Mascot ver2

Peptides were identified using tandem MS (MS² or MS/MS) experiments performed with a LTQ linear ion trap mass spectrometer (LTQ Orbitrap XL with ETD, Thermo Fisher, San Jose, CA) over a 70 min gradient. Product ion spectra were acquired in a data-dependent mode and the five most abundant ions were selected for the product ion analysis per scan event. The MS/MS *.raw data files were converted to *.mgf files and then submitted to MASCOT ver2.3 (Matrix Science, London, UK) for peptide identification. The maximum number of missed cleavage was set at 4 with the mass tolerance for precursor ions +/−0.6 Da and for fragment ions ± 8 ppm. Oxidation of methionine was selected for variable modification. Pepsin was used for digestion and no specific enzyme was selected in the MASCOT during the search. Peptides included in the peptide set used for HDX detection had a MASCOT score of 20 or greater. The MS/MS MASCOT search was also performed against a decoy (reverse) sequence and false positives were ruled out. The MS/MS spectra of all the peptide ions from the MASCOT search were further manually inspected and only the unique charged ions with the highest MASCOT score were used in estimating the sequence coverage and included in HDX peptide set.

Peptides were identified using tandem MS (MS² or MS/MS) experiments performed with a LTQ linear ion trap mass spectrometer (LTQ Orbitrap XL with ETD, ThermoFisher, San Jose, CA) over a 70 min gradient. Product ion spectra were acquired in a data-dependent mode and the five most abundant ions were selected for the product ion analysis per scan event. The MS/MS *.raw data files were converted to *.mgf files and then submitted to MASCOT ver2.3 (Matrix Science, London, UK) for peptide identification. The maximum number of missed cleavage was set at four with the mass tolerance for precursor ions ± 0.6 Da and for fragment ions ± 8 ppm. Oxidation to Methionine was selected for variable modification. Pepsin was used for digestion and no specific enzyme was selected in the MASCOT during the search. Peptides included in the peptide set used for HDX detection had a MASCOT score of 20 or greater. The MS/MS MASCOT search was also performed against a decoy (reverse) sequence and false positives were ruled out. The MS/MS spectra of all the peptide ions from the MASCOT search were further manually inspected and only the unique charged ions with the highest MASCOT score were used in estimating the sequence coverage and included in HDX peptide set.

To identify the ubiquitination sites of EGFR mediated by ZNRF1 and CBL, EGFR was isolated by SDS-PAGE followed by in-gel enzymatic digestion with a mixture of trypsin and chymotrypsin. The digested peptides were analyzed by nanoflow LC-MS/MS on an LTQ-FT hybrid mass spectrometer (Thermo Fisher Scientific) equipped with a nano-electrospray ion source (New Objective, Inc., Woburn, MA, United States) in positive ion mode. The liquid chromatography system used was the Agilent 1100 HPLC with the Famos Autosampler (LC Packings, Amsterdam, Netherlands). The digested peptide samples were subjected to nanoflow-LC-MS/MS as described previously (Chang et al., 2019 (link)). All experimental RAW files were converted to MGF format by MSConvert (ProteoWizard ver. 3.0.9134) (Chambers et al., 2012 (link)) and then submitted for MS/MS ion search on Mascot (ver. 2.3) (MatrixScience, Boston, MA, United States). The protein sequences of Homo sapiens from UniprotKB¹ were used for MS/MS data analysis. The search parameters of error tolerance of precursor ions and the MS/MS fragment ions in spectra were 10 ppm and 0.6 Da, respectively. The variable post-translational modifications of search parameters in Mascot include ubiquitination of lysine (GlyGly), carbamidomethylation of cysteine, the oxidation of methionine, and phosphorylation of serine/threonine/tyrosine.