DOI: 10.25345/c5md7w
title: MassIVE MSV000085126 - MudPIT analyses of the pyruvate dehydrogenase complex (PDC) isolated from Saccharomyces cerevisiae by FLAG-affinity purification and Size Exclusion Chromatography
title: Yeast protein extractions and subcellular fractionation Yeast cells expressing FLAG tagged proteins were grown in YPD medium at 30oC to OD600 of 0.8. 12 Liters YPD culture of cells were pelleted and washed in SB buffer. The pellet was resuspended in SB buffer and treated with 2 mg/ml Zymolyase 20T at 23oC for 45 min. The resulting spheroplasts were collected by centrifugation at 2,710 g for 5 min and washed twice with SB buffer. The spheroplasts were then resuspended in 20 ml of homogenization buffer (10 mM Tris-HCl pH 7.4, 0.6 M sorbitol, 1 mM EDTA, 0.2% (w/v) BSA) and homogenized with 8 strokes of a revolving pestle at full speed. The homogenized spheroplasts were centrifuged at 1,500 g for 5 min and the resulting pellets were collected for nuclear extracts while the supernatant was collected for mitochondrial extracts. The pellet enriched for nucleus was washed with NP buffer (0.34 M sucrose, 20 mM Tris pH 7.5, 50 mM KCl, 5 mM MgCl2) and resuspended and homogenized in H350 buffer (40 mM HEPES-KOH pH7.5, 10% glycerol, 350 mM KCl, 2 mM MgCl2, 1 mM EDTA, 0.02% NP40, 1 ug/ml pepstatin A, 2 ug/ml leupeptin, 1 mM PMSF) with 25 strokes. The crude extract was incubated with 125 U of Benzonase on a roller for 1 hour in 4oC, then clarified by ultracentrifugation at 45,000 rpm for 90 min after following a centrifugation at 20,000 g for 15 min. FLAG affinity purification Clarified extracts were incubated with anti-FLAG M2 agarose (Sigma Aldrich) at 4oC for 4 hrs and washed two times with H350 and once with E100 buffer (25 mM HEPES-KOH pH7.5, 10 % glycerol, 100 mM KCl, 2 mM MgCl2, 1 mM EDTA, 0.02% NP40, 1 ug/ml pepstatin A, 2 ug/ml leupeptin, 1 mM PMSF). The FLAG tagged proteins were eluted with E100 buffer with 400 ug/ml 3xFLAG peptide. Size-exclusion chromatography Eluates from FLAG purification were loaded onto a Superose 6 size-exclusion column (GE17-5172-01, Amersham Bioscience) equilibrated in superose 6 buffer (350 mM NaCl, 40 mM HEPES pH7.5, 5 % glycerol, 0.1 % Tween 20) for 350 mM NaCl condition. The 500 ul fractions eluted from the column were analyzed by western blots and multidimensional protein identification technology (MudPIT). Multidimensional Protein Identification Technology Proteins isolated from FLAG-AP of (Halo-E1-alpha and E1-alpha-Halo) were precipitated with 20% trichloroacetic acid (TCA) and the resulting pellet was washed once with 10% TCA and twice with cold acetone. TCA-precipitated protein pellet was solubilized using Tris-HCl pH 8.5 and 8 M urea, followed by addition of TCEP (Tris(2-carboxyethyl)phosphine hydrochloride; Pierce) and CAM (chloroacetamide; Sigma) were added to a final concentration of 5 mM and 10 mM, respectively. Proteins were digested using Endoproteinase Lys-C at 1:100 w/w (Roche) at 37oC overnight. The samples were brought to a final concentration of 2 M urea and 2 mM CaCl2 and a second digestion was performed overnight at 37oC using trypsin (Promega) at 1:100 w/w. The reactions were stopped using formic acid (5% final). The samples were loaded on a split-triple-phase fused-silica micro-capillary column and placed in-line with a linear ion trap mass spectrometer (LTQ, Thermo Scientific), coupled with a Quaternary Agilent 1100 Series HPLC system. A fully automated 10-step chromatography run was carried out. Each full MS scan (400-1600 m/z) was followed by five data-dependent MS/MS scans. The number of the micro scans was set to 1 both for MS and MS/MS. The settings were as follows: repeat count 2; repeat duration 30 s; exclusion list size 500 and exclusion duration 120 s, while the minimum signal threshold was set to 100. The MS/MS data set was searched using ProLuCID (v. 1.3.3) against a database consisting of 36,628 Homo sapiens NR proteins (NCBI -released June 10, 2016), 193 usual contaminants, and, to estimate false discovery rates (FDRs), randomized amino acid sequences derived from each NR protein entry. To account for alkylation by CAM, 57 Da were added statically to the cysteine residues. To account for the oxidation of methionine to methionine sulfoxide, 16 Da were added as a differential modification to the methionine residue. Peptide/spectrum matches were sorted and selected to an FDR less than 5% at the peptide and protein levels, using DTASelect v1.9 in combination with swallow, an in-house software.