Interactome analysis of ACAD11 in HEK293 cells
Affinity purification - mass spectrometry analysis of N-terminally FLAG-tagged ACAD11 transietly expressed in HEK293 cells.
Sample Processing Protocol
HEK293 cells transfected with either a control pcDNA3.1 DNA plasmid (EV) or N-terminally FLAG-tagged ACAD11 in pcDNA3.1 DNA plasmid were lysed in 0.3% CHAPS, 150 mM NaCl, 5 % glycerol in 50 mM Tris (pH 7.5) containing protease inhibitor cocktail (Roche; 11873580001) by passing through a 22- and 27-gauge needle at 4°C. Cellular debris was removed by centrifugation at 20,000 x g, 10 min at 4°C and quantified by BCA (Thermo Fisher; 23225). Two milligram of protein was incubated with 40 µl of μMACS Anti-DYKDDDDK beads (Miltenyi Biotech; 130-101-591) for 45 min with rotation at 4°C. The suspension was separated with a μMACS column and magnetic separator, and washed with lysis buffer containing only 0.01% CHAPS followed by lysis buffer containing no CHAPS. Proteins were eluted with 2 M urea in 50 mM Tris (pH 7.5) containing 1 mM DTT, 5 mM IAA and 125 ng of trypsin (Promega; V5111), and digested overnight at room temperature. Peptides were acidified to 1% TFA and desalted using Styrene Divinylbenzene - Reversed-Phase Sulfonate (SDB-RPS) microcolumns and eluted with 80% acetonitrile in 2% ammonium hydroxide followed by vacuum concentration.
Data Processing Protocol
Peptides were analyzed on an Easy nLC-1200 coupled to a Q-Exactive HF mass spectrometer in positive mode. Peptides were separated using an in-house packed 75 μm x 50 cm pulled column (1.9 μm particle size, C18AQ; Dr Maisch, Germany) with a gradient of 2 – 30% acetonitrile containing 0.1% FA over 90 min (ACAD11 interactome) or 180 min (oligonucleotide treated livers) at 300 nl/min at 55°C. An MS1 scan was acquired from 300 – 1650 m/z (60,000 resolution, 3e6 AGC, 50 ms injection time) followed by MS/MS data-dependent acquisition with HCD (1e5 AGC, 25 ms injection time, 27% normalized collision energy, 1.2 m/z quadrupole isolation width). LFQ data were processed with MaxQuant (v188.8.131.52) using Andromeda against the UniProt human database. All parameters were default and quantification performed with MaxLFQ including match between runs [PMID: 24942700].
Parker BL, Calkin AC, Seldin MM, Keating MF, Tarling EJ, Yang P, Moody SC, Liu Y, Zerenturk EJ, Needham EJ, Miller ML, Clifford BL, Morand P, Watt MJ, Meex RCR, Peng KY, Lee R, Jayawardana K, Pan C, Mellett NA, Weir JM, Lazarus R, Lusis AJ, Meikle PJ, James DE, de Aguiar Vallim TQ, Drew BG. An integrative systems genetic analysis of mammalian lipid metabolism. Nature. 2019 PubMed: 30814737