4.3. Proximity-Dependent Biotin Identification (BioID) Assay

Cloning and construction of the Tet-on inducible BioID construct were previously described [21]. Briefly, the pRetroX-mycBioID-MCS vector was cloned from the pcDNA3.1mycBioID plasmid (Addgene plasmid #35700) and pRetroX-Tight-Pur (Clontech). The full-length human MEN1 cDNA from pBABE hygro MEN1 WT (Addgene plasmid #11024) was cloned into pRetroX-mycBioID-MCS at Not1 and Mlu I site by the Gibson reaction (New England Biolabs, Ipswich, MA, USA) to obtain pRetroX-mycBirA-MEN1. Engineered parental T47D and MCF7 cell lines that stably express a Tet repressor were infected with mycBirA-MEN1 retrovirus and stable cell lines were obtained under the selection of G418 (600 μg/mL for T47D and 300 μg/mL for MCF-7) and puromycin (1 μg/mL). To induce mycBirA-menin protein expression, stable T47D and MCF-7 cells were incubated in the medium with 2 μg/mL of doxycycline for 24 h and then in the medium with 50 mM biotin for another 24 h. For WES, the total lysate was collected by the RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing the protease/phosphatase inhibitor (Thermo Fisher Scientific, Waltham, MA, USA); the cytoplasmic and nuclear lysates were isolated by NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Waltham, MA, USA). For LC–MS/MS, cells were lysed by the RIPA buffer containing protease/phosphatase inhibitors. Free biotins were filtered out by 10 kDa filters (Sigma-Aldrich, St. Louis, MO, USA) and the biotin-labeled proteins were captured by incubation with 200 μL of streptavidin magnetic beads (Thermo Fisher Scientific, Waltham, MA, USA) for 5 h at 4 °C and collected by a magnetic stand. The beads were subject to 5 washes with 1 mL of RIPA buffer for 10 min at 4 °C and then 4 washes with PBS for 10 min at room temperature (RT). About 10% of the PBS-beads mixture was boiled for 10 min to release the proteins and the supernatant proteins were analyzed using WES. After removing the supernatant from the remaining mixture, beads were kept at −80 °C until further mass spectrometry analysis in the Proteomics Core at the Sanford-Burnham Prebys Medical Discovery Institute.

The total sample peptide amount was evaluated by Pierce Quantitative Colorimetric Peptide Assay, 500 Assays (Thermo Fisher Scientific, Waltham, MA, USA) before loaded into LC–MS. To reconstitute dried samples, 2% acetonitrile and 0.1% formic acid were used and reconstituted samples were analyzed by LC–MS/MS using a Proxeon EASY nanoLC system (Thermo Fisher Scientific, Waltham, MA, USA) coupled to an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). An analytical C18 Acclaim PepMap column (75 µm × 500 mm, 2 µm particles; Thermo Fisher Scientific) was used to separate peptides at a rate of 300 nL min⁻¹ for over 121 min: 1–6% B in 1 min, 6–23% B in 56 min, 23–34% B in 37 min, 34–48% B in 26 min, and 48–98% B in 1 min (A = FA 0.1%; B = 80% ACN: 0.1% FA).

Operation of the mass spectrometer was set up in data-dependent acquisition mode. Spectra of the first spectrometer (MS1) were measured by Orbitrap analyzer with a resolution of 60,000 (automatic gain control (AGC) target of 1e6 and a mass range from 350 to 1450 m/z). Collision-induced dissociation method was used to generate fragments for the second spectrometer (MS2) and data was acquired by the linear ion trap (AGC target of 1e4, isolation window of 2 m/z, and a normalized collision energy of 35). The duration of dynamic exclusion was enabled with 30 s.

MaxQuant software (v1.5.5.1) was used to process MS raw files with most of the default settings, and the Andromeda search engine was used to search against the human UniProt database and GMP cRAP sequences (the common contaminants database). Variable modifications were set to methionine oxidation and N-terminal acetylation, while fixed modification was set to carbamidomethylation. The minimum ratio count was set to two for label-free protein quantification, and peptides for quantification were set to unique and razor. Proteins and peptides were selected based on the false discovery rate (FDR) set to 0.01, with a minimum length of seven amino acids.

We analyzed the label free quantitation (LFQ) intensity and defined the biotinylated differential proteins (menin associated proteins, MAPs) as ≥3-fold in doxycycline⁺/biotin⁺ cells compared to the doxycycline⁺/biotin⁻ cells’ counterpart. The heat map was generated using log(LFQ intensity+1). The network analysis was performed by R package igraph and the distance was defined as the ratio between the LFQ intensity of each MAPs and menin.