MassIVE MSV000083465

Complete Public PXD012807

MudPIT analyses of the proteins co-purified with the Interactor of Little Elongation Complex ELL subunit 1 (FLAG-ICE1), WT MED26, and D2G8 NTD-mutant MED26, affinity-purified from HEK293T cell lines

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Description

Protein Complexes Purification: We generated 293FRT cell lines that stably express FLAG-tagged ICE1 (Interactor of Little Elongation Complex ELL subunit 1; KIAA0947). We extracted nuclear proteins in the presence of Benzonase to digest nucleic acids, including both DNA and RNA, and purified the little elongation complex (LEC) through anti-FLAG affinity purification using anti-FLAG M2 agarose. Cells only expressing the FLAG epitope-tag were analyzed in parallel as negative controls. A MED26 hypomorphic HEK293T cell line D2G8 was generated by applying multiple small guide RNAs (sgRNAs) complementary to the MED26 gene to induce random sequence insertions and/or deletions. This cell line expresses mutant MED26 lacking the NTD. Mediator was purified from cells expressing either WT MED26 or D2G8 MED26 via its ability to bind the transcriptional activation domain of ATF6-alpha. 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/C5J629] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Mediator ; MED26 ; little elongation complex (LEC) ; Interactor of Little Elongation Complex ELL subunit 1 (ICE1)

Contact

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

Publications

Takahashi H, Ranjan A, Chen S, Suzuki H, Shibata M, Hirose T, Hirose H, Sasaki K, Abe R, Chen K, He Y, Zhang Y, Takigawa I, Tsukiyama T, Watanabe M, Fujii S, Iida M, Yamamoto J, Yamaguchi Y, Suzuki Y, Matsumoto M, Nakayama KI, Washburn MP, Saraf A, Florens L, Sato S, Tomomori-Sato C, Conaway RC, Conaway JW, Hatakeyama S.
The role of Mediator and Little Elongation Complex in transcription termination.
Nat Commun. 2020 Feb 26;11(1):1063. Epub 2020 Feb 26.

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