{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T02:45:46Z","timestamp":1760237146456,"version":"build-2065373602"},"reference-count":31,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2020,3,10]],"date-time":"2020-03-10T00:00:00Z","timestamp":1583798400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["P30 DK063491"],"award-info":[{"award-number":["P30 DK063491"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Proteomes"],"abstract":"<jats:p>A subsection of integral membrane proteins partition into chloroform during a chloroform\/methanol\/water extraction primarily designed to extract lipids. Traditionally, these proteins were called proteolipids due to their lipid-like properties; the c-subunit of the ATP synthase integral FO component is the best known due to its abundance. In this manuscript, we investigate purification of proteolipid proteins away from lipids for high-resolution mass spectrometry. Size-exclusion chromatography on silica beads using a chloroform\/methanol\/aqueous formic acid (4\/4\/1; v\/v) mobile phase allowed the separation of larger proteins (&gt;3 kDa) from lipids (&lt;1.5 kDa) and analysis by online electrospray ionization mass spectrometry. Fraction collection for mass spectrometry was limited by presence of plasticizers and other contaminants solubilized by chloroform. Drying down of the protein sample followed by resuspension in formic acid (70%) allowed reverse-phase chromatography on a polymeric support at elevated temperature, as described previously. Fractions collected in this way could be stored for extended periods at \u221280 \u00b0C without adducts or contaminants. Top\u2013down mass spectrometry enabled the definition of PsaI as a novel proteolipid of spinach thylakoid membrane. Proteolipid preparation worked similarly when total membranes from mouse brains were extracted with chloroform. While it might be tempting to use the described extraction, we prefer to broaden the meaning of the term, whereby the proteolipidome is defined as a novel biological membrane proteome that includes the full complement of membrane proteins, their binding partners\/ligands and their tightly bound structural lipids that constitute each protein\u2013lipid complex\u2019s functional unit; that is, a complete description of a biological membrane.<\/jats:p>","DOI":"10.3390\/proteomes8010005","type":"journal-article","created":{"date-parts":[[2020,3,10]],"date-time":"2020-03-10T11:59:36Z","timestamp":1583841576000},"page":"5","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Targeting a Subset of the Membrane Proteome for Top\u2013Down Mass Spectrometry: Introducing the Proteolipidome"],"prefix":"10.3390","volume":"8","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2763-7733","authenticated-orcid":false,"given":"Julian","family":"Whitelegge","sequence":"first","affiliation":[{"name":"The Pasarow Mass Spectrometry Laboratory, Jane and Terry Semel Institute of Neuroscience and Human Behavior, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2020,3,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"720","DOI":"10.1126\/science.175.4023.720","article-title":"The fluid mosaic model of the structure of cell membranes","volume":"175","author":"Singer","year":"1972","journal-title":"Science"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1054","DOI":"10.1002\/(SICI)1522-2683(20000401)21:6<1054::AID-ELPS1054>3.0.CO;2-8","article-title":"Membrane proteins and proteomics: Un amour impossible?","volume":"21","author":"Santoni","year":"2000","journal-title":"Electrophoresis"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1002\/pmic.200300543","article-title":"A detergent- and cyanogen bromide-free method for integral membrane proteomics: Application to Halobacterium purple membranes and the human epidermal membrane proteome","volume":"4","author":"Blonder","year":"2004","journal-title":"Proteomics"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"532","DOI":"10.1038\/nbt819","article-title":"A method for the comprehensive proteomic analysis of membrane proteins","volume":"21","author":"Wu","year":"2003","journal-title":"Nat. Biotechnol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"4613","DOI":"10.1021\/ac0700225","article-title":"Shotgun analysis of integral membrane proteins facilitated by elevated temperature","volume":"79","author":"Speers","year":"2007","journal-title":"Anal. Chem."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1077","DOI":"10.1111\/j.1432-1033.1992.tb17144.x","article-title":"Sequence analysis of photoaffinity-labelled peptides derived by proteolysis of photosystem-2 reaction centres from thylakoid membranes treated with [14C]azidoatrazine","volume":"207","author":"Whitelegge","year":"1992","journal-title":"Eur. J. Biochem."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"24251","DOI":"10.1074\/jbc.C300197200","article-title":"Calcium-triggered membrane fusion proceeds independently of specific presynaptic proteins","volume":"278","author":"Szule","year":"2003","journal-title":"J. Biol. Chem."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"444","DOI":"10.1074\/mcp.M500234-MCP200","article-title":"Toward the complete membrane proteome: High coverage of integral membrane proteins through transmembrane peptide detection","volume":"5","author":"Fischer","year":"2006","journal-title":"Mol. Cell. Proteomics"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"816","DOI":"10.1074\/mcp.M200045-MCP200","article-title":"Full subunit coverage liquid chromatography electrospray ionization mass spectrometry (LCMS+) of an oligomeric membrane protein: Cytochrome b(6)f complex from spinach and the cyanobacterium Mastigocladus laminosus","volume":"1","author":"Whitelegge","year":"2002","journal-title":"Mol. Cell. Proteomics"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"2558","DOI":"10.1021\/ac303064a","article-title":"Integral membrane proteins and bilayer proteomics","volume":"85","author":"Whitelegge","year":"2013","journal-title":"Anal. Chem."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2814","DOI":"10.1073\/pnas.1820450116","article-title":"Mass spectrometry: From plasma proteins to mitochondrial membranes","volume":"116","author":"Robinson","year":"2019","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_12","first-page":"419","article-title":"Analysis of proteolipid proteins of human and bovine brains","volume":"35","author":"Chatagnon","year":"1953","journal-title":"Bull. Soc. Chim. Biol. (Paris)"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"391","DOI":"10.1016\/0014-5793(76)80154-8","article-title":"Assembly of the mitochondrial membrane system XVI. Modified form of the ATPase proteolipid in oligomycin-resistant mutants of Saccharomyces cerevisiae","volume":"65","author":"Tzagoloff","year":"1976","journal-title":"FEBS Lett."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"12167","DOI":"10.1021\/bi00096a029","article-title":"Helical structure and folding of subunit c of F1F0 ATP synthase: 1H NMR resonance assignments and NOE analysis","volume":"32","author":"Girvin","year":"1993","journal-title":"Biochemistry"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2205","DOI":"10.1021\/pr0607031","article-title":"Increased coverage in the transmembrane domain with activated-ion electron capture dissociation for top-down Fourier-transform mass spectrometry of integral membrane proteins","volume":"6","author":"Zabrouskov","year":"2007","journal-title":"J. Proteome Res."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"10695","DOI":"10.1073\/pnas.96.19.10695","article-title":"Toward the bilayer proteome, electrospray ionization-mass spectrometry of large, intact transmembrane proteins","volume":"96","author":"Whitelegge","year":"1999","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"4237","DOI":"10.1021\/bi000150m","article-title":"Proteomics on full-length membrane proteins using mass spectrometry","volume":"39","author":"Whitelegge","year":"2000","journal-title":"Biochemistry"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1423","DOI":"10.1002\/pro.5560070619","article-title":"Electrospray-ionization mass spectrometry of intact intrinsic membrane proteins","volume":"7","author":"Whitelegge","year":"1998","journal-title":"Protein Sci."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1016\/0003-2697(84)90782-6","article-title":"A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids","volume":"138","author":"Wessel","year":"1984","journal-title":"Anal. Biochem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"497","DOI":"10.1016\/S0021-9258(18)64849-5","article-title":"A simple method for the isolation and purification of total lipides from animal tissues","volume":"226","author":"Folch","year":"1957","journal-title":"J. Biol. Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"911","DOI":"10.1139\/y59-099","article-title":"A rapid method of total lipid extraction and purification","volume":"37","author":"Bligh","year":"1959","journal-title":"Can. J. Biochem. Physiol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"3644","DOI":"10.1002\/pmic.201000190","article-title":"Data-directed top-down Fourier-transform mass spectrometry of a large integral membrane protein complex: Photosystem II from Galdieria sulphuraria","volume":"10","author":"Thangaraj","year":"2010","journal-title":"Proteomics"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"46","DOI":"10.1074\/mcp.M100007-MCP200","article-title":"The chloroplast grana proteome defined by intact mass measurements from liquid chromatography mass spectrometry","volume":"1","author":"Gomez","year":"2002","journal-title":"Mol. Cell. Proteomics"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"919","DOI":"10.1016\/j.plaphy.2004.11.005","article-title":"Mass spectrometry for high throughput quantitative proteomics in plant research: Lessons from thylakoid membranes","volume":"42","author":"Whitelegge","year":"2004","journal-title":"Plant. Physiol. Biochem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"440","DOI":"10.3390\/proteomes3040440","article-title":"Proteomics Is Analytical Chemistry: Fitness-for-Purpose in the Application of Top-Down and Bottom-Up Analyses","volume":"3","author":"Coorssen","year":"2015","journal-title":"Proteomes"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"11564","DOI":"10.1073\/pnas.192449199","article-title":"Plant proteomics: BLASTing out of a MudPIT","volume":"99","author":"Whitelegge","year":"2002","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"806","DOI":"10.1021\/ja973655h","article-title":"Top down versus bottom up protein characterization by tandem high-resolution mass spectrometry","volume":"121","author":"Kelleher","year":"1999","journal-title":"J. Am. Chem. Soc."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1016\/j.jprot.2017.02.002","article-title":"Top-down proteomics: Where we are, where we are going?","volume":"175","author":"Fornelli","year":"2018","journal-title":"J. Proteomics"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"172","DOI":"10.1038\/nature13419","article-title":"Membrane proteins bind lipids selectively to modulate their structure and function","volume":"510","author":"Laganowsky","year":"2014","journal-title":"Nature"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"829","DOI":"10.1126\/science.aau0976","article-title":"Protein assemblies ejected directly from native membranes yield complexes for mass spectrometry","volume":"362","author":"Chorev","year":"2018","journal-title":"Science"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Hirst, J., Kunji, E.R.S., and Walker, J.E. (2019). Comment on \u201cProtein assemblies ejected directly from native membranes yield complexes for mass spectrometry\u201d. Science, 366.","DOI":"10.1126\/science.aaw9830"}],"container-title":["Proteomes"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2227-7382\/8\/1\/5\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T09:05:45Z","timestamp":1760173545000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2227-7382\/8\/1\/5"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,3,10]]},"references-count":31,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2020,3]]}},"alternative-id":["proteomes8010005"],"URL":"https:\/\/doi.org\/10.3390\/proteomes8010005","relation":{},"ISSN":["2227-7382"],"issn-type":[{"type":"electronic","value":"2227-7382"}],"subject":[],"published":{"date-parts":[[2020,3,10]]}}}