Mass Spec Molecular Oncology Charpentier et al., 2021
These files contain the full analysed mass spec data of the results presented in Charpentier et al., 2021 Molecular Oncology. We focused on MELOE-1, a melanoma neoantigen whose expression is restricted to tumor cells as a result of lineage-specific transcription and tumor-specific IRES-dependent translation of the polycistronic meloe RNA (Godet et al., 2008 and Bobinet et al., 2013). Our previous study on MELOE-1 translation reported the presence of an IRES activity located around 250nt upstream of MELOE-1 ORF (Carbonnelle et al., 2013). In the present work, we looked for the ITAF(s) that could bind to this region. To this aim, we in vitro transcribed the 275nt RNA sequence upstream of MELOE-1 ORF, biotinylated it and coupled it to a streptavidin BIAcore chip. We then prepared whole cell lysates from three melanoma cell-lines M117, M134 and M170, ran them on the RNA-coated BIAcore chip (40 rounds of 5 min injections) and then recovered the eluted material for mass spectrometry analysis. references: Godet Y et al. MELOE-1 is a new antigen overexpressed in melanomas and involved in adoptive T cell transfer efficiency. J Exp Med 2008;205(11):2673–2682. Bobinet M et al. Overexpression of Meloe Gene in Melanomas Is Controlled Both by Specific Transcription Factors and Hypomethylation. PLoS ONE 2013;8(9):e75421. Carbonnelle D et al. The Melanoma Antigens MELOE-1 and MELOE-2 Are Translated from a Bona Fide Polycistronic mRNA Containing Functional IRES Sequences. PLoS ONE 2013;8(9).
Steps to reproduce
In vitro transcription MELOE-1 intercistronic region IR1215-1490 (275nt IRES region)  was cloned into a pBSSK vector under the control of a T7 promoter. It was linearized with NotI and in vitro transcribed according to the MegaShort Script Kit (Ambion) protocol. RNA (0.2 nmol) was biotinylated using the 5’end modification kit and biotin maleimide (Vector Laboratories) following the manufacturer’s guidelines. Micro-recovery analysis for further MS identification The biosensor used in this study was a Biacore T200 instrument (GE Healthcare). Streptavidin Research Grade Sensor Chips (carboxy-methyl-dextran derivatized with streptavidin surface) and HBS-N (0.01 HEPES, pH 7.4, 0.15 M NaCl) running buffer were also purchased from GE Healthcare and used in all BIA/MS experiments. All SPR experiments were performed at a flow rate of 5 µl/min at 37°C. The 275nt biotinylated RNA sequence upstream of MELOE-1 ORF was coupled onto the streptavidin surface following the standard biotin-streptavidin coupling protocol (according to supplier’s procedures) to achieve a residual coupling response of around 1500 RU. Fresh whole cell lysates (10Mm Tris HCl pH=7.4, 1.5Mm MgCl2, 1Mm KCl, 0.5Mm DTT, 0.05% NP40 supplemented with protease inhibitor) from three melanoma cell-lines M117, M134 and M170 were diluted 10-fold in HBS-N and injected over the RNA-coated chip for 5 minutes. Following the end of sample injection, we proceeded to a micro-recovery method to elute bound molecules from the biosensor i.e., we injected a small amount (1µL) of elution solvent (30mM NaOH) separated from the running buffer by two air bubbles. Using this sandwich elution, bound molecules were recovered without dispersion. Each lysate sample was run for 40 cycles/rounds using this micro-recovery procedure in order to recover enough bound materials for further mass spectrometry identification. Recovered material from 40 rounds micro-recovery procedures eluted in Low Binding tubes (Protein LoBind tubes, Eppendorf) were frozen and subsequently analyzed by nanoliquid chromatography coupled with tandem mass spectrometry (NanoLC-MS/MS).