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. 1998 Mar;7(3):758–764. doi: 10.1002/pro.5560070325

Mass spectrometric analysis of integral membrane proteins: application to complete mapping of bacteriorhodopsins and rhodopsin.

L E Ball 1, J E Oatis Jr 1, K Dharmasiri 1, M Busman 1, J Wang 1, L B Cowden 1, A Galijatovic 1, N Chen 1, R K Crouch 1, D R Knapp 1
PMCID: PMC2143964  PMID: 9541408

Abstract

Integral membrane proteins have not been readily amenable to the general methods developed for mass spectrometric (or internal Edman degradation) analysis of soluble proteins. We present here a sample preparation method and high performance liquid chromatography (HPLC) separation system which permits online HPLC-electrospray ionization mass spectrometry (ESI-MS) and -tandem mass spectrometry (MS/MS) analysis of cyanogen bromide cleavage fragments of integral membrane proteins. This method has been applied to wild type (WT) bacteriorhodopsin (bR), cysteine containing mutants of bR, and the prototypical G-protein coupled receptor, rhodopsin (Rh). In the described method, the protein is reduced and the cysteine residues pyridylethylated prior to separating the protein from the membrane. Following delipidation, the pyridylethylated protein is cleaved with cyanogen bromide. The cleavage fragments are separated by reversed phase HPLC using an isopropanol/acetonitrile/aqueous TFA solvent system and the effluent peptides analyzed online with a Finnigan LCQ Ion Trap Mass Spectrometer. With the exception of single amino acid fragments and the glycosylated fragment of Rh, which is observable by matrix assisted laser desorption ionization (MALDI)-MS, this system permits analysis of the entire protein in a single HPLC run. This methodology will enable pursuit of chemical modification and crosslinking studies designed to probe the three dimensional structures and functional conformational changes in these proteins. The approach should also be generally applicable to analysis of other integral membrane proteins.

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Selected References

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  1. Bairoch A., Apweiler R. The SWISS-PROT protein sequence data bank and its new supplement TREMBL. Nucleic Acids Res. 1996 Jan 1;24(1):21–25. doi: 10.1093/nar/24.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Balashov S. P., Govindjee R., Kono M., Imasheva E., Lukashev E., Ebrey T. G., Crouch R. K., Menick D. R., Feng Y. Effect of the arginine-82 to alanine mutation in bacteriorhodopsin on dark adaptation, proton release, and the photochemical cycle. Biochemistry. 1993 Oct 5;32(39):10331–10343. doi: 10.1021/bi00090a008. [DOI] [PubMed] [Google Scholar]
  3. Balashov S. P., Imasheva E. S., Ebrey T. G., Chen N., Menick D. R., Crouch R. K. Glutamate-194 to cysteine mutation inhibits fast light-induced proton release in bacteriorhodopsin. Biochemistry. 1997 Jul 22;36(29):8671–8676. doi: 10.1021/bi970744y. [DOI] [PubMed] [Google Scholar]
  4. Barnidge D. R., Dratz E. A., Sunner J., Jesaitis A. J. Identification of transmembrane tryptic peptides of rhodopsin using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Protein Sci. 1997 Apr;6(4):816–824. doi: 10.1002/pro.5560060408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Biemann K. Appendix 5. Nomenclature for peptide fragment ions (positive ions). Methods Enzymol. 1990;193:886–887. doi: 10.1016/0076-6879(90)93460-3. [DOI] [PubMed] [Google Scholar]
  6. Duffin K. L., Lange G. W., Welply J. K., Florman R., O'Brien P. J., Dell A., Reason A. J., Morris H. R., Fliesler S. J. Identification and oligosaccharide structure analysis of rhodopsin glycoforms containing galactose and sialic acid. Glycobiology. 1993 Aug;3(4):365–380. doi: 10.1093/glycob/3.4.365. [DOI] [PubMed] [Google Scholar]
  7. Hargrave P. A., McDowell J. H., Feldmann R. J., Atkinson P. H., Rao J. K., Argos P. Rhodopsin's protein and carbohydrate structure: selected aspects. Vision Res. 1984;24(11):1487–1499. doi: 10.1016/0042-6989(84)90311-0. [DOI] [PubMed] [Google Scholar]
  8. Knapp D. R., Oatis J. E., Jr, Papac D. I. Small-scale manual multiple peptide synthesis system. Application to phosphopeptide synthesis. Int J Pept Protein Res. 1993 Sep;42(3):259–263. doi: 10.1111/j.1399-3011.1993.tb00140.x. [DOI] [PubMed] [Google Scholar]
  9. McDowell J. H., Kühn H. Light-induced phosphorylation of rhodopsin in cattle photoreceptor membranes: substrate activation and inactivation. Biochemistry. 1977 Sep 6;16(18):4054–4060. doi: 10.1021/bi00637a018. [DOI] [PubMed] [Google Scholar]
  10. Oesterhelt D., Stoeckenius W. Isolation of the cell membrane of Halobacterium halobium and its fractionation into red and purple membrane. Methods Enzymol. 1974;31:667–678. doi: 10.1016/0076-6879(74)31072-5. [DOI] [PubMed] [Google Scholar]
  11. Tarr G. E., Crabb J. W. Reverse-phase high-performance liquid chromatography of hydrophobic proteins and fragments thereof. Anal Biochem. 1983 May;131(1):99–107. doi: 10.1016/0003-2697(83)90140-9. [DOI] [PubMed] [Google Scholar]

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