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


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.


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



supported by the Stanley Medical Research Institute and the Wellcome Trust.