title: Efficient peptide recovery from secreted recombinant MHC-I molecules expressed via mRNA transfection.
abstract: Most current methods for identifying peptides that are bound to a distinct MHC-I product in a given cell sample utilize detergent solubilization of membrane proteins followed by immunoaffinity purification. Since detergent traces and cell debris hamper subsequent peptide analysis, exceedingly large cell samples are often required. To avoid the use of detergents, truncated MHC-I heavy chains have recently been expressed by stable DNA transfection or retroviral transduction, resulting in the secretion of soluble MHC-I complexes to the growth medium. The electroporation of in vitro-transcribed mRNA achieves remarkable efficacy and uniformity of gene expression in numerous cell types, exhibiting exceedingly fast kinetics. We reasoned that mRNA transfection offers a simple, fast and widely applicable alternative to current gene delivery protocols for expressing secreted MHC-I products in cells of interest. 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-2K(d) in the mouse SP2/0 B cell myeloma. The level of MHC-I complexes secreted by these cells peaked within less than 24h 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.
authors.affiliation: Laboratory of Immunology, MIGAL - Galilee Research Institute, South Industrial Zone, Kiryat Shmona 11016, Israel; The Lautenberg Center for General and Tumor Immunology, The Hebrew University Hadassah Medical School, Jerusalem 91120, Israel.
authors.forename: Daphna
authors.initials: D
authors.lastname: Lazarus
authors.affiliation: Laboratory of Immunology, MIGAL - Galilee Research Institute, South Industrial Zone, Kiryat Shmona 11016, Israel; Department of Medicine, Hadassah University Hospital, Faculty of Medicine, Hebrew University, Jerusalem 91120, Israel.
authors.forename: Hadas
authors.initials: H
authors.lastname: Weinstein-Marom
authors.affiliation: Laboratory of Immunology, MIGAL - Galilee Research Institute, South Industrial Zone, Kiryat Shmona 11016, Israel.
authors.forename: Sigal
authors.initials: S
authors.lastname: Fishman
authors.affiliation: Laboratory of Immunology, MIGAL - Galilee Research Institute, South Industrial Zone, Kiryat Shmona 11016, Israel; Department of Biotechnology, Tel-Hai College, Upper Galilee, 12210, Israel.
authors.forename: Rami
authors.initials: R
authors.lastname: Yossef
authors.affiliation: Laboratory of Immunology, MIGAL - Galilee Research Institute, South Industrial Zone, Kiryat Shmona 11016, Israel; Department of Biotechnology, Tel-Hai College, Upper Galilee, 12210, Israel.
authors.forename: Dotan
authors.initials: D
authors.lastname: Zuri
authors.affiliation: Department of Biology, Technion - Israel Institute of Technology, Haifa 32000, Israel.
authors.forename: Eilon
authors.initials: E
authors.lastname: Barnea
authors.affiliation: Department of Biology, Technion - Israel Institute of Technology, Haifa 32000, Israel.
authors.forename: Arie
authors.initials: A
authors.lastname: Admon
authors.affiliation: Laboratory of Immunology, MIGAL - Galilee Research Institute, South Industrial Zone, Kiryat Shmona 11016, Israel; Department of Biotechnology, Tel-Hai College, Upper Galilee, 12210, Israel.
authors.forename: Alon
authors.initials: A
authors.lastname: Margalit
authors.affiliation: Laboratory of Immunology, MIGAL - Galilee Research Institute, South Industrial Zone, Kiryat Shmona 11016, Israel; Department of Biotechnology, Tel-Hai College, Upper Galilee, 12210, Israel. Electronic address: gidi@migal.org.il.
authors.forename: Gideon
authors.initials: G
authors.lastname: Gross
mesh_terms: D000818:Animals; D002460:Cell Line; D005434:Flow Cytometry; D015870:Gene Expression; D015395:Histocompatibility Antigens Class I / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D006801:Humans; D051379:Mice; D010455:Peptides / Q000276:immunology*; D012333:RNA, Messenger / Q000235:genetics*; D011994:Recombinant Proteins; D014162:Transfection
chemical_list: D015395:Histocompatibility Antigens Class I; D010455:Peptides; D012333:RNA, Messenger; D011994:Recombinant Proteins
keywords: Immunoaffinity purification; MHC-I peptidome; Mass-spectrometry; Soluble MHC-I; mRNA electroporation