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The Lyme disease agent, <jats:italic>Borrelia burgdorferi<\/jats:italic>, is one of an increasing number of bacterial pathogens that incorporates cholesterol onto its membrane, and form cholesterol glycolipid domains that possess all the hallmarks of eukaryotic lipid rafts. In this study, we isolated lipid rafts from cultured <jats:italic>B. burgdorferi<\/jats:italic> as a detergent resistant membrane (DRM) fraction on density gradients, and characterized those molecules that partitioned exclusively or are highly enriched in these domains. Cholesterol glycolipids, the previously known raft\u2010associated lipoproteins OspA and OpsB, and cholera toxin partitioned into the lipid rafts fraction indicating compatibility with components of the DRM. The proteome of lipid rafts was analyzed by a combination of LC\u2010MS\/MS or MudPIT. Identified proteins were analyzed <jats:italic>in silico<\/jats:italic> for parameters that included localization, isoelectric point, molecular mass and biological function. The proteome provided a consistent pattern of lipoproteins, proteases and their substrates, sensing molecules and prokaryotic homologs of eukaryotic lipid rafts. This study provides the first analysis of a prokaryotic lipid raft and has relevance for the biology of <jats:italic>Borrelia<\/jats:italic>, other pathogenic bacteria, as well as for the evolution of these structures. All MS data have been deposited in the ProteomeXchange with identifier PXD002365 (<jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"http:\/\/proteomecentral.proteomexchange.org\/dataset\/PXD002365\">http:\/\/proteomecentral.proteomexchange.org\/dataset\/PXD002365<\/jats:ext-link>).<\/jats:p>","DOI":"10.1002\/pmic.201500093","type":"journal-article","created":{"date-parts":[[2015,8,10]],"date-time":"2015-08-10T07:53:02Z","timestamp":1439193182000},"page":"3662-3675","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":28,"title":["The lipid raft proteome of <i>Borrelia burgdorferi<\/i>"],"prefix":"10.1002","volume":"15","author":[{"given":"Alvaro","family":"Toledo","sequence":"first","affiliation":[{"name":"Department of Molecular Genetics and Microbiology Stony Brook University  Stony Brook NY USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alberto","family":"P\u00e9rez","sequence":"additional","affiliation":[{"name":"Laufer Center for Physical and Quantitative Biology Stony Brook University  Stony Brook NY USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"James L.","family":"Coleman","sequence":"additional","affiliation":[{"name":"Department of Molecular Genetics and Microbiology Stony Brook University  Stony Brook NY USA"},{"name":"New York State Department of Health Stony Brook University  Stony Brook NY USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jorge L.","family":"Benach","sequence":"additional","affiliation":[{"name":"Department of Molecular Genetics and Microbiology Stony Brook University  Stony Brook NY USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2015,9,28]]},"reference":[{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.7043737"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.1056\/NEJM198303313081302"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.mi.31.100177.000513"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1128\/JB.01288-08"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.91.25.12130"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.R000005200"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.1007\/s002329900397"},{"key":"e_1_2_8_9_1","doi-asserted-by":"publisher","DOI":"10.1242\/jcs.111.1.1"},{"key":"e_1_2_8_10_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbamem.2008.03.016"},{"key":"e_1_2_8_11_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0006-3495(97)78067-6"},{"key":"e_1_2_8_12_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0955-0674(00)00239-8"},{"key":"e_1_2_8_13_1","doi-asserted-by":"publisher","DOI":"10.1242\/jcs.00840"},{"key":"e_1_2_8_14_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1600-0854.2004.0162.x"},{"key":"e_1_2_8_15_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chom.2010.09.001"},{"key":"e_1_2_8_16_1","doi-asserted-by":"crossref","first-page":"4130","DOI":"10.1128\/iai.63.10.4130-4137.1995","article-title":"Experimental immunization with Borrelia burgdorferi induces development of antibodies to gangliosides","volume":"63","author":"Garcia\u2010Monco J. 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