Posttranslational modification of CTNNBL1 protein
Description
Posttranslational modification of and ubiquitinated peptides in CTNNBL1 during HIV-1 infection. Posttranslational modification analysis of CTNNBL1 protein by phosphorylation, acetylation, ubiquitination, succinylation, crotonylation, and methylation
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Gel pieces were destained in 50 mM NH4HCO3 in 50% ACN (v/v) until clear for in-gel tryptic digestion. Gel pieces were dehydrated with 100 μL of 100% ACN for 5 min, liquid removed, rehydrated in 10 mM DTT, and incubated at 37°C for 60 min. Gel pieces were again dehydrated in 100% ACN, liquid removed, and rehydrated with 55 mM IAA. The samples were incubated at room temperature in the dark for 45 min. Gel pieces were washed with 50 mM NH4HCO3 and dehydrated with 100% ACN. The pieces were rehydrated with 10 ng/μL trypsin and resuspended in 50 mM NH4HCO3 on ice for 1 h. Excess liquid was removed, and gel pieces were digested with trypsin at 37°C overnight. Peptides were extracted with 50% ACN/5% FA, followed by 100% ACN. Peptides were dried to completion and resuspended in 2% ACN/0.1% FA. Tryptic peptides were dissolved in 0.1% FA and directly loaded onto a reverse-phase precolumn (Acclaim PepMap 100; Thermo Fisher Scientific). Peptide separation used a reverse-phase analytical column (Acclaim PepMap RSLC; Thermo Fisher Scientific). Elution used a 6% to 25% gradient solvent B (0.1% FA in 98% ACN) over 16 min, 25% to 40% over 6 min, and 80% over 4 min then holding at 80% for the last 4 min. Flow rate was constant at 320 nL/min using an EASY-nLC 1000 UPLC system. Peptides were subjected to nanospray ionization, followed by tandem MS (MS/MS) with Q-Exactive (Thermo Fisher Scientific) coupled online to the UPLC. Intact peptides were detected in an orbitrap at a resolution of 70,000. Peptides were selected for MS/MS using a normalized collision energy setting of 28; ion fragments were detected at a resolution of 17,500. A data-dependent procedure alternated between 1 and 20 MS/MS scans was employed for the top 20 precursor ions above a threshold ion count of 5E3 in the MS survey scan with 15 s dynamic exclusion. The electrospray voltage was 2.0 kV. AGC was used to prevent the overfilling of the orbitrap; 5E4 ions were accumulated for the generation of MS/MS spectra. The m/z scan range was from 350 to 1800. The fixed first mass was set to 100 m/z. MS/MS data were processed with the Maxquant search engine (version 1.5.2.8). MS/MS were searched against the CTNNBL1 sequence. Trypsin/P was specified as the cleavage enzyme, allowing up to four missing cleavages. The mass error was set to 10 ppm for precursor ions and 0.02 Da for ion fragments. Cys carbamidomethylation was specified as a fixed modification, and methionine oxidation, lysine ubiquitination, serine, threonine, and tyrosine phosphorylation, and protein N-term acetylation were specified as variable modifications. The site localization probability was set to >0.75.