DOI: 10.25345/c5r39s
title: MassIVE MSV000085125 - MudPIT analyses of the pyruvate dehydrogenase complex (PDC) isolated from human
title: Mammalian protein extraction and Halo purification For Halo purification, HEK293T cells at 40% confluency in a 100 mm plate were transfected with 10ug of plasmid containing ORF of PDHA1-HA-Halo in 1X BBS with 125 mM CaCl2. Cells were harvested by scraping in PBS following PBS wash after 24 hours from transfection. For non-transfected cells, cells were harvested at 100% confluency. The cell pellet was resuspended in 1 ml of Hypotonic buffer (50 mM Tris-HCl pH 7.5, 0.1% NP-40) and centrifuged at 4000 rpm for 5 min. The resulting pellet was resuspended in 1 ml of lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 % Triton X-100, 0.1 % Na-deoxycholate) and clarified by centrifugation at 12,000 rpm for 30 min. The crude extract in the supernatant was treated with 125 U of benzonase for 20 minutes in 4oC, then clarified by centrifugation at 12,000 rpm for 10 minutes. The clarified extract was either directly loaded onto Superose 6 column or subjected to Halo-purification by adding with 100 ul of Halo beads suspension incubated overnight in 4oC on a rotator. The beads were washed 3 times then subjected for protein elution with 50 U of AcTEV protease (Invitrogen, 12575-015) in PBS buffer by 8 hr incubation in 4oC on a rotator. Multidimensional Protein Identification Technology Proteins isolated from Halo-AP of N- and C-terminally tagged human PDHA1 (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.