Computational protocol: Constitutive activated Cdc42-associated kinase (Ack) phosphorylation at arrested endocytic clathrin-coated pits of cells that lack dynamin

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Protocol publication

[…] Equal numbers of DKO fibroblasts and WT controls were grown in heavy (supplemented with l-lysine [13C6]) and light (supplemented with regular l-lysine) SILAC DMEM medium (Thermo Fisher Pierce, Rockford, IL). The cells were grown for at least five cell divisions in heavy medium to ensure sufficient incorporation before adding 4-hydroxytamoxifen to generate DKO cells. Cell lysis and phosphopeptide enrichment were performed according to the PhosphoScan kit protocol (Cell Signaling, Beverly, MA), except that the lysates were subjected to digestion using Lys-C protease (lysyl endopeptidase; Wako, Richmond, VA). Phosphopeptides were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) on an LTQ-Orbitrap Discovery hybrid linear ion trap (Thermo Fisher Pierce, Rockford, IL) using a top-10 method. For each scan cycle, one high-resolution full MS scan was acquired in the Orbitrap mass analyzer, and up to 10 parent ions were chosen based on intensity for MS/MS analysis in the linear ion trap.MS/MS spectra were searched using the SEQUEST (Eng et al., ) algorithm (v.27, rev.13) against a composite mouse database (IPI v3.60) and its reversed complement. Search parameters specified lys-C digestion, a mass tolerance of 25 ppm, a static modification of 57.02146 Da on cysteine, and dynamic modifications of 15.99491 Da on methionine, 6.02013 Da on lysine, and 79.96633 Da on serine, threonine, and tyrosine. Search results were filtered to a 1% peptide false discovery rate by restricting the mass tolerance window, and setting thresholds for Xcorr and dCn. For all resulting peptides, a heavy/light abundance ratio was calculated using Vista. The data were further filtered to require a signal-to-noise ratio ≥ 3 for both heavy and light versions of each peptide. The confidence of phosphorylation site assignment was measured by applying the Ascore algorithm (Beausoleil et al., ). […]

Pipeline specifications

Software tools PhosphoScan, Comet, Ascore
Application MS-based untargeted proteomics
Organisms Mus musculus, Saccharomyces cerevisiae