Methods for Proteomics of Synechocystis sp. PCC 6803

 

     Two complementary strategies are being employed in order to fully characterize changes in the Synechocystis proteome. 2D-electrophoresis provides a simple way to visualize many of the more abundant proteins of the cell and changes of abundance, as well as post-translational modifications that alter mobility. Proteins are identified after proteolysis and mass spectrometry of derived peptides. Alternatively, intact protein mass profiles generated by liquid chromatography – mass spectrometry (LC-MS) are obtained defining the native covalent state of a gene product and heterogeneity associated with it, revealing subtle covalent modifications that are undetectable in 2D-gel systems. Sub-fractionation techniques are used to enrich less abundant members of the proteome. By combining LC-MS with fraction collection, using a liquid-flow splitter, the benefits of both identification and intact mass measurement are combined (LC-MS+; Figure 1). Peptide sequences with masses that deviate from those predicted by genome translation are located and available for structural determination by tandem mass spectrometric techniques toward complete description of primary structure and the proteome (Whitelegge et al, 2002a).

     Intrinsic membrane proteins are coded for by around one third of genomic open-reading frames and support a diverse array of functions critical to the health and success of the cell, especially in the case of photosynthetic organisms such as Synechocystis because nearly all the critical energy conversion processes and electron transport are embedded in the thylakoid membrane. While some membrane proteins are not recovered in 2D-gel experiments, reverse-phase and size-exclusion HPLC coupled with ESI-MS allow analysis of the full complement of intrinsic membrane proteins providing a route to complete coverage in proteomics (Whitelegge, 2002; 2003; Whitelegge et al 1998; 1999; le Coutre et al 2000; Turk et al 2000; Gómez et al 2002; Whitelegge et al, 2002b). In this approach the column eluent passes through a UV detector and the line is split for simultaneous ESI-MS and fraction collection (LC-MS+; Figure 1). Fractions are used for mass and sequence tag experiments to confirm identity of eluting proteins and for localization of post-translational alterations (Zhang et al 2001; Whitelegge et al, 2002b). Intact mass proteomics, using LC-MS+, provides the most thorough protein analysis available in proteomics, allowing detection of the earliest sites of protein modification during oxidative damage, for example, and is entirely compatible with nearly all membrane proteins for complete proteome coverage. To improve depth of coverage, a preliminary round of non-denaturing chromatography is inserted into the workflow allowing fractionation of intact complexes for information on functional associations (Figure 2).

     An LC-MS+ experiment with an intact protein profile is shown in Figure 3.

     ‘Shotgun’ proteomics experiments, where the entire proteome is fragmented to peptides prior to analysis, is providing high-throughput capability with the disadvantage that many gene products are tracked by the presence of single peptides. Protocols for ‘shotgun’ analysis of Synechocystis are under development.


References
  • Gómez SM, Nishio JN, Faull KF, Whitelegge JP. (2002) The chloroplast grana proteome defined by intact mass measurements from LC-MS. Molecular and Cellular Proteomics 1, 45-59.

  • Le Coutre J., Whitelegge J.P., Gross A., Turk E., Wright E.M., Kaback H.R. and Faull K.F. (2000) ‘Proteomics on Full-Length Membrane Proteins Using Mass Spectrometry’ Biochemistry 39, 4237-4242.

  • Turk E, Kim O, le Coutre J, Whitelegge JP, Eskandari S, Lam JT, Kreman M, Zampighi G, Faull KF, Wright EM. (2000) Molecular characterization of Vibrio parahaemolyticus vSGLT: a model for sodium-coupled sugar cotransporters. Journal of Biological Chemistry 275, 25711-25716.

  • Whitelegge JP (2002) Plant proteomics: BLASTing out of a MudPIT. Proceedings of the National Academy of Sciences U.S.A. 99, 11564-11566.

  • Whitelegge JP (2003) Thylakoid membrane proteomics. Photosynthesis Research, in the press.

  • Whitelegge JP, Gundersen C, Faull KF (1998) 'Electrospray-Ionization Mass Spectrometry of Intact Intrinsic Membrane Proteins.' Protein Science 7, 1423-1430.

  • Whitelegge J.P., le Coutre J., Lee J.C., Engel C.K., Privé G.G., Faull K.F., and Kaback H.R. (1999) ‘Towards the Bilayer Proteome, Electrospray-Ionization Mass Spectrometry of Large Intact Transmembrane Proteins’ Proceedings of the National Acadamy of Sciences U.S.A. 96, 10695-10698.

  • Whitelegge JP, Gómez SM, Aguilera R, Roberson RW, Vermaas WF, Crother TR, Champion CI, Nally JE, Blanco DR, Lovett MA, Miller JN, Faull KF. (2002a) Identification of Proteins and Intact Mass Measurements in Proteomics. Applied Genomics and Proteomics 1, 85-94.

  • Whitelegge JP, R. Aguilera, H. Zhang, R. Taylor, W.A Cramer (2002b) Full subunit coverage liquid chromatography electrospray-ionization mass spectrometry (LCMS+) of an Oligomeric Membrane Protein Complex: Cytochrome b6f Complex from Spinach and the Cyanobacterium, M. laminosus. (2002). Molecular and Cellular Proteomics 1, 816-827.

  • Zhang H, Whitelegge JP, Cramer WA. (2001) Ferredoxin: NADP+ oxidoreductase is a subunit of the chloroplast cytochrome b6f complex. J Biol Chem 276, 38159-38165.

  • http://massspec.chem.ucla.edu/

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