Computational protocol: Identifying the substrate proteins of U-box E3s E4B and CHIP by orthogonal ubiquitin transfer

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Protocol publication

[…] After washing, the streptavidin beads were spun down, residual urea was removed, and liquid chromatography coupled to tandem MS (LC-MS/MS) on an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) was performed at the Emory Integrated Proteomics Core (EIPC) according to previously published methods (, ). Collected spectra were searched using Proteome Discoverer 2.0 against human UniProt database (90,300 target sequences). Searching parameters included fully tryptic restriction and a parent ion mass tolerance (±20 parts per million). Methionine oxidation (+15.99492 Da), asparagine and glutamine deamidation (+0.98402 Da), lysine ubiquitination (+114.04293 Da), and protein N-terminal acetylation (+42.03670) were variable modifications (up to three were allowed per peptide); cysteine was assigned a fixed carbamidomethyl modification (+57.021465 Da). Percolator was used to filter the PSMs to a false discovery rate of 1%. […]

Pipeline specifications

Software tools Proteome Discoverer, Percolator
Application MS-based untargeted proteomics
Organisms Homo sapiens
Diseases Ovarian Neoplasms
Chemicals Acetaldehyde