Pull-down of Mlh3-Flag from yeast meiotic cells and analysis of pulled-down partner proteins

Description: Description from Proteome Xchange: "Mlh3 internally tagged with Flag was pulled down from synchronous meiotic cells at t = 4 h in meiosis, in order to identify its interacting partners involved in meiotic crossover formation"

Sample Processing Protocol from PRIDE: "2.1010 cells of VBD1674 strain at 4 h in meiosis were harvested, washed two times with ice-cold TNG buffer (50 mM Tris/HCl pH 8; 150 mM NaCl, 10% Glycerol; 1 mM PMSF; 1X Complete Mini EDTA-Free (Roche); 1X PhosSTOP (Roche)) and flash-frozen in liquid nitrogen. Frozen cells were mechanically ground in liquid nitrogen with the 6775 Freezer/Mill cryogenic grinder (SPEX SamplePrep). The resulting powder was resuspended in 25 mL of lysis buffer (50 mM Tris/HCl pH 7.5; 1 mM EDTA; 0.5% NP40; 10% glycerol; 150 mM NaCl; 1X Complete Mini EDTA-Free (Roche); 1X PhosSTOP (Roche), 210 U/mL benzonase (Sigma)) and incubated 1 h at 4°C with rotation. The lysate was cleared by centrifugation at 8000 g for 10 min and pre-cleared by pre-incubated with 100 μl Mouse IgG−Agarose beads (A0919 Sigma) without antibody for 2 h at 4°C on wheel. The pre-cleared lysate was incubated with 100 μl of washed and buffer equilibrated anti-Flag magnetic beads (Sigma-Aldrich, St. Louis, MO) for 2 h at 4°C. The beads were washed once with lysis buffer and three times with washing buffer (20 mM Tris/HCl pH 7.5; 0.5 mM EDTA; 0.1% tween; 10% glycerol; 150 mM NaCl; 5 mM MgCl2; 0.5 mM PMSF; 1X Complete Mini EDTA-Free (Roche, Switzerland); 1X PhosSTOP (Roche)). Proteins were eluted with 5 bed volume of elution buffer (20 mM Tris/HCl pH 8; 0.5 mM EDTA; 0.1% tween; 10% glycerol; 150 mM NaCl; 5 mM MgCl2; 0.5 mM PMSF; 1X Complete Mini EDTA-Free (Roche); 1X PhosSTOP (Roche); 100 μg/mL Flag peptide) for 2 h at 4°C. Proteins were separated by SDS-PAGE, stained with colloidal blue, and bands covering the entire lane were excised for each sample. In-gel digestion was performed overnight by using trypsin/LysC (Promega, Madison, WI). Peptides extracted from each band were analyzed by nanoLC-MS/MS using an Ultimate 3000 system (Dionex, Thermo Scientific, Waltham, MA) coupled to a TripleTOFTM 6600 mass spectrometer (ABSciex)."

Subject of Study

Saccharomyces cerevisiae

Subject(s)

Meiosis

MutL Proteins

Proteome

Recombination, Genetic

Access via ProteomeXchange
Dataset files accessible through FTP [file transfer protocol] link.
Accession #: PXD014180

Access via PRIDE
Dataset files accessible through FTP link.
Accession #: PXD014180
Access Restrictions: Free to All
Access Instructions: Public PXD datasets can be browsed over at ProteomeCentral.
All public datasets available in PRIDE Archive in the repository can be accessed via the website, Web Service (for programmatic access), and the PRIDE Inspector stand-alone tool.
Associated Publications: Sanchez A, Adam C, Rauh F, et al. Exo1 recruits Cdc5 polo kinase to MutLγ to ensure efficient meiotic crossover formation. Proceedings of the National Academy of Sciences of the United States of America. 2020; 117(48):30577-30588.
Equipment Used: TripleTOF 6600 System
Mass Spectrometer
Software Used: Mascot
For identification, characterisation and quantitation of proteins using mass spectrometry data.

myProMS
Mass spectrometry(MS)-based proteomics data management and analysis software.
Dataset Format(s): DAT, MGF
Dataset Size: 3.85 GB (MGF), 5.1 GB (DAT)

Data Catalog Record Updated: 2021-06-23