DOI: 10.1002/pro.5560070619. Electrospray‐ionization mass spectrometry of intact intrinsic membrane proteins

Electrospray‐ionization mass spectrometry of intact intrinsic membrane proteins

10.1002/pro.5560070619

Crossref journal-article

Wiley

Protein Science (311)

Abstract

AbstractMembrane proteins drive and mediate many essential cellular processes making them a vital section of the proteome. However, the amphipathic nature of these molecules ensures their detailed structural analysis remains challenging. A versatile procedure for effective electrospray‐ionization mass spectrometry (ESI‐MS) of intact intrinsic membrane proteins purified using reverse‐phase chromatography in aqueous formic acid/isopropanol is presented. The spectra of four examples, bacteriorhodopsin and its apoprotein from Halobacterium and the Dl and D2 reaction‐center subunits from spinach thylakoids, achieve mass measurements that are within 0.01% of calculated theoretical values. All of the spectra reveal lesser quantities of other molecular species that can usually be equated with covalently modified subpopulations of these proteins. Our analysis of bovine rhodopsin, the first ESI‐MS study of a G‐protein coupled receptor, yielded a complex spectrum indicative of extensive molecular heterogeneity. The range of masses measured for the native molecule agrees well with the range calculated based upon variable glycosylation and reveals further heterogeneity arising from other covalent modifications. The technique described represents the most precise way to catalogue membrane proteins and their post‐translational modifications. Resolution of the components of protein complexes provides insights into native protein/protein interactions. The apparent retention of structure by bacteriorhodopsin during the analysis raises the potential of obtaining tertiary structure information using more developed ESI‐MS experiments.

Bibliography

Whitelegge, J. P., Gundersen, C. B., & Faull, K. F. (1998). Electrosprayâionization mass spectrometry of intact intrinsic membrane proteins. Protein Science, 7(6), 1423â1430. Portico.

Authors 3

Julian P. Whitelegge (first)
Cameron B. Gundersen (additional)
Kym F. Faull (additional)

References 53 Referenced 177

10.1038/370621a0
10.1002/pro.5560060408
10.1007/BF00277062
10.1007/BF00034943
10.1002/bms.1200220608
10.1146/annurev.biochem.66.1.717
10.1021/a1960021u
10.1016/S0021-9258(18)55400-4 / J Biol Chem / A novel metabolic form of the 32 kDa‐Dl protein in the grana‐localized reaction center of photosystem II by Callahan FE (1990)
10.1126/science.1411504
10.1002/rcm.1290021111
10.1002/j.1460-2075.1989.tb08338.x
10.1042/bst0240912
10.1126/science.2675315
10.1093/glycob/4.5.633
10.1016/S0021-9258(19)86876-X / J Biol Chem / Rhodopsin carbohydrate. Structure of small oligosaccharides attached at two sites near the NH2 terminus by Fukuda MN (1979)
10.1073/pnas.76.1.227
10.1146/annurev.bi.56.070187.003151
10.1042/bj2840603
10.1042/bst0190929
10.1016/S0021-9673(01)88415-6
10.1093/nar/12.23.8819
10.1016/S0021-9258(18)33492-6 / J Biol Chem / Orientation of retinal in bacteriorhodopsin as studied by cross‐linking using a photosensitive analog of retinal by Huang KS (1982)
10.1006/abio.1996.0480
10.1016/0014-5793(88)81039-1
10.1021/ac00171a028
10.1073/pnas.90.4.1166
10.1126/science.274.5287.536
10.1006/abio.1993.1455
10.1016/S0021-9258(18)50382-3
10.1016/S0021-9258(19)57275-1
10.1111/j.1432-1033.1973.tb03214.x
10.1016/0076-6879(74)31072-5
10.1128/MCB.4.11.2556
10.1021/bi00708a031
10.1038/372646a0
10.1016/S0958-1669(97)80151-6
10.1002/jms.1190301012
10.1002/(SICI)1097-0231(19970228)11:4<418::AID-RCM795>3.0.CO;2-R
10.1016/S0006-3495(92)81699-5
10.1006/abio.1993.1418
10.1021/bi00039a016
10.1074/jbc.272.7.3935
10.1074/jbc.272.52.33153
10.1002/j.1460-2075.1986.tb04464.x
{'key': 'e_1_2_1_46_1', 'first-page': '273', 'article-title': 'Chromatographic purification and determination of the carboxy‐terminal sequences of photosystem‐II reaction center protein‐Dl, and protein‐D2', 'volume': '31', 'author': 'Takahashi Y', 'year': '1990', 'journal-title': 'Plant Cell Physiol'} / Plant Cell Physiol / Chromatographic purification and determination of the carboxy‐terminal sequences of photosystem‐II reaction center protein‐Dl, and protein‐D2 by Takahashi Y (1990)
10.1515/znc-1987-0616
WhiteleggeJP.1989.The role of protein phosphorylation in photosynthetic light acclimation[PhD thesis]. London: University of London.
{'key': 'e_1_2_1_49_1', 'first-page': '241', 'volume-title': 'The handbook of photosynthesis', 'author': 'Whitelegge JP', 'year': '1996'} / The handbook of photosynthesis by Whitelegge JP (1996)
10.1111/j.1432-1033.1992.tb17144.x
{'key': 'e_1_2_1_51_1', 'first-page': '583', 'volume-title': 'Mass based cyanogen bromide mapping of methionine oxidation sites in proteins', 'author': 'Whitelegge JP', 'year': '1997'} / Mass based cyanogen bromide mapping of methionine oxidation sites in proteins by Whitelegge JP (1997)
10.1016/S0021-9258(17)43178-4
10.1002/pro.5560020404
10.1073/pnas.79.24.7699

Dates

Type	When
Created	16 years, 6 months ago (Feb. 10, 2009, 1:22 a.m.)
Deposited	1 year, 9 months ago (Oct. 28, 2023, 4:45 p.m.)
Indexed	2 weeks, 6 days ago (Aug. 6, 2025, 9:19 a.m.)
Issued	27 years, 2 months ago (June 1, 1998)
Published	27 years, 2 months ago (June 1, 1998)
Published Online	16 years, 7 months ago (Dec. 31, 2008)
Published Print	27 years, 2 months ago (June 1, 1998)

Funders 0

None

BibTeX

@article{Whitelegge_1998, title={Electrospray‐ionization mass spectrometry of intact intrinsic membrane proteins}, volume={7}, ISSN={1469-896X}, url={http://dx.doi.org/10.1002/pro.5560070619}, DOI={10.1002/pro.5560070619}, number={6}, journal={Protein Science}, publisher={Wiley}, author={Whitelegge, Julian P. and Gundersen, Cameron B. and Faull, Kym F.}, year={1998}, month=jun, pages={1423–1430} }

JSON

{
  "indexed": {
    "date-parts": [
      [
        2025,
        8,
        6
      ]
    ],
    "date-time": "2025-08-06T13:19:57Z",
    "timestamp": 1754486397065
  },
  "reference-count": 53,
  "publisher": "Wiley",
  "issue": "6",
  "license": [
    {
      "start": {
        "date-parts": [
          [
            2008,
            12,
            31
          ]
        ],
        "date-time": "2008-12-31T00:00:00Z",
        "timestamp": 1230681600000
      },
      "content-version": "vor",
      "delay-in-days": 3866,
      "URL": "http://onlinelibrary.wiley.com/termsAndConditions#vor"
    }
  ],
  "content-domain": {
    "domain": [],
    "crossmark-restriction": false
  },
  "published-print": {
    "date-parts": [
      [
        1998,
        6
      ]
    ]
  },
  "abstract": "<jats:title>Abstract</jats:title><jats:p>Membrane proteins drive and mediate many essential cellular processes making them a vital section of the proteome. However, the amphipathic nature of these molecules ensures their detailed structural analysis remains challenging. A versatile procedure for effective electrospray\u2010ionization mass spectrometry (ESI\u2010MS) of intact intrinsic membrane proteins purified using reverse\u2010phase chromatography in aqueous formic acid/isopropanol is presented. The spectra of four examples, bacteriorhodopsin and its apoprotein from <jats:italic>Halobacterium</jats:italic> and the Dl and D2 reaction\u2010center subunits from spinach thylakoids, achieve mass measurements that are within 0.01% of calculated theoretical values. All of the spectra reveal lesser quantities of other molecular species that can usually be equated with covalently modified subpopulations of these proteins. Our analysis of bovine rhodopsin, the first ESI\u2010MS study of a G\u2010protein coupled receptor, yielded a complex spectrum indicative of extensive molecular heterogeneity. The range of masses measured for the native molecule agrees well with the range calculated based upon variable glycosylation and reveals further heterogeneity arising from other covalent modifications. The technique described represents the most precise way to catalogue membrane proteins and their post\u2010translational modifications. Resolution of the components of protein complexes provides insights into native protein/protein interactions. The apparent retention of structure by bacteriorhodopsin during the analysis raises the potential of obtaining tertiary structure information using more developed ESI\u2010MS experiments.</jats:p>",
  "DOI": "10.1002/pro.5560070619",
  "type": "journal-article",
  "created": {
    "date-parts": [
      [
        2009,
        2,
        10
      ]
    ],
    "date-time": "2009-02-10T06:22:01Z",
    "timestamp": 1234246921000
  },
  "page": "1423-1430",
  "source": "Crossref",
  "is-referenced-by-count": 177,
  "title": "Electrospray\u2010ionization mass spectrometry of intact intrinsic membrane proteins",
  "prefix": "10.1002",
  "volume": "7",
  "author": [
    {
      "given": "Julian P.",
      "family": "Whitelegge",
      "sequence": "first",
      "affiliation": []
    },
    {
      "given": "Cameron B.",
      "family": "Gundersen",
      "sequence": "additional",
      "affiliation": []
    },
    {
      "given": "Kym F.",
      "family": "Faull",
      "sequence": "additional",
      "affiliation": []
    }
  ],
  "member": "311",
  "published-online": {
    "date-parts": [
      [
        2008,
        12,
        31
      ]
    ]
  },
  "reference": [
    {
      "key": "e_1_2_1_2_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/370621a0"
    },
    {
      "key": "e_1_2_1_3_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/pro.5560060408"
    },
    {
      "key": "e_1_2_1_4_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1007/BF00277062"
    },
    {
      "key": "e_1_2_1_5_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1007/BF00034943"
    },
    {
      "key": "e_1_2_1_6_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/bms.1200220608"
    },
    {
      "key": "e_1_2_1_7_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1146/annurev.biochem.66.1.717"
    },
    {
      "key": "e_1_2_1_8_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/a1960021u"
    },
    {
      "key": "e_1_2_1_9_1",
      "doi-asserted-by": "crossref",
      "first-page": "15357",
      "DOI": "10.1016/S0021-9258(18)55400-4",
      "article-title": "A novel metabolic form of the 32 kDa\u2010Dl protein in the grana\u2010localized reaction center of photosystem II",
      "volume": "265",
      "author": "Callahan FE",
      "year": "1990",
      "journal-title": "J Biol Chem"
    },
    {
      "key": "e_1_2_1_10_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.1411504"
    },
    {
      "key": "e_1_2_1_11_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/rcm.1290021111"
    },
    {
      "key": "e_1_2_1_12_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/j.1460-2075.1989.tb08338.x"
    },
    {
      "key": "e_1_2_1_13_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1042/bst0240912"
    },
    {
      "key": "e_1_2_1_14_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.2675315"
    },
    {
      "key": "e_1_2_1_15_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/glycob/4.5.633"
    },
    {
      "key": "e_1_2_1_16_1",
      "doi-asserted-by": "crossref",
      "first-page": "8201",
      "DOI": "10.1016/S0021-9258(19)86876-X",
      "article-title": "Rhodopsin carbohydrate. Structure of small oligosaccharides attached at two sites near the NH2 terminus",
      "volume": "254",
      "author": "Fukuda MN",
      "year": "1979",
      "journal-title": "J Biol Chem"
    },
    {
      "key": "e_1_2_1_17_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.76.1.227"
    },
    {
      "key": "e_1_2_1_18_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1146/annurev.bi.56.070187.003151"
    },
    {
      "key": "e_1_2_1_19_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1042/bj2840603"
    },
    {
      "key": "e_1_2_1_20_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1042/bst0190929"
    },
    {
      "key": "e_1_2_1_21_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0021-9673(01)88415-6"
    },
    {
      "key": "e_1_2_1_22_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1093/nar/12.23.8819"
    },
    {
      "key": "e_1_2_1_23_1",
      "doi-asserted-by": "crossref",
      "first-page": "13616",
      "DOI": "10.1016/S0021-9258(18)33492-6",
      "article-title": "Orientation of retinal in bacteriorhodopsin as studied by cross\u2010linking using a photosensitive analog of retinal",
      "volume": "257",
      "author": "Huang KS",
      "year": "1982",
      "journal-title": "J Biol Chem"
    },
    {
      "key": "e_1_2_1_24_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1006/abio.1996.0480"
    },
    {
      "key": "e_1_2_1_25_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0014-5793(88)81039-1"
    },
    {
      "key": "e_1_2_1_26_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/ac00171a028"
    },
    {
      "key": "e_1_2_1_27_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.90.4.1166"
    },
    {
      "key": "e_1_2_1_28_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1126/science.274.5287.536"
    },
    {
      "key": "e_1_2_1_29_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1006/abio.1993.1455"
    },
    {
      "key": "e_1_2_1_30_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0021-9258(18)50382-3"
    },
    {
      "key": "e_1_2_1_31_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0021-9258(19)57275-1"
    },
    {
      "key": "e_1_2_1_32_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1111/j.1432-1033.1973.tb03214.x"
    },
    {
      "key": "e_1_2_1_33_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/0076-6879(74)31072-5"
    },
    {
      "key": "e_1_2_1_34_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1128/MCB.4.11.2556"
    },
    {
      "key": "e_1_2_1_35_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/bi00708a031"
    },
    {
      "key": "e_1_2_1_36_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1038/372646a0"
    },
    {
      "key": "e_1_2_1_37_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0958-1669(97)80151-6"
    },
    {
      "key": "e_1_2_1_38_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/jms.1190301012"
    },
    {
      "key": "e_1_2_1_39_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/(SICI)1097-0231(19970228)11:4<418::AID-RCM795>3.0.CO;2-R"
    },
    {
      "key": "e_1_2_1_40_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0006-3495(92)81699-5"
    },
    {
      "key": "e_1_2_1_41_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1006/abio.1993.1418"
    },
    {
      "key": "e_1_2_1_42_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1021/bi00039a016"
    },
    {
      "key": "e_1_2_1_43_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1074/jbc.272.7.3935"
    },
    {
      "key": "e_1_2_1_44_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1074/jbc.272.52.33153"
    },
    {
      "key": "e_1_2_1_45_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/j.1460-2075.1986.tb04464.x"
    },
    {
      "key": "e_1_2_1_46_1",
      "first-page": "273",
      "article-title": "Chromatographic purification and determination of the carboxy\u2010terminal sequences of photosystem\u2010II reaction center protein\u2010Dl, and protein\u2010D2",
      "volume": "31",
      "author": "Takahashi Y",
      "year": "1990",
      "journal-title": "Plant Cell Physiol"
    },
    {
      "key": "e_1_2_1_47_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1515/znc-1987-0616"
    },
    {
      "key": "e_1_2_1_48_1",
      "unstructured": "WhiteleggeJP.1989.The role of protein phosphorylation in photosynthetic light acclimation[PhD thesis]. London: University of London."
    },
    {
      "key": "e_1_2_1_49_1",
      "first-page": "241",
      "volume-title": "The handbook of photosynthesis",
      "author": "Whitelegge JP",
      "year": "1996"
    },
    {
      "key": "e_1_2_1_50_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1111/j.1432-1033.1992.tb17144.x"
    },
    {
      "key": "e_1_2_1_51_1",
      "first-page": "583",
      "volume-title": "Mass based cyanogen bromide mapping of methionine oxidation sites in proteins",
      "author": "Whitelegge JP",
      "year": "1997"
    },
    {
      "key": "e_1_2_1_52_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1016/S0021-9258(17)43178-4"
    },
    {
      "key": "e_1_2_1_53_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1002/pro.5560020404"
    },
    {
      "key": "e_1_2_1_54_1",
      "doi-asserted-by": "publisher",
      "DOI": "10.1073/pnas.79.24.7699"
    }
  ],
  "container-title": "Protein Science",
  "original-title": [],
  "language": "en",
  "link": [
    {
      "URL": "https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1002%2Fpro.5560070619",
      "content-type": "unspecified",
      "content-version": "vor",
      "intended-application": "text-mining"
    },
    {
      "URL": "https://onlinelibrary.wiley.com/doi/pdf/10.1002/pro.5560070619",
      "content-type": "unspecified",
      "content-version": "vor",
      "intended-application": "similarity-checking"
    }
  ],
  "deposited": {
    "date-parts": [
      [
        2023,
        10,
        28
      ]
    ],
    "date-time": "2023-10-28T20:45:07Z",
    "timestamp": 1698525907000
  },
  "score": 1,
  "resource": {
    "primary": {
      "URL": "https://onlinelibrary.wiley.com/doi/10.1002/pro.5560070619"
    }
  },
  "subtitle": [],
  "short-title": [],
  "issued": {
    "date-parts": [
      [
        1998,
        6
      ]
    ]
  },
  "references-count": 53,
  "journal-issue": {
    "issue": "6",
    "published-print": {
      "date-parts": [
        [
          1998,
          6
        ]
      ]
    }
  },
  "alternative-id": [
    "10.1002/pro.5560070619"
  ],
  "URL": "http://dx.doi.org/10.1002/pro.5560070619",
  "relation": {},
  "ISSN": [
    "0961-8368",
    "1469-896X"
  ],
  "subject": [],
  "container-title-short": "Protein Science",
  "published": {
    "date-parts": [
      [
        1998,
        6
      ]
    ]
  }
}