Proteomic analysis of Psmd9 knockdown in the liver
We have shown that decreasing psmd9 expression effects lipid metabolism in vivo and were interested in investigating the pathways by which psmd9 elicited these changes. To investigate these mechanisms, we performed proteomic analyses on the livers from C57 and DBA mice that had been treated with antisense oligonucleotides targeted to psmd9.
Sample Processing Protocol
Frozen liver (10 mg) was homogensized in 6 M urea, 2 M thiourea, 50 mM triethylammonium bicarbonate (TEAB) pH 8.0 containing protease inhibitor cocktail (Roche; 11873580001) by tip-probe sonication and centrifuged at 16,000 x g, 10 min at 4°C. Lysates were precipitated with 6 volumes of acetone overnight at -30°C. Protein pellets were centrifuged at 10,000 x g, 10 min at 4°C resuspended in 6 M urea, 2 M thiourea, 50 mM TEAB, pH 7.9 and quantified by Qubit fluorescence (Thermo Fisher; Q33212). Concentrations were normalized and 100 µg of protein reduced with 10 mM dithiothreitol for 60 min at 25ºC followed by alkylation with 25 mM iodoacetamide for 30 min at 25ºC in the dark. The reaction was quenched to a final concentration of 20 mM dithiothreitol and digested with lysyl endopeptidase Lys-C (Wako Pure Chemical Industries; 125-05061) at 1:50 enzyme to substrate ratio for 2 h at 25ºC. The mixture was diluted 5-fold with 25 mM TEAB and digested with trypsin (Promega; V5111) at 1:50 enzyme to substrate ratio for 16 h at 30ºC. The peptide mixture was acidified to a final concentration of 2% formic acid, 0.1% trifuoroacetic acid (TFA) and centrifuged at 16,000 x g for 15 min. Peptides were desalted using hydrophilic lipophilic balance – solid phase extraction (HLB-SPE) 96-well plates (Waters; 186000128) followed by elution with 50% acetonitrile, 0.1% TFA and dried by vacuum centrifugation. For proteomic analysis of adenovirus infected livers by label-free quantification (LFQ), purified peptides were resuspended in 2% acetonitrile, 0.1% TFA quantified by Qubit and normalized to 1 µg / 3 µl. A pooled sample was fractionated into 12 fractions using amide-80 HILIC [PMID: 21127490]. One microgram of each replicate was analysed by single shot in addition to the fractionated peptides.
Data Processing Protocol
Peptides were analyzed on a Easy nLC-1200 coupled to Q-Exative HF mass spectrometer in positive mode. For proteomic analysis of antisense oligonucleotide treated livers by LFQ, 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 180 min at 300 nl/min at 55°C. An MS1 scan was acquired from 300 – 1500 m/z (60,000 resolution, 3e5 AGC, 50 ms injection time) followed by MS/MS data-dependent acquisition with HCD and detection in the orbitrap (1e5 AGC, 25 ms injection time, 27% normalized collision energy, 1.4 m/z quadrupole isolation width). LFQ data from the adenovirus treated livers were processed with MaxQuant (v22.214.171.124) using Andromeda [PMID; 19029910] against the UniProt mouse database (March 2017, 59,038). All settings were default with precursor-ion and product-ion tolerance set to 20 ppm and 0.02 Da, respectively. Full trypsin specificity was employed with a maximum of 2-missed cleavages and peptides searched with oxidation of methionine and acetylation on protein N-terminus set as variable modifications, and carbamidomethylation of cysteine set as fixed modification. All data was searched as a single batch with PSMs and protein FDR set to 1% using a target decoy approach. The match between runs and label-free quantification (MaxLFQ) options were selected and included matching into the 12 pooled fractionated peptides [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