Recombinant MBP-tagged PER2 was expressed and purified as described above. Three micrograms of MBP-PER2 was buffer exchanged into 25 mM ammonium bicarbonate (pH 8.0). The sample was dried using a speed-vac, reconstituted in 25 mM ammonium bicarbonate (pH 8.0):50% acetonitrile, and incubated at 37°C for 1 hour. Trypsin, Arg-C, or Asp-N (500 ng each; Promega) was added, and samples were digested overnight at 37°C. The resulting peptides were dried and reconstituted in 25 mM ammonium bicarbonate:5% acetonitrile. Samples were loaded onto a C18 column (2-μm particles, 25-cm by 75-μm inner diameter) and eluted using a 2-hour acetonitrile gradient into a Q Exactive HF-X mass spectrometer, equipped with a nanospray source (flow at 350 nl/min). Full MS resolutions were set to 60,000 at 200 m/z (mass/charge ratio), full MS automatic gain control (AGC) target was 3 × 106, and mass range was set to 300 to 1400. AGC target value for fragment spectra was set at 1 × 105, intensity threshold was set at 2 × 105, and isolation width was at 1.3 m/z. Normalized collision energy was set at 28% (34). The mass spectra from each sample were searched against the UniProt human database and a custom database containing the sequence of our MBP-PER2 construct using Proteome Discoverer (version 2.2). Precursor mass tolerance was set to 10 parts per million, fragment mass tolerance was set at 0.02 Da, Delta Cn of 0.05, false discovery rate of 0.01, minimum peptide length of 6, and a minimum number of peptides of 2.

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