In-gel digestion was performed by using chymotrypsin/AspN, AspN/typsin/Lys-C, or chymotrypsin/trypsin/LysC mix (Promega). Obtained peptides were analyzed by nanoflow reverse-phase LC followed by MS, using an LTQ XL ion trap mass spectrometer (Thermo Fisher). A capillary reverse-phase HPLC-MS/MS system is composed of an Agilent 1100 series gradient pump equipped with Valco C2 valves with 150-μm ports and an LTQ XL ion trap mass spectrometer equipped with an XYZ nanoelectrospray ionization source (AMR). Samples were automatically injected using the PAL system (CTC analytics) directly using a peptide L-trap column (Trap and Elute mode, Chemical Evaluation Research Institute) attached to an injector valve for desalting and concentrating peptides. After washing the trap with solvent C, the peptides were loaded into a separation capillary reverse-phase column (MAGIC C18 packed with gel particles of 3 μm in diameter and 200-Å pore size, 150 mm by 0.2 mm; Michrom BioResources) by switching the valve. The eluents used were as follows: A, 100% water containing 0.1% formic acid, and B, 100% acetonitrile containing 0.1% formic acid. The column was developed at a flow rate of 1 μl/min with a concentration gradient of acetonitrile: from 5% B to 40% B in 20 min, then from 40% B to 95% B in 1 min, sustaining 95% B for 3 min, from 95% B to 5% B in 1 min, and finally re-equilibrating with 5% B for 20 min. Xcalibur 2.2 system (Thermo) was used to record peptide spectra over the mass range of m/z 350 to 1500 and MS/MS spectra in information-dependent data acquisition over the mass range of m/z 150 to 2000. Repeatedly, MS spectra were recorded followed by three data-dependent CID-MS/MS spectra generated from three highest intensity precursor ions. Multiple charged peptides were chosen for MS/MS experiments because of their excellent fragmentation characteristics.

Searches were performed by using the Mascot server (ver. 2.4.0, Matrix Science) against mouse PER2, CRY1, CLOCK, or BMAL1 peptide sequence for phosphorylation site identification. Search parameters were set as follows: enzyme selected as used with three maximum missing cleavage sites, a mass tolerance of 2 Da for peptide tolerance, 0.8 Da for MS/MS tolerance, fixed modification of carbamidomethyl (C), and variable modification of oxidation (M) and phosphorylation (S, T, Y). The maximum expectation value for accepting individual peptide ion scores [−10*log(p)] was set to ≤0.05. Phosphorylation site information returned from Mascot was manually inspected and filtered to obtain confirmed phosphorylation lists of CID-MS/MS.

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