Myc-Affinity Capture and on-Beads Crosslinking
For each Saccharomyces cerevisiae strain (C-terminally myc-tagged Scc2 subunit of the cohesin loading factor complex or wild-type BY4741), 2x 3 L YPD medium (add 300 ml 20% Glucose to each 3 L of YPD before using) were separately inoculated in a 5L flask with 15 ml overnight culture and incubated at 30C with shaking until OD600 reached 1.5. Cell pellets were resuspended in up to 30 ml in BH0.15 extraction buffer (25 mM HEPES, pH 7.5, 2 mM MgCl2, 0.1 mM EDTA, 0.5 mM EGTA-KOH, 15% Glycerol, 0.1% NP-40, 150 mM KCl) with freshly added 100x protease inhibitor and 5mM DTT. Cells were lysed using liquid nitrogen and dry ice.
Pierce Anti-c-Myc Magnetic Beads (150 ul) were washed with 1 ml of BH0.15 extraction buffer. The supernatant was discarded, and the washed beads were added to the protein lysate and incubated on a rotating wheel overnight at 4C. Proteins bound to anti-Myc beads were washed twice with 15 ml BH0.15. Beads were washed with 1 ml of pre-elution rinse buffer (50 mM HEPES, pH 7.5, 75 mM KCl, 1 mM EGTA). Proteins bound to anti-myc beads were crosslinked by adding 150 ul pre-elution rinse buffer complemented with protease inhibitor cocktail and DTT with 0.6 ul of 250 mM disuccinimidyl sulfoxide (DSSO) to a final concentration of 1mM and let to crosslink at room temperature for 40 min. The crosslinking reaction was quenched by adding 7.5 ul of 1 M NH4CO3 (final 50 mM) and rotating at RT for 15 min.
On-Beads Protein Digestion
Crosslinked proteins bound to anti-myc beads were washed twice with 500 ul 10 mM Tris-HCl pH7.5, 150 mM NaCl, then denatured, reduced and digested with 100 ul 50 mM Tris-HCl pH7.5, 2 M urea, 1 mM TCEP (Pierce), 5 ul Trypsin at 0.1 ug/ ul (Sequencing Grade Modified Trypsin; Promega), at 30 C for 30 min. The supernatant was collected. Another 60 ul of 50 mM Tris-HCl pH7.5, 2 M urea, 5 mM 2-Chloroacetamide (CAM, Sigma) were added to the beads to alkylate free cysteines, and the resulting supernatant combined to the first one. CAM was next added to 2.5 mM and the reaction incubated in the dark at RT for 30 min. CaCl2 was added to 2 mM along with 5 ul of Trypsin, and the digestion was let to proceed at 37C overnight. The digestion was quenched by adding formic acid to 5%. The combined supernatants from these multiple steps constitute the first elution (E1). These digestion steps were repeated once, and the collected supernatants combined as the second elution (E2).
Digested peptides were analyzed on an Orbitrap Fusion Lumos mass spectrometer equipped with a FAIMS Pro interface coupled to a Dionex Ultimate 3000 RSCLnano System. Peptides were loaded (5 or 10ul for the Scc2-myc digests and 5 or 66ul for the wild-type BY4741 negative control digests) on an Acclaim PepMap 100 C18 0.3 mm i.D. x 5 mm length trap cartridge with loading pump at 2 ul/min via autosampler. A 75 um i.d. analytical microcapillary column was packed in-house with 250 mm of 1.9 um ReproSil-Pur C18-AQ resin. Column temperature was maintained at 40C. The organic solvent solutions were water/acetonitrile/formic acid at 95:5:0.1 (v/v/v) for buffer A (pH 2.6) and at 20:80:0.1 (v/v/v) for buffer B. The chromatography gradient was a 25 min column equilibration step in 2% B; a 3 min ramp to reach 10% B; 90 min from 10 to 40 % B; 6 min to reach 95% B; a 9 min wash at 95% B; 0.1 min to 2% B; followed by a 12 min column re-equilibration step in 2% B. The nano pump flow rate was set to 180 nL/min. Orbitrap Fusion Lumos was set up with peptide identification method as: full MS1 resolution 120,000; ITMS2 isolation window 1.4 m/z, ITMS2 max ion injection time 50 ms, ITMS2 CID 35% with normal scan. FAIMS compensation voltages (CVs) were set up as -40V, -60V, and -80V.
MS Dataset Processing
Collected MS/MS spectra were searched with the ProLuCID algorithm against a database of 12276 protein sequences combining 6010 non-redundant Saccharomyces cerevisiae proteins (NCBI, 2017-05-16 release), 193 common contaminants, and their corresponding 6138 randomized amino acid sequences. All cysteines were considered as fully carboxamidomethylated (+57 Da statically added), while methionine oxidation was searched as a differential modification. DTASelect v1.9 and swallow, an in-house developed software, were used to filter ProLuCID search results at given FDRs at the spectrum, peptide, and protein levels. Here, all controlled FDRs were less than 1%.
[dataset license: CC0 1.0 Universal (CC0 1.0)]
Keywords: cohesin loading factor ; SCC2 ; DSSO crosslinking
(in alphabetical order)
|Laurence Florens, The Stowers Institute for Medical Research, USA|
Mattingly M, Seidel C, Muñoz S, Hao Y, Zhang Y, Wen Z, Florens L, Uhlmann F, Gerton JL.
Mediator recruits the cohesin loader Scc2 to RNA Pol II-transcribed genes and promotes sister chromatid cohesion.
Curr Biol. 2022 Jul 11;32(13):2884-2896.e6. Epub 2022 Jun 1.