MassIVE MSV000087493

Description

To identify proteins interacting with Pof8, cell-free extracts of strains FP1547 harboring 3xFLAG epitope-tagged Pof8 and untagged control (FP1546) were diluted to 5 mg/ml in 8 ml of lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM sodium chloride, 0.1% (v/v) NP40 and 10% (v/v) glycerol) plus complete EDTA-free protease inhibitor cocktail (Roche), 0.5 mM PMSF, 1 mM EDTA, and 0.1 mM DTT and incubated with 500 ul of EZview Red Flag-agarose 50% slurry equilibrated in lysis buffer. The mix was incubated for 8 hours at 4 C with gentle rotation. Agarose beads were collected by centrifugation at 300 x g for 5 min with low brake and washed 5 times for 5 minutes with 5 ml of wash buffer (50 mM Tris-HCl pH 7.5, 250 mM sodium chloride, 0.1% (v/v) NP40 and 10% (v/v) glycerol) at 4 C with gentle rotation. Proteins were eluted from beads by incubation for 30 min with 500 ul elution buffer (50 mM Tris- HCl pH 7.5, 250 mM sodium chloride, 0.1% (v/v) NP40 and 10% (v/v) glycerol, and 100 ug/ml 3xFLAG peptide) at 4 C. The elution step was repeated two more times and the eluates were pooled. A 4 ul aliquot of the first eluate was used for Silver stained SDS PAGE. The elutes were treated with 0.1U of benzonase for 30 min at 37 C and further diluted with the same volume of 100 mM Tris-HCl, pH 8.5. The samples were then split in three 400ul aliquots, mixed with 100 ul of 100% TCA and incubated at 4 C overnight. After centrifugation at 14,000 rpm for 30 min at 4 C, the pellets were washed twice with 500 ul cold acetone and centrifuged at 14,000 rpm for 10 min after each wash. Air dried pellets were resuspended in 30 ul 100 mM Tris-HCl, pH 8.5, 8M Urea and pooled. 4.5 ul of 0.1M TCEP was added to the pooled solution to a final concentration of 5 mM and incubated for 30 min at room temperature. After the incubation, 1.8 ul of 0.5 M CAM was added and incubated for 30 min at RT in the dark. Proteins were then digested with 0.1 ug/ul Lys-C at 37 C overnight. The samples were then diluted to 2M Urea with 100 mM Tris-HCl, pH8.5, added with CaCl2 to 2 mM and digested with 0.1 ug/ul Trypsin at 37 C overnight. Finally, 90% formic acid was added to a final concentration of 5%. and samples were analyzed by MudPIT. Tandem mass (MS/MS) spectra were interpreted using ProLuCID against the NCBI database of S. pombe proteins supplemented with 177 sequences from common contaminants (human keratins, IgGs, proteolytic enzymes). To estimate relative protein levels, (dNSAFs) were calculated for each detected protein. dNSAF values take into account peptides shared between multiple proteins and the length of the protein for normalization. [doi:10.25345/C58R6V] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: La-related protein (LARP) super family ; Telomerase

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Publications

Páez-Moscoso DJ, Ho DV, Pan L, Hildebrand K, Jensen KL, Levy MJ, Florens L, Baumann P.
A putative cap binding protein and the methyl phosphate capping enzyme Bin3/MePCE function in telomerase biogenesis.
Nat Commun. 2022 Feb 25;13(1):1067. Epub 2022 Mar 25.