MHC peptide elution mass spectrometry

Two healthy donor-derived Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs), expressing a number of common HLA class I alleles (LCL 1 = HLA-A*01:01, −A*03:01, -B*08:01, -B*35:01 and LCL 2 = HLA-A*02:01, −A*11:01, -B*07:02, -B*44:02), were retrovirally transduced with a pMX-mCALR-IRES-eGFP or pMX-eGFP alone vector. GFP+ LCLs were sorted to a high purity and expanded to approximately 5 × 10⁸ cells before being washed with PBS and snap-frozen. Subsequent immunoprecipitation of HLA class I molecules, elution of bound peptides, and analysis of peptides by liquid chromatography tandem mass spectrometry (LC-MS/MS) was carried out as previously described [11]. Briefly, cell pellets were ground using a Retsch mixer mill (MM400) and then lysed in lysis buffer (0.5% IGEPAL CA-630 [Sigma-Aldrich], 50 mM Tris-HCl [pH 8.0], 150 mM NaCl, and protease inhibitors [complete protease inhibitor cocktail; Roche Life Science]) for 1 h at 4 °C. Lysate was pre-cleared by passing over unconjugated protein A sepharose [GE Healthcare] and then MHC:peptide complexes were captured by protein A-coupled pan-MHC class I-specific antibody W6/32. Dissociation of heavy chain, beta-2 microglobulin and peptide was then achieved using 10% acetic acid. Eluted MHC-peptides were subsequently purified by fractionation using reversed phased HPLC (RP-HPLC; Chromolith Speed Rod (Merck)) using an ÄKTAmicro HPLC system [GE Healthcare], as described elsewhere [11].

Fractionated peptides were vacuum concentrated using a Labconco Centrivac concentrator and resuspended to a final volume of 20 μL in 0.1% formic acid in water prior to LC-MS/MS analysis using an Eksigent NanoUltra cHiPLC system [SCIEX] (microfluidic trap column (200 μm × 0.5 mm ChromXP C18-CL [3 μm, 120 Å]) at a flow rate of 5 μL/min; microfluidic analytical column (75 μm × 15 cm ChromXP C18-CL [3 μm, 120 Å]) at a flow rate of 300 nL/min across a gradient of increasing concentration of acetonitrile in water supplemented with 0.1% formic acid) coupled to a TripleTOF® 5600+ mass spectrometer [SCIEX). Each scan cycle consisted of MS1 spectra acquired for 200 msec and the top 20 precursor ions fragmented using rolling collision energy and acquired for 150 msec. Mass spectra were searched using the Paragon™ algorithm of ProteinPilot™ v5 software [SCIEX]. A custom database was assembled using the combined Uniprot reference proteomes of Homo sapiens (v2016_06), enhanced green fluorescent protein, and mCALR sequences. Through the use of a decoy database, data were subjected to a 5% false discovery rate (FDR) cut-off.