RECENT FINDINGS OF A
PROTEOMIC ANALYSIS OF ANTERIOR CINGULATE CORTEX IN MAJOR PSYCHIATRIC DISORDERS &
FUTURE DIRECTIONS IN THIS AREA
David
Cotter, Michael Dunn
Alterations in neuronal and glial cell density and size have been observed in
the anterior cingulate cortex (ACC) in schizophrenia, major depressive disorder
(MDD) and bipolar disorder (BPD). The basis for these changes are unknown but
may be revealed through proteomic analysis. In the first part of this talk we
present the results of a study in which two dimensional gel electrophoresis and
mass spectrometry were used to compare and identify disease-specific protein
changes in schizophrenia, MDD, and BPD in the ACC. We applied immobilized pH
gradients (IPG) 4-7 and 6-9 on the Stanley Foundation Brain Consortium brain
series (comprising 15 subjects per group from each of MDD, BPD and
schizophrenia). Gel image analysis was undertaken using Progenesis 2003.1
(NonLinear Dynamics). Data was analyses by ANCOVA.
In the IPG 4-7 gels, 33
spots, present in 40 cases or more, were found to be differentially expressed in
the disease groups. Of these 17 have been identified using peptide mass
profiling by MALDI-TOF-MS. These include altered expression of DRP 1, DRP 2,
DRP 3, two forms of creatine kinase, succinyl-coenzyme A, tubulin
a4,
tubulin b1,
tubulin b5,
GR75 mitochondrial protein, IEFS and nuclear ribonuclearprotein K, VAB-2. In
the IPG 6-9 gels, 18 spots were found to be differentially expressed in the
disease groups. Of these 3 have been identified so far. These are carbonic
anhydrase I, flavin reductase and neuronal protein NP25. Altered expression of
two forms of fructose bisphosphate aldolase (identified as a candidate protein
in a previous proteomic investigation) were observed at trend level. These
findings replicate and extend previous observations of altered protein
expression in psychiatric disorders. Some of our findings are novel, and the
potential role of these proteins in the pathophysiology of these brain disorders
will need to be explored further.
In the
second half of the presentation we discuss certain problems associated with 2-DE
that constrain its applicability to analysis of all types of sample and classes
of proteins. These problems and some approaches to overcome them will be
reviewed. The first problem is that of proteomics coverage. This can be
addressed by the use of narrow pH range gels to “zoom in” on particular regions
of the proteome. This will be particularly useful when applied in conjunction
with enrichment procedures such as those employing laser capture
microdissection. Another major problem is the large number of quantitative
comparisons of individual 2-D protein profiles that need to be made to generate
meaningful data. This is being approached by multiplexing methods (e.g.
DIGE) that allow multiple samples to be run on the same 2-D gel. A further
problem in global proteomics using 2-DE is the very high dynamic range of
protein abundance, estimated at 106 for cells and tissues. This is
beyond the dynamic range of 2-DE, with an estimated maximum dynamic range of 104.
Reproducible sample fractionation methods will therefore be essential to enrich
low-abundance proteins for proteomic studies. Finally, more hydrophobic
proteins such as represented by integral membrane proteins are generally
under-represented in 2_d gel separations.
These
limitations of 2-DE have stimulated interest in the so called “gel free”,
proteomic technologies. In particular, there is considerable interest in
combining liquid chromatography (LC) with MS in so-called “shotgun” approaches
in which a tryptic digest of the sample is separated by one or more dimensions
of LC and introduced into a tandem mass spectrometer for sequence-based
identification. However, a major limitation of this approach is that it
provides no information on quantitative abundance or post-translational
modifications of the identified proteins. The problem of quantitation is being
addressed by the development of MS-based techniques in which stable isotopes are
used to differentiate between two populations of proteins. The most widely used
such method is the isotope-coded affinity tag (ICAT). Although these approaches
are promising, caveats are (a) their quantitative reproducibility needs to be
established, (b) the dynamic range of the ICAT technique seems to be no better
than 2-DE and (c) there is evidence that it can be complementary to a 2-DE
approach in identifying a different subset of proteins from a given sample.
Finally, there is much interest in the development of antibody and protein
arrays for quantitative expression profiling, but considerable work remains to
be carried out before this approach can be routinely used in proteomic
investigations.
Research
supported by the Stanley Medical Research Institute and the Wellcome Trust.