Centricons with a 10 kda cut off membrane

For H-2K^b isolation, sarcoma cell lines d42m1-T3 and F244 were each expanded to 5 x 10⁸ cells. Prior to harvesting, cells were stimulated with 300 U ml⁻¹ IFN-γ for 48 hours to increase MHC expression. Detachment of cells was facilitated by incubation with 100 U ml⁻¹ Collagenase IV (Gibco) in PBS for 10 min at 37° C in a 5% CO₂ incubator. Cells were washed twice with PBS and cell pellets were subsequently snap frozen. MHC class I molecules were isolated as previously described^{40 (link)}. In brief, cell pellets were taken up in 1 ml of lysis buffer (1.2% CHAPS (Applichem, Darmstadt, Germany), 1x Protease Inhibitor Cocktail (Roche) in PBS) and homogenized by sonication. Lysates were cleared from remaining cell debris by centrifugation (2,500g, 30 min) and passing through a 0.2 μm filter (Sartorius, Goettingen, Germany). MHC class I molecules were isolated by immunoaffinity purification using H-2K^b-specific antibody Y3 covalently coupled to cyanogen bromide-activated sepharose 4B (GE Healthcare). MHC molecules were eluted with 0.2% trifluoroacetic acid (TFA) and released peptides were further isolated by ultrafiltration through centricons with a 10 kDa cut-off membrane (Millipore, Schwalbach, Germany). Prior to LC-MS analysis peptides were desalted using C18 Zip Tips (Millipore) according to manufacturer’s instructions and volumes were adjusted by vacuum centrifugation.