1-AP; Proteintech Group), BRD9 (61537; Active Motif Rabbit), and normal Rabbit IgG (2729; Cell signaling technology). MKL-1 or WaGa suspension cells were harvested in EBC lysis buffer. INSM1 was also immunopurified from MKL-1 cells treated with the GSK-LSD1 inhibitor for 3 days. For all IPs, clarified cell extract (100-300 mg) was incubated overnight at 4oC with 20ug antibodies crosslinked to 30 mg protein G agarose beads by dimethyl pimelimidate. Beads were washed with high salt buffer 5x, eluted with 0.2 M glycine pH 3 and neutralized with 1 M Tris pH 8.0. Proteins were precipitated with 1/5 TCA overnight at 4oC and washed 2x with cold acetone. Multidimensional Protein Identification Technology: TCA-precipitated protein pellets were with Tris-HCl pH 8.5 8 M urea, followed by addition of TCEP (Pierce) and chloroacetamide (Sigma) to a final concentration of 5 mM and 10 mM, respectively. Proteins were digested using Endoproteinase Lys-C (Roche) at 37oC overnight. The samples were brought to a final concentration of 2 M urea and 2 mM CaCl2 and a second digestion was performed overnight at 37oC using trypsin (Promega). Digested peptides were loaded on a split-triple-phase fused-silica micro-capillary column and placed in-line with a linear ion trap mass spectrometer (LTQ, Thermo Scientific), coupled with a Quaternary Agilent 1100 Series HPLC system. A fully automated 10-step chromatography run was carried out. Each full MS scan (400-1600 m/z) was followed by five data-dependent MS/MS scans. The number of the micro scans was set to 1 both for MS and MS/MS. The settings were as follows: repeat count 2; repeat duration 30 s; exclusion list size 500 and exclusion duration 120 s, while the minimum signal threshold was set to 100. MS Data Processing: The MS/MS data set was searched using ProLuCID (v. 1.3.3) against 36628 non-redundant Homo sapiens proteins (downloaded from NCBI RefSeq 2016-06-10), 193 usual contaminants, and, to estimate false discovery rates (FDRs), 36821 randomized amino acid sequences derived from each NR protein. To account for alkylation by CAM, 57 Da were added statically to the cysteines. To account for oxidation, 16 Da were added as a differential modification to methionines. Peptide/spectrum matches were sorted and selected to an FDR less than 5% at the peptide and protein levels, using DTASelect in combination with swallow, an in-house software. [doi:10.25345/C5KW48] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Merkel cell carcinoma (MCC) ; Merkel cell polyomavirus (MCV) ; transcriptional repressor complex ; LSD1, histone demethylase ; non-canonical BAF ; BRD9, BET family protein

Contact

Principal Investigators:
(in alphabetical order) 		Laurence Florens, The Stowers Institute for Medical Research, USA
Submitting User: laflorens

Publications

Park DE, Cheng J, McGrath JP, Lim MY, Cushman C, Swanson SK, Tillgren ML, Paulo JA, Gokhale PC, Florens L, Washburn MP, Trojer P, DeCaprio JA.
Merkel cell polyomavirus activates LSD1-mediated blockade of non-canonical BAF to regulate transformation and tumorigenesis.
Nat Cell Biol. 2020 May;22(5):603-615. Epub 2020 Apr 13.

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