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
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Laurence Florens
PROVIDER: MSV000083364 | MassIVE | Fri Jan 25 12:41:00 GMT 2019
SECONDARY ACCESSION(S): PXD012516
REPOSITORIES: MassIVE
Publications
Merkel cell polyomavirus activates LSD1-mediated blockade of non-canonical BAF to regulate transformation and tumorigenesis.
Park Donglim Esther DE Cheng Jingwei J McGrath John P JP Lim Matthew Y MY Cushman Camille C Swanson Selene K SK Tillgren Michelle L ML Paulo Joao A JA Gokhale Prafulla C PC Florens Laurence L Washburn Michael P MP Trojer Patrick P DeCaprio James A JA
Nature cell biology 20200413 5
Merkel cell carcinoma (MCC)-a neuroendocrine cancer of the skin-is caused by the integration of Merkel cell polyomavirus and persistent expression of large T antigen and small T antigen. We report that small T antigen in complex with MYCL and the EP400 complex activates the expression of LSD1 (KDM1A), RCOR2 and INSM1 to repress gene expression by the lineage transcription factor ATOH1. LSD1 inhibition reduces the growth of MCC in vitro and in vivo. Through a forward-genetics CRISPR-Cas9 screen, ...[more]
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2018-06-28 | MSV000082520 | MassIVE
Project description:Yeast protein extractions and subcellular fractionation
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Clarified extracts were incubated with anti-FLAG M2 agarose (Sigma Aldrich) at 4oC for 4 hrs and washed two times with H350 and once with E100 buffer (25 mM HEPES-KOH pH7.5, 10 % glycerol, 100 mM KCl, 2 mM MgCl2, 1 mM EDTA, 0.02% NP40, 1 ug/ml pepstatin A, 2 ug/ml leupeptin, 1 mM PMSF). The FLAG tagged proteins were eluted with E100 buffer with 400 ug/ml 3xFLAG peptide.
Size-exclusion chromatography
Eluates from FLAG purification were loaded onto a Superose 6 size-exclusion column (GE17-5172-01, Amersham Bioscience) equilibrated in superose 6 buffer (350 mM NaCl, 40 mM HEPES pH7.5, 5 % glycerol, 0.1 % Tween 20) for 350 mM NaCl condition. The 500 ul fractions eluted from the column were analyzed by western blots and multidimensional protein identification technology (MudPIT).
Multidimensional Protein Identification Technology
Proteins isolated from FLAG-AP of (Halo-E1-alpha and E1-alpha-Halo) were precipitated with 20% trichloroacetic acid (TCA) and the resulting pellet was washed once with 10% TCA and twice with cold acetone. TCA-precipitated protein pellet was solubilized using Tris-HCl pH 8.5 and 8 M urea, followed by addition of TCEP (Tris(2-carboxyethyl)phosphine hydrochloride; Pierce) and CAM (chloroacetamide; Sigma) were added to a final concentration of 5 mM and 10 mM, respectively. Proteins were digested using Endoproteinase Lys-C at 1:100 w/w (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) at 1:100 w/w. The reactions were stopped using formic acid (5% final). The samples 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. The MS/MS data set was searched using ProLuCID (v. 1.3.3) against a database consisting of 36,628 Homo sapiens NR proteins (NCBI -released June 10, 2016), 193 usual contaminants, and, to estimate false discovery rates (FDRs), randomized amino acid sequences derived from each NR protein entry. To account for alkylation by CAM, 57 Da were added statically to the cysteine residues. To account for the oxidation of methionine to methionine sulfoxide, 16 Da were added as a differential modification to the methionine residue. Peptide/spectrum matches were sorted and selected to an FDR less than 5% at the peptide and protein levels, using DTASelect v1.9 in combination with swallow, an in-house software.
2020-03-20 | MSV000085126 | MassIVE
Project description:Gene expression profiles of Bacillus subtilis strain AG174 were compared as a function of steady-state external pH during growth with aeration. Aerated overnight cultures were diluted 1:500 and incubated in 250 mL baffled flasks containing potassium-modified Luria-Bertani medium (LBK) buffered with 50 mM HOMOPIPES at pH 6.0, pH 7.0, and pH 9.0. Tubes were incubated at 37°C with aeration (260 rpm) to an optical density at 600 nm of 0.2. For each of the three pH conditions, RNA was isolated from five independent cultures.
2009-01-13 | GSE11810 | GEO