FIG 1.

Deep ligand sequencing and validation of peptide ligands recovered from class I HLA of HIV-1-infected CD4⁺ T cells. (A) Deep ligand sequencing work flow whereby SUP-T1 CD4⁺ T cells are infected with HIV-1_NL4-3. Soluble HLA is purified by immunoaffinity chromatography, and HLA is separated from bound peptides by acid boil and size exclusion filtration (3-kDa cutoff). Directly eluted peptides are then fractionated at high pH. mAU, milli-absorbance units; RP-HPLC, reverse-phase high-pressure liquid chromatography. (B) Fractions are further separated at low pH and directly injected into an AB Sciex 5600 mass spectrometer to generate LC-MS spectra and corresponding information dependent acquisition (IDA)-generated MS/MS spectra. PEAKS software, using a 1% false discovery rate, assigns the ligand sequence for synthesis. (C) Finally, ligands undergo a three-step validation process to include comparison of synthetic MS/MS spectra, IC₅₀ determination by competitive binding assay, and the extent of T cell reactivity by ELISPOT assay testing against patient PBMCs.