Computational protocol: Cooperative Interaction between Phosphorylation Sites on PERIOD Maintains Circadian Period in Drosophila

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Protocol publication

[…] Protein samples were excised from the Coomassie-blue gel and subjected to in-gel digestion with sequencing grade 12.5 ng/µl trypsin (Promega) in 50 mM ammonium bicarbonate pH 8.2 overnight at 37°C. Peptides were extracted with 50% acetonitrile (MeCN), 5% formic acid (FA) and then dried. Dried peptides were resuspended in 10 µL of 5%MeCN, 4% formic acid (FA), and 2 µL was loaded onto a pulled fused silica microcapillary column packed with C18 reverse-phase resin (Magic C18AQ; 5-µm particles; 200-Å pore size; Michrom Bioresources) using a Famos autosampler (LC Packings). Once loaded, the peptides were separated using an Agilent 1100 series binary pump across a 45 min linear gradient of 6–28% CH3CN in 0.125% HCOOH at a flow rate of 600 nL/min.Mass spectra were acquired in an LTQ-Orbitrap hybrid mass spectrometer (Thermo Fisher) over the entire run using ten MS/MS scans following each survey scan. Raw data were searched against the HA-Per amino acid sequence using Sequest software with no enzyme specificity and a 1.1 Da mass tolerance. The search parameters used for post-translational modification included a static modification of 57.02146 Da on cysteine (carboxyamidomethylation) and dynamic modifications of 15.99491 Da on methionine (oxidation) and 79.96633 Da on serine, threonine, and tyrosine (phosphorylation). The probability of correct phosphorylation site localization was determined for every site in each peptide using the AScore algorithm as described in . […]

Pipeline specifications

Software tools Comet, Ascore
Application MS-based untargeted proteomics
Organisms Drosophila melanogaster, Homo sapiens