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