DOI: 10.6084/m9.figshare.14071499
title: MS RAW DATA CTPS.xlsx

description: <b>Tandem affinity purification (TAP) and Liquid chromatography - Mass spectrometry (LC-MS) Analyses</b> We used a Cts1-flag-3HA strain for our TAP and LC-MS analyses. For the TAP assay we used the FLAG-HA tandem affinity purification kit manufactured by Sigma (TP0010), and performed the protocol according to the manufacturer’s specifications. Briefly, Cts1-flag-3HA cells were cultured in rich media (YE4S) until they reached exponential phase. 50x10<sup>7</sup> cells were collected and the cell lysate was extracted, as described in the co-IP protocol. The cell lysate was then incubated with anti-FLAG M2 resin rotating overnight at 4<sup>0</sup>C, ensuring efficient binding. The supernatant was then removed carefully and the resin was washed with RIPA buffer (Sigma, R0278) containing protease inhibitors (Sigma, P8215), in order to remove any unbound protein. The first elution of the protein complex bound on the resin was done by using 3XFLAG peptide, and in a following step the eluate was bound to anti-HA resin slurry. In the second elution of the protein complex, the anti-HA slurry was washed with TBS (50mM Tris-Cl, ph=7.5; 150mM NaCl), to remove any unbound protein. The final elution was done by using TBS with 100mM ammonium bicarbonate, and the sample was subsequently digested with trypsin overnight. Peptides were then separated and analyzed on an Easy-nLC 1000 system coupled to a Q Exactive HF (both - Thermo Scientific). About 2 µg of peptides were separated in an home-made column (75 µm x 15 cm) packed with C18 AQ (5 µm, 300Å, Michrom BioResources, Auburn, CA, USA) at a flow rate of 300 nL/min. Mobile phase A (0.1% formic acid in 2% ACN) and mobile phase B (0.1% formic acid in 98% ACN) were used to establish a 60 min gradient comprised of 2 min of 5% B, 40 min of 5-30% B, 6 min of 30-45% B, 2 min of 45-90% B and 10 min of 90% B. Peptides were then ionized by electrospray at 2.3 kV. A full MS spectrum (300-1800 m/z range) was acquired at a resolution of 120,000 at m/z 200 and a maximum ion accumulation time of 20 ms. Dynamic exclusion was set to 30 s. Resolution for HCD MS/MS spectra was set to 30,000 at m/z 200. The AGC setting of MS and MS2 were set at 3E6 and 1E5, respectively. The 20 most intense ions above a 1.3E4 counts threshold were selected for fragmentation by HCD with a maximum ion accumulation time of 80 ms. Isolation width of 1.6 m/z units was used for MS2. Single and unassigned charged ions were excluded from MS/MS. For HCD, normalized collision energy was set to 30%. The underfill ratio was defined as 1%. The raw data were processed and searched with MaxQuant 1.5.4.1 with MS tolerance of 4.5 ppm, and MS/MS tolerance of 20 ppm. The Uniprot Schizosaccharomyces pombe proteome database and the database for proteomics contaminants from MaxQuant were used for database searches. Reversed database searches were used to evaluate false discovery rate (FDR) of peptide and protein identifications. Two missed cleavage sites of trypsin were allowed. Oxidation (M), Acetyl (Protein N-term), deamidation (NQ) and GGE (K) were set as variable modifications. The FDR of both peptide identification and protein identification is set to be 1%. The option of “Second peptides”, “Match between runs” and “Dependent peptides” was enabled. Label-free quantification was used to quantify the difference of protein abundances between different samples. Each sheet on the xls file corresponds to a different trial.<br>