DOI: 10.25345/c5zw1955x
title: MassIVE MSV000099342 - MudPIT analyses of the proteins affinity purified with C. albicans TFIID and SAGA subunits
title: Affinity Purification C. albicans TAF12L-FLAG (SKC3), TBP-TAP (ISC33), TAF11-TAP (ISC49), and control (SN87) strains were precultured for 14h in YPD and diluted into 6L of YPD and grown to mid-log phase. Cells were collected by centrifugation, washed, and resuspended in either 20ml H350 buffer (25 mM HEPES-KOH, pH=7.5, 350mM KCl, 2 mM MgCl2, 1 mM EDTA, 10% glycerol, 0.02% NP40) for FLAG samples, or 12ml TAP extraction buffer (40 mM HEPES-KOH, pH 7.5, 10% Glycerol, 350 mM NaCl, 0.1% Tween-20) with protease inhibitors (1mM PMSF, 1ug/ml pepstatin A, 2ug/ml leupeptin, and 100ul Sigma Yeast Protease Inhibitor). Cells were lysed in a BeadBeater at 4C, and heparin (0.5mg for FLAG extracts, or 1mg for TAP extracts) and 125 units benzonase were added, centrifuged at 45000 rpm for 1.5h, and the supernatants were collected. For FLAG purification, 400ul pre-washed anti-FLAG M2 affinity agarose gel was added to the SKC3 and SN87 lysates and incubated with rotation at 4C for 3-5h. Proteins were eluted with 250ug/ml 3xFLAG peptide, incubated for 30 min at 4C, repeated 3x. The lysates from TAP-tagged (ISC33 and ISC49) and untagged control (SN87) strains were incubated with 400ul IgG-Sepharose and incubated for 2h on a rotator. Beads were collected by gentle centrifugation at 600-1200 rpm. Bead fractions were washed 5x with TAP extraction buffer at 4C. Beads were incubated with 1ml TEV cleavage buffer containing 10ul AcTEV (Invitrogen), incubated with rotation at 4C for 16h, and eluates were collected. Next, the eluate was added to 25ul pre-washed Calmodulin-Sepharose beads in calmodulin binding buffer (CBB; 10 mM Tris, pH 8.0, 1 mM MgOAc,1 mM Imidazole, 2 mM CaCl2, 0.1% NP-40, 10% Glycerol, 0.3M NaCl and protease inhibitors) containing 1M CaCl2 and incubated at 4C for 3h on a rotator, and washed 5x with CBB (0.15 M NaCl). Proteins were eluted 7x with 200ul calmodulin elution buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 1mM MgOAc, 1mM imidazole, 2mM EGTA, 0.1% NP-40, 10% Glycerol, protease inhibitors), and the fractions collected. The eluted samples were precipitated using 20% (w/v) TCA, washed with cold acetone and air-dried. Multidimensional Protein Identification Technology (MudPIT) TCA-precipitated protein pellets were resuspended in 100 mM Tris-HCl, pH 8.5, 8 M urea, reduced with 5 mM TCEP, and alkylated with 10 mM IAM. Endoproteinase Lys-C was added to 0.5ug for at least 6 hours at 37C, then the sample was diluted to 2 M urea with 100 mM Tris-HCl, pH 8.5. Calcium chloride was added to 2 mM and the digestion with 0.5ug trypsin proceeded O/N at 37C. The reaction was quenched by adding formic acid to 5% and the peptide mixture was loaded onto a 100um fused silica microcapillary column packed with 8 cm of reverse phase material and connected to a 250um FS column packed with 3 cm of 5um Strong Cation Exchange material, followed by 2 cm of C18 reverse phase. The loaded microcapillary column was placed in-line with a Quaternary Agilent 1100 series HPLC pump. Overflow tubing was used to decrease the flow rate from 0.1 ml/min to about 200-300 nl/min. Fully automated 10-step chromatography runs were carried out. Three different elution buffers were used: A: 5% acetonitrile, 0.1% formic acid; B: 80% ACN, 0.1% FA; and C: 0.5 M ammonium acetate, 5% ACN, 0.1% FA. Peptides were sequentially eluted from the SCX to the RP by increasing salt steps, followed by organic gradient. The application of a 2.5 kV distal voltage electrosprayed the eluting peptides directly into a LTQ linear ion trap mass spectrometer equipped with a nano-LC electrospray ionization source. Full MS spectra were recorded on the peptides over a 400 to 1,600 m/z range, followed by five MS/MS events sequentially generated in a data-dependent manner at 35% collision energy. SEQUEST was used to match MS/MS spectra to peptides in a consisting of 9269 non-redundant proteins derived from all orf translation of Candida albicans SC5314 Assembly 22, 354 usual, and, to estimate false discovery rates, 9623 randomized amino acid sequences derived from each non-redundant protein entry. PSMs were only retained if they had a DeltCn of at least 0.08 and and minimum XCorr of 1.8 for +1, 2.0 for +2, and 3.0 for +3 spectra. Peptides had to be fully-tryptic and at least 7 AAs long. Combining all runs, proteins had to be detected by at least 2 such peptides, or 1 peptide with 2 independent spectra. Under these criteria the final FDRs at protein and peptide levels were less than 1%. DTASelect was used to select and sort PSMs. Peptide hits from multiple runs were compared using CONTRAST. Spectral counts were normalized as dNSAFs.