MassIVE MSV000086978

Description

Sample description: Anti-PD-L1 co-immunoprecipitation samples from cultured wild-type, PD-L1-VavCre or PD-L1-LysMCre bone marrow cells. Samples are: KO-VavCre-1 (775112) v.s. WT-1 (775113); KO-LysMCre-2 (775114) v.s. WT-2 (775115). Among these samples, KO-VavCre-1 and KO-LysMCre-2 are the respective blank controls (from knockout mice). Three 10-cm plates of bone marrow cells from each genotype (wild-type or PD-L1-VavCre, and wild-type or PD-L1-LysMCre) of 2-month-old male littermate mice at a density of 2.8 * 107 cells/plate were cultured in the presence of 40 ng/mL of mouse M-CSF for 3 days. Cells were washed with PBS and resuspend in lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM PMSF, 1x protease inhibitor cocktail) for 4 hours at 4C. The lysate was centrifuged at 12,000 g for 10 min. The supernatant was mixed with 2 micro G of anti-PD-L1 antibody and incubated for overnight at 4C. Protein A/G agarose (Santa Cruz) was added and incubated for 2 hours at 4C. Agarose was washed with lysis buffer for four times. The samples were separated in SDS-PAGE gel (10-mm length per lane). Coomassie blue stained gel slices were analyzed by mass spectrometry. Samples were digested overnight with trypsin (Pierce) following reduction and alkylation with DTT and iodoacetamide (Sigma-Aldrich). The samples then underwent solid-phase extraction cleanup with an Oasis HLB plate (Waters) and were subsequently dried and reconstituted into 10 microL of 2% ACN, 0.1% TFA. 2 microL of these samples were injected onto a QExactive HF mass spectrometer coupled to an Ultimate 3000 RSLC-Nano liquid chromatography system. Samples were injected onto a 75 micro M i.d., 15-cm long EasySpray column (Thermo) and eluted with a gradient from 0-28% buffer B over 90 min with a flow rate of 250 nL/min. Buffer A contained 2% (v/v) ACN and 0.1% formic acid in water, and buffer B contained 80% (v/v) ACN, 10% (v/v) trifluoroethanol, and 0.1% formic acid in water. The mass spectrometer operated in positive ion mode with a source voltage of 2.2 kV and an ion transfer tube temperature of 275 C. MS scans were acquired at 120,000 resolution in the Orbitrap and up to 20 MS/MS spectra were obtained for each full spectrum acquired using higher-energy collisional dissociation (HCD) for ions with charges 2-8. Dynamic exclusion was set for 20 second after an ion was selected for fragmentation. [doi:10.25345/C5H217] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: PD-L1, breast cancer, melanoma, osteoclast, bone metastasis, immunotherapy

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