Efficient peptide recovery from secreted recombinant MHC-I molecules expressed via mRNA transfection

To test this assumption we used mRNA to express soluble derivatives of HLA-A2 in the human AF10 B cell myeloma and 624mel melanoma and H-2Kd in the mouse SP2/0 B cell myeloma. The level of MHC-I complexes secreted by these cells peaked within less than 24 h post-transfection and they could be affinity-purified directly from the culture medium in considerably greater yields when compared to nonionic detergent lysates on a cell-to-cell basis. Mass-spectrometry analysis of eluted peptides revealed larger pools in the secreted material than in lysates with substantial overlap in composition. Our results introduce mRNA transfection as a powerful tool for determining the cell's MHC-I peptidome, which can be potentially applied to a broad range of cell types.