Diurnal regulatory landscape of the mouse liver nucleus
To dissect how diurnal rhythms affect key functions such as transcription or chromatin remodeling, we quantified the temporal nuclear accumulation of proteins and phosphoproteins from mouse liver by SILAC-based MS. Protein extracts from isotope labelled mice liver nuclei were used as a reference and mixed with extracts from animals collected every 3h for 45h total. Total protein levels were analysed together with phosphopeptides after enrichment (see separate dataset for phospho data).
Sample Processing Protocol
After reduction/alkylation of cysteines, proteins were precipitated, resuspended in 8M urea and digested with trypsin overnight. Peptides were desalted on C18 cartridges and fractionated (12 fractions) by off-gel focusing. After cleanup, fractions were analysed by nano-LC-MS/MS on a LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher, Bremen, Germany) interfaced to a Dionex RSLC 3000 nanoHPLC system (Dionex, Sunnyvale, CA, USA). Two pairs of fractions with low peptide content (fr.7+8 and 9+10) were pooled for analysis. Peptides were separated on a C18 Pepmap nanocolumn (75 μm ID x 25 cm, 2.0 μm, 100Åµ, Dionex) with a gradient from 5 to 85 % acetonitrile (total time: 120 min) and a flow rate of 0.3 ul/min. Full MS scans were performed at 60’000 resolution. Data-dependent acquisition targeted the 20 most intense precursor ions for CID fragmentation in the LTQ linear trap (dynamic exclusion 120s).
Data Processing Protocol
MS data were analyzed and quantified with MaxQuant 126.96.36.199, using Andromeda as search software against UniProt (release 2012_02) database restricted to Mouse (Mus musculus) taxonomy and a custom database containing usual contaminants (digestion enzymes, keratins, etc). Cleavage specificity was trypsin/P (cleavage after K, R, including KP and RP) with two missed cleavages. Mass tolerances were of 6 ppm for the precursor and 0.5 Da for CID tandem mass spectra. The iodoacetamide derivative of cysteine was specified as a fixed modification, and oxidation of methionine and protein N-terminal acetylation were specified as variable modifications. Protein identifications were filtered at 1% FDR established by MaxQuant against a reversed sequence database. A minimum of one unique peptide was necessary to discriminate sequences which shared peptides.
Wang J, Mauvoisin D, Martin E, Atger F, Galindo AN, Dayon L, Sizzano F, Palini A, Kussmann M, Waridel P, Quadroni M, Dulić V, Naef F, Gachon F. Nuclear Proteomics Uncovers Diurnal Regulatory Landscapes in Mouse Liver. Cell Metab. 2016 Oct 31. pii: S1550-4131(16)30534-4 PubMed: 27818260