Identification of ubiquitination site in BCL-XL after PROTAC DT2216 treatment
The availability of a suitable lysine for PROTAC-mediated ubiquitination can also determine the degradative ability of a protein target and the selectivity of a PROTAC. PROTAC DT2216 selectively triggers ubiquitination and degradation of BCL-XL. In this study, we identify K87 is the key ubiquitinated lysine under DT2216 treatment through LC-MS/MS.
Sample Processing Protocol
Enrichment of FLAG-tagged protein for mass spectrometry. HEK293T cells were co-transfected with Flag-BCL-XL(pDL2009) and HA-Ub vectors. Proteins were extracted by using IP lysis buffer and then subjected to immunoprecipitation using Pierce™ Anti-DYKDDDDK Affinity Resin (Cat. No. A36801, Thermal Fisher Scientific) according to the manufacturer’s protocol. Anti-DYKDDDDK Affinity Resin was washed with 1X TBS three times and then added to protein samples, and the mixture was incubated at 4 °C with rotation for 4 h. The immunoprecipitated samples were centrifuged at 1000×g for 1 min at 4 °C, and then washed three times with 1X TBS. Immunoprecipitated samples were eluted with 0.1 M glycine HCl (pH 2.8). Sample preparation and LC-MS/MS. Purified proteins were reduced, alkylated, and digested using filter-aided sample preparation58. Resulting peptides were then separated by reverse phase XSelect CSH C18 2.5 um resin (Waters) on an in-line 150 x 0.075 mm column using an UltiMate 3000 RSLCnano system (Thermo Fisher Scientific). Peptides were eluted using a 90 min gradient from 97:3 to 60:40 buffer A:B ratio A (buffer A: 0.1% formic acid and 0.5% acetonitrile in water, buffer B: 0.1% formic acid in acetonitrile). Eluted peptides were ionized by electrospray (2.15 kV) followed by MS/MS analysis using higher-energy collisional dissociation (HCD) on an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) in top-speed data-dependent mode. MS data were acquired using the FTMS analyzer in profile mode at a resolution of 240,000 over a range of 375 to 1500 m/z. Following HCD activation, MS/MS data were acquired using the ion trap analyzer in centroid mode and normal mass range with precursor mass-dependent normalized collision energy between 28.0 and 31.0.
Data Processing Protocol
Proteomics data analysis. Proteins were identified by database search against human Uniprot database (73911 proteins, March 16, 2019) using Mascot with a parent ion tolerance of 3 ppm and a fragment ion tolerance of 0.5 Da. Methionine oxidation (+15.99492 Da), protein N-terminal acetylation (+42.03670) and lysine ubiquitination (+114.04293 Da) were variable modifications; cysteine was assigned a fixed carbamidomethyl modification (+57.021465 Da). Percolator was used to filter the peptide spectrum matches to a false discovery rate of 1%. Scaffold (Proteome Software) was used to verify MS/MS based peptide and protein identifications. Protein identifications were accepted if they could be established with less than 1.0% false discovery and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm59