DOI 10.1007/s00702-006-0513-7 J Neural Transm (2006) 113: 1041–1054

Proteomics of the human brain: sub-proteomes might hold the key to handle brain complexity Review F. Tribl1;3 , K. Marcus2 , G. Bringmann3 , H. E. Meyer2, M. Gerlach4 , and P. Riederer1 1

The National Parkinson Foundation (NPF) Research Laboratories, Miami, FL, USA, and Department of Clinical Neurochemistry, Clinic and Polyclinic for Psychiatry & Psychotherapy, Bayerische Julius-Maximilians-Universit€at W€urzburg, W€urzburg, 2 Medizinisches Proteom-Center, Ruhr-Universit€at Bochum, Bochum, 3 Institute of Organic Chemistry, and 4 Clinical Neurochemistry, Clinic and Polyclinic for Child & Adolescent Psychiatry & Psychotherapy, Bayerische Julius-Maximilians-Universit€at W€urzburg, W€urzburg, Germany Received September 1, 2005; accepted February 24, 2006 Published online July 13, 2006; # Springer-Verlag 2006

Summary. Proteomics is a promising approach, which provides information about the expression of proteins and increasingly finds application in life science and disease research. Meanwhile, proteomics has proven to be applicable even on post mortem human brain tissue and has opened a new area in neuroproteomics. Thereby, neuroproteomics is usually employed to generate large protein profiles of brain tissue, which mostly reflect the expression of highly abundant proteins. As a complementary approach, the focus on sub-proteomes would enhance more specific insight into brain function. Sub-proteomes are accessible via several strategies, including affinity pull-down approaches, immunoprecipitation or subcellular fractionation. The extraordinary potential of subcellular proteomics to reveal even minute differences in the protein constitution of related cellular organelles is exemplified by a recent global description of neuromelanin granules from

the human brain, which could be identified as pigmented lysosome-related organelles. Keywords: Proteomics, human brain, neuromelanin, mass spectrometry, sub-proteomes, lysosome-related, subcellular. From genomics to proteomics Even though critics point out that the sequencing of the human genome did not meet the expectations of DNA-based molecular biology, it is a scientific milestone, as it provides the valuable data on which functional genomics and proteomics are now successfully based on (Owens and Breithaupt, 2002). Functional genomics has been widely applied to unravel gene expression of biological systems via the analysis of mRNA species. With the introduction of cDNA microarrays to neuroscience, a tool was provided that markedly contributed to a deeper understanding of complex topics, such as the evolution

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of the human brain (Preuss et al., 2004), or provided new insight especially in the pathogenesis of brain disorders (Gr€ unblatt, 2004). Comparative microarray studies allow a simultaneous monitoring of changes in gene expression due to disease related processes. However, these data do not consistently reflect the expression of the actual effectors in a biological system, the proteins: rather than the moderate quantitative correlation of mRNA transcripts and the corresponding protein (Gygi et al., 1999), the most substantial reason is the multiple protein isoforms a single mRNA species mostly codes for. Such protein isoforms are generated by more than 320 currently known types of protein modification following translation (Aebersold and Goodlett, 2001). These posttranslational modifications of proteins, e.g., acetylation, glycosylation, methylation, palmitoylation, phosphorylation, ubiquitination, etc., thus establish the huge variety of different protein species, which establish a far more complex cellular picture than suggested by mRNA expression. It is thus beneficial to directly study gene expression at the protein level. Various robust technologies are yet available to address the complexity of a given proteome. Protein expression profiling The term ‘proteome’ was first introduced in 1994 by Marc Wilkins in order to describe the ‘protein equivalent of a genome’. A proteome thus describes all the proteins expressed in a given biological system (e.g., an organ, tissue, cell) under defined physiological conditions at a given timepoint, such as a specific developmental stage (Lottspeich, 1999). A proteome is highly dynamic and undergoes substantial changes dependent on the respective environmental inputs. Proteomics includes (1) several protein separation techniques such as isoelectric focussing, gel electrophoretic or liquid chromatographic approaches in combination with

(2) techniques to identify and characterize proteins, most frequently by mass spectrometry (MS), but also by amino acid analysis or Edman sequencing (Meyer et al., 1986; Kellner et al., 1999; Larsen and Roepstorff, 2000; Aebersold and Goodlett, 2001; Aebersold and Mann, 2003). The core methodology in proteomics still is two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), which holds the capacity to separate complex mixtures consisting of several hundreds of proteins (Klose, 1975; O’Farrell, 1975). 2-D PAGE has been up-scaled to separate several thousands of proteins on large gels, which emerges as a very potent tool to display the proteome, e.g., of an entire mouse brain (Klose et al., 2002). Meanwhile, this technique has been applied to monitor global protein changes during brain development (Kaindl et al., 2005) or to assist the characterization of the Parkin –=– mouse, a genetic model for Parkinson’s dieseas (PD) (Palacino et al., 2004). The major technical drawbacks of 2-D PAGE are found in the difficulties to properly resolve certain classes of proteins, such as hydrophobic membrane proteins (Santoni et al., 2000) or proteins with a pI beyond the range of pI 3–10, as usually applied in 2-D PAGE. Alternatively, gel-free separation approaches have been adapted, in particular a multi-dimensional protein identification technology (MudPIT) (Wolters et al., 2001), which generally couples liquid chromatography (LC) for peptide separation with electrospray ionization tandem mass spectrometry (ESI-MS=MS). A major advantage of LC lies in the possibility to directly transfer the analytes from the capillary to the mass spectrometer. This opens up the possibility to automated high-throughput analyses. As a technology to follow DNA microarrays, the development of protein microarrays as platforms for profiling and functional proteomics is aspired (L€ uking et al., 2005). Functional protein arrays are under way to screen for enzymatic activities and protein

Proteomics of the human brain

functions, e.g., the interaction of proteins with different binding partners rangeing from proteins and nucleic acids to lipids and lowMW compounds. Comparative analyses to monitor the changes of a proteome, e.g., after pharmacological treatment, or due to pathobiochemical, disease-related processes, have been facilitated by sophisticated protein labelling or tagging methods. Difference gel electrophoresis (DIGE) allows the simultaneous separation and detection of at least two samples, each previously modified by a different fluorecence tag, on the same 2-D gel (Unlu et al., 1997). A comparative gel-free technology was introduced with isotope-coded affinity tags (ICAT) (Gygi et al., 1999), which allows the display of differentially expressed proteins by LC-MS=MS. Both approaches are limited to those proteins which contain the respective reactive amino acid for chemical modification. Nevertheless, a variety of methods including protein-tagging have been introduced to open up the possibility for quantitative proteomics (Ong and Mann, 2005). A comprehensive selection of descriptive literature and tutorials is available, which not only offer reliable proteomics protocols, but also address the problems and pitfalls arising in biological sample handling as well as cover various practical aspects of MS-based proteomics (Herbert, 1999; Chambers et al., 2000; Binz et al., 2003; Davidsson et al., 2003; Wu and Yates, 2003; Choudhary and Grant, 2004; Fountoulakis, 2004; Marcus et al., 2004; Paulson et al., 2005). In the following section we aim to illustrate aspects of neuroproteomics especially applied to investigate human brain tissue, an area of research that still is in its infancy. Proteomics of the cerebrospinal fluid is reviewed elsewhere (Yuan and Desiderio, 2005). Neuroproteomics of the human brain Animal models in neuroscience have significantly contributed to our current knowledge

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about brain functions in higher mammals. Generally, as any biological model system, animal models allow the investigation of a given process in a segregatable, reductionist way. Most animal models, however, exhibit considerable drawbacks when it comes to modelling of complex human diseases, in particular disorders associated with the brain (Dodd et al., 1988; Gerlach and Riederer, 1996; Marcotte et al., 2001). Human brain autopsy tissue, by contrast, holds a not yet assessable potential to uncover diseaserelated molecular aberrations. Post mortem brain tissue, however, is limited and specific criteria are to be considered, such as neurochemical factors, gender, age, pre-mortal effects, e.g., medication, ischemia, hypoxia or the duration of the agonal state, etc., equally as the post mortem delays (Hynd et al., 2003) to facilitate insights into a direct understanding of human brain pathology. In addition, it has to be considered that a post mortem analysis represents the final stage of a disease process. There is an increasing body of data which demonstrate the applicability of current state-of-the-art technologies to investigate human autopsy brain tissue: apart from the routine investigations of autopsy brain tissue via immunohistochemistry, numerous enzymatic activities persist in post mortem tissue, but moreover, mRNA can be retrieved for large-scale cDNA microarray applications (Buesa et al., 2004; Gr€ unblatt et al., 2004). Recent investigation of psychiatric and neurodegenerative disorders have revealed valuable new insights into the respective pathomechanisms (Marcotte et al., 2003; Gr€ unblatt et al., 2004; Mimmack et al., 2004; Ryan et al., 2004). Complementarily, the investigation of the human brain with proteomic techniques has been started. With regard to a proper evaluation of disease-related changes in protein levels compared to protein changes due to the post mortem delay, the progressive monitoring of protein variances over time is crucial. A study undertaken by Fountoulakis

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et al. specifically addressed this important issue and monitored the protein alterations in rat brains at 23
C for several post mortem time points up to 72 hours (Fountoulakis et al., 2001). Intriguingly, most alterations seem to occur after 48 hours and the list of increased (including 14-3-3 proteins, serum albumin, synaptotagmin and transferrin) or decreased protein levels (including glial fibrillary acidic protein, neurofilaments and heat shock proteins) seems to be unexpectedly short. 2-D gel-based proteomics has consequently been applied to study disease-related aberration in the human brain in several psychiatric disorders (Johnston-Wilson et al., 2000; Voshol et al., 2003), especially in schizophrenia (Edgar et al., 1999a, b, 2000), but has also found considerable application in the field of neurodegenerative disorders. Early investigations on Alzheimer’s disease (AD) in four cortical regions suggested slightly increased levels of 14-3-3 protein isoforms compared to age- and post mortem delay-mached controls (Fountoulakis et al., 1999). These proteins, however, are sensitive to post mortem changes (Fountoulakis et al., 2001). A recent study on AD identified proteins associated with key pathogenic events (Schonberger et al., 2001), including impaired cholinergic neurotransmission, an altered lipid metabolism or an elevation of cellular stress (Krapfenbauer et al., 2003). Investigations on specific protein modifications in AD revealed an aberrant glycosylation pattern in this disorder (Kanninen et al., 2004), nitrated and oxidized proteins (Korolainen et al., 2002; Butterfield and Castegna, 2003; Castegna et al., 2003). The concept of oxidative stress in neurodegenerative disorders is not only supported in studies using tissue from AD patients, but was recently reiterated in a protein profile of the substantia nigra pars compacta (Basso et al., 2004), a brain area which almost selectively degenerates in PD. In a solid comparative study on Huntington’s disease (HD) in the human brain and

in a mouse model the group of Klose was able to demonstrate a significant decline of a1-antitrypsin and aB-crystalline as the disease progresses (Zabel et al., 2002; Zabel and Klose, 2004). In the HD mouse model, protein alternations were additionally traceable in peripheral organs. This not only points at the far-reaching consequences of neurodegenerative disorders, but also opens the possibility to monitor the disease progression by peripheral markers. The shortcoming of global brain protein profiles 2-D protein maps have been established with the ambitious aim to untangle the proteome of the human brain. In most brain protein profiles, only a few hundreds of proteins have been characterized (Langen et al., 1999). These protein profiles, however, have so far been established from entire brain tissue and reflect the innate complexity due to the presence of different cell types. Although the human brain consists of various anatomically discernible and grossly dissectible nuclei, most brain nuclei still may constitute a rather complex entity, which consists of a variety of different cell types. To give an example, the human substantia nigra (SN) pars compacta is established by several different cell types arranged in cell clusters (McRitchie et al., 1995), but only a few of the SN cells represent the dopaminergic neurons, which selectively degenerate in PD (Bj€ orklund and Lindvall, 1984; Braak and Braak, 1986; Hirsch et al., 1988; Kastner et al., 1992) (Fig. 1). Whenever gross-dissected brain areas are subjected to global analyses, the most abundant proteins, e.g., proteins engaged in ‘housekeeping’ processes, in the cell skeleton, scaffolding proteins, etc., will be obviously visualized by a conventional 2-D PAGE system (Langen et al., 1999), in contrast to those proteins that are specifically expressed in a few, maybe disease-associated cells. Such a considerable diluting or mask-

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Fig. 1. Human substantia nigra neurons – A, B Most dopaminergic neurones in the substantia nigra pars compacta can be detected on the basis of the brown pigment neuromelanin, which is clearly visible in the cytoplasm of these cells (A; 100-fold magnification, B; 200-fold magnification)

ing effect at the expense of ‘low abundance proteins’ might be a substantial drawback of global tissue profiling. Aware of this issue various strategies have thus been devised to effectively increase the output of information from such complex samples: either by application of 2-D large gel electrophoresis (Klose, 1999) or by fractionation. Fractionation to elucidate brain function Since a proteomic analysis of specific nerve cells from primary cultures, e.g., cortical neuron preparations, is rather restricted to animal models (Yu et al., 2004), fractionation may thus be an indispensable demand to address the complexity and the cellular heterogeneity of the human brain in an appropriate way. There are a variety of strategies to reduce tissue complexity, including differential centrifugation to enrich proteins of various subcellular membranes additional to cytosolic proteins (Klose, 1999), the separated work-up of chromatographic or electrophoretic fractions, but also laser capture microdissection (LCM) (Sitek et al., 2005),

or the isolation of sub-proteomes. Most of these approaches have been used on animal tissue, but should also be applicable to the human brain. Laser capture microdissection LCM is an elegant approach mainly utilised in functional genomics (Van Deerlin et al., 2002; Standaert, 2005), which allows a laserbeam-mediated dissection of small, defined regions or even single cells populations. LCM recently provided valuable insights into the cellular diversity of certain populations of catecholaminergic neurons found in four different brain areas of the rat (SN, locus coeruleus, ventral tegmental area, A13) (Grimm et al., 2004). This study revealed selectively different patterns of mRNA expression, which might explain their specific roles in schizophrenia, addiction, and PD. LCM would be a promising approach even in neuroproteomics (Mouledous et al., 2003a, b), although the lack of protein amplification systems analogous to PCR for nucleic acid amplification might currently retard a more widespread application of LCM in neuroproteomics.

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Isolation of sub-proteomes Alternatively, the ‘dissection’ of specific subproteomes, such as specific protein classes, cellular organelles or protein complexes, gives unique possibilities to identify those proteins usually missing in most global 2-D brain protein profiles. Immunoprecipitation or affinity pulldown strategies are suitable approaches to isolate particular subsets of proteins, which exhibit specific post-translational modifications, e.g., phosphoproteins. The enrichment of phosphopeptides via immobilized metal ion affinity chromatography (IMAC) has recently been applied to untangle the phosphoproteome of murine post-synaptic densities (Trinidad et al., 2005) or to get new insights into hyperphosphorylation of certain proteins in AD (D’Ambrosio et al., 2005). Affinitybased approaches have also been applied to screen for specific binding partners of the metabotropic glutamate receptor 5 protein complex (mGluR5) (Farr et al., 2004) or a-synuclein (Zhou et al., 2004). The isolation of specific organelles seems to be the most potent approach to reveal important insights into cellular functions. Proteins associated with specific organelles can be enriched and isolated by subcellular fractionation (Whittaker and Sheridan, 1965) and investigated by proteomics (Jung et al., 2000; Huber et al., 2003). This approach has contributed not only to a thorough description of various cellular organelles, but provided new insights into their biogenesis and cross-talk to different other cellular structures (Taylor et al., 2003). With respect to the central nervous system relevant structures have been investigated, such as synaptic vesicles (Coughenour et al., 2004) or post-synaptic densities, which receive and transduce the synaptic information (Jordan et al., 2004; Li et al., 2005). Moreover, differential proteomics enabled important insights into temporal axoplasmatic protein changes following axonal damage, e.g., new candidates of retro-

grade injury signalling molecules (Perlson et al., 2004; Jimenez et al., 2005). Mitochondrial dysfunctions have been reported to occur in PD, in particular a significant reduction of the complex I of the mitochondrial electron transport chain (Mizuno et al., 1989; Reichmann and Riederer, 1989; Schapira et al., 1989). The oxidation of dopamine seems to be an important factor in this pathogenic process (Berman and Hastings, 1999). A new approach based on the isolation of these important cellular organelles was developed to enhance the detailed investigation of complex I aberrations in PD mitochondria and provided evidence for an altered redox state of cysteine residues (Schilling et al., 2005). We have recently applied a sequential fractionation strategy to isolate neuromelanin (NM) granules, rare pigment organelles found in some dopaminergic neurons of the human SN, for subcellular proteomics (Tribl et al., 2005) (Figs. 2, 3). The isolation of this target structure was designed as a top–down approach based on two consecutive centrifugation steps, which gradually reduce the sample complexity first from SN tissue to pigmented cell bodies and further down to the organellar level.

Fig. 2. Morphological evaluation of the organelle isolation – A The transmission electron microscopic inspection of a substantia nigra tissue homogenate reveals a neuromelanin granule (arrow) among various cellular organelles and vesicular structures. B The step-wise enrichment of neuromelanin granules via density gradient centrifugation leads to the isolation of these structures in very high purity (adapted from Tribl et al., 2005)

Proteomics of the human brain

Fig. 3. Organellar marker proteins for molecular quality control – Western blot analyses allow the evaluation of the isolates at the molecular level. The comparison of proteins extracted from neuromelanin granules (NM) with substantia nigra tissue homogenate (SN) as a positive control reveals the absence or presence of particular organelle marker proteins. The Golgi complex [a] (GM130), mitochondria [a] (Mcl-1), early endosomal compartments [b] (EEA1), the plasma membrane [b] (VLA-2a) and the nucleus [e] (nucleoporin p62) are absent, while lysosomes and lysosome-related organelles are be detected on the basis of [c] cathepsin B and the lysosome-associated membrane protein 1 [h] (Lamp-1). Dynamin [f] and clathrin [g] are not only involved in the vesicular traffic, but furthermore are suggested to be associated to endosomal compartments. The melanogenic chaperone calnexin [d], which is usually found in the endoplasmic reticulum and in oculocutaneous melanosomes, is present, while the endoplasmic reticulum [i] (BiP=grp) cannot be detected. (MM, molecular mass; adapted from Tribl et al., 2005)

The neuromelanin granule, a new member of lysosome-related organelles in the human brain as revealed by subcellular proteomics NM granules are pigmented compartments mainly found in catecholaminergic neurones of the midbrains of primates. NM granules harbor the polyphenolic pigment NM, which gives name to the brain areas locus coeruleus and SN (Fedorow et al., 2005).

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For a long time, NM has been regarded as a cellular waste product of the catecholamine catabolism, until the findings of increased iron in the SN (Riederer et al., 1985, 1988; Dexter et al., 1987), the substantial binding of iron to artificial dopamine melanin (Ben-Shachar et al., 1991) and the extraordinary affinity of iron to the NM backbone (Jellinger et al., 1992) were uncovered. Nowadays, NM granules are regarded as compartments specific for iron storage in the pigmented neurons of the locus coeruleus and the SN and are supposed to play a role in the pathogenesis of PD (Mann and Yates, 1983; Marsden, 1983; Hirsch et al., 1988; Smythies, 1996; Shamoto-Nagai et al., 2004). In particular, the investigation of NM granules has so far been hampered by the lack of an appropriate in vitro system. The brains of most laboratory animals, such as mice, rats and rabbits, are devoid of NM granules. Attempts to generate NM in an in vitro cellular system unfortunately gave rise to the formation of pigment-containing autophagic vacuoles rather than structures resembling genuine NM granules (Sulzer et al., 2000; Gomez-Santos et al., 2003). The occurrence of NM granules is phylogenetically restricted to the brains of primates and the amount of NM is constantly increasing the nearer the species is related to man (Marsden, 1961). In humans the amount of NM granules is rapidly established during infancy, which is followed by a period of pigment maturation (Fedorow et al., 2005). Due to the absence of an appropriate model organism to investigate SN pigmentation and the scarcity of these organelles in the human brain, subcellular proteomics have proven the most efficient way to tackle these structures. To illustrate the exceptional power of proteomics on the subcellular level, about 70 proteins could be identified in human NM granules from the SN, most of which are classified ‘lysosomal proteins’, proteins engaged in the vesicular traffic, but also a

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Table 1. The direct comparison of the subcellular proteome of lysosomes with those of autophagosomes, melanosomes, and neuromelanin granules facilitates the insights into the diversity of lysosome-related organelles. Despite common features each organelle exhibits a specific subset of proteins, which seems to be fundamental to the respective morphology or function Protein

Lysosome

Phagosome

Phase I & II melanosome

Neuromelanin granule

Lysosomal Proteins LAMP1 LAMP2 LIMP II LAMP3 V-ATPase A V-ATPase B V-ATPase D V-ATPase E Niemann-Pick C1 cathepsin L cathepsin Z cathepsin B nicastrin prosaposin tripeptidyl-peptidase I

X X X – X X X X X X X X X X ?

X X X – ? X – X – X X ? – – –

X X – X X X X X X – – X X X X

X X X X X X – X – – X X – X X

Immune System MHC I alpha

X

X

–

–

Endoplasmic Reticulum BiP=grp78 protein disulfidisomerase (ER60) ribophorin I calnexin ER59 cytochrome P450 calreticulin

X X X – – – –

X X – X X X X

X X X X – – X

– – X X – – X

Others peroxiredoxin 1 PI-4-K type 2 hsp60 prohibitin

X X ? X

X – X X

– X X –

X X X –

Apoptosis VDAC1 alix TRAIL annexin 5

X – – –

X X X X

X – – –

X – – –

Transport SNAP-alpha rab7 rab2 rab5C

X X X X

X X X X

– X – –

X – – – (continued)

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Table 1 (continued) Protein

Lysosome

Phagosome

Phase I & II melanosome

Neuromelanin granule

ubiquitin NIPSNAP1 20 ,30 -cNucleotide-3-phospho-diesterase

X X –

X – –

X – X

X X X

Melanogenesis tyrosinase TRP1 TRP2 tyrosine hydroxylase Identified proteins

– – – – – 215

– – – – – 117

X X X X – 68

? – – – X 72

Adapted from a table initially presented by Bagshaw et al. (2005), with the particular consideration of protein profiles published elsewhere (Garin et al., 2001; Basrur et al., 2003; Bagshaw et al., 2005; Tribl et al., 2005)

few mitochondrial proteins and proteins originating from the endoplasmic reticulum (ER). Interestingly, proteins from the ER have recently also been localized in phagosomes (Garin et al., 2001), melanosomes (Basrur et al., 2003), and neutrophil granules (Lominadze et al., 2005). Phagosomes are organelles in macrophages engaged in tissue remodelling and the elimination of apoptotic cells as well as pathogens, while melanosomes are lysosome-related organelles, which establish the oculocutaneous pigmentation on the basis of melanin. Neutrophil granules are lysosome-related secretory compartments which mediate chemotaxis, phagocytosis and defense against-by microbes circulating neutrophils. When comparing a selection of these NM granular proteins with protein profiles of conventional lysosomes (Bagshaw et al., 2005), phagosomes (Garin et al., 2001) and melanosomes (Basrur et al., 2003), common features of these organellar proteomes become apparent (Table 1). Moreover, this comparison of subcellular proteomes pinpoints the unique arrangement of proteins each organelle is established of. The differences in the protein composition, however, contribute to the unique features of the respective organelle, such as morphology or cellular function.

NM granules are lysosome-related organelles that share lysosomal aspects, but – in analogy to phagosomes or melanosomes – exhibit striking differences from the protein profile of conventional lysosomes. It is the exceptional power of subcellular proteomics to reveal even minute differences in related subcellular proteomes, which finally form the basis of the amazing diversity of cellular organelles. Conclusion and outlook Proteomics is a rapidly developing methodology, which increasingly finds application in life science and disease research of the post-genomic era. Proteomics has proved its potential to allow even deeper insights into (patho-)biological processes than gained by pure genomic approaches. Nevertheless, proteomics applied on entire tissue is currently very likely to reveal just the tip of an iceberg. The search for approaches to reasonably handle the vast morphological complexity and the enormous functional heterogeneity of the nervous system, which is due to a highly heterogeneous protein expression, is a major challenge that neuroproteomics will have to face. Fractionation – a confinement and focus on smaller sub-proteomes being part

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of a larger entity – might thus be crucial, not only to handle the enormous complexity of the brain, but rather to provide more stringent information far beyond the expression data of mostly ‘housekeeping’ proteins. It is especially the need for new drug targets and disease markers for an earlier and more concise diagnosis of diseases which might be delineated and offered by proteomics not only at the level of whole tissue, but also by investigation of specific sub-proteomes. Acknowledgements F.T. was recipient of the Ph.D. stipend DOC from the Austrian Academy of Sciences. This work was supported by the BrainNet Europe II and the BMBFproject ‘Human Brain Proteome Project’ (HUPO). Financial support from the BMBF (to H.E.M., grant 031U102F), the Deutsche Parkinson Vereinigung, and the Fond der Chemischen Industrie (to G.B.) is gratefully acknowledged. The authors were supported by the Federal Ministry of Education and Research (BMBF) (F€ orderkennzeichen 01GR0440) in the framework of the National Genome Research Network (NGFN). This research was completed within ‘The National Parkinson Foundation Center of Excellence Research Laboratories’, Miami, FL, USA, at the Clinic and Polyclinic for Psychiatry and Psychotherapy of the University of W€ urzburg (awarded to P.R.).

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Yuan X, Desiderio DM (2005) Proteomics analysis of human cerebrospinal fluid. J Chromatogr B Analyt Technol Biomed Life Sci 815: 179–189 Zabel C, Chamrad DC, Priller J, Woodman B, Meyer HE, Bates GP, Klose J (2002) Alterations in the mouse and human proteome caused by Huntington’s disease. Mol Cell Proteomics 1: 366–375 Zabel C, Klose J (2004) Influence of Huntington’s disease on the human and mouse proteome. Int Rev Neurobiol 61: 241–283 Zhou Y, Gu G, Goodlett DR, Zhang T, Pan C, Montine TJ, Montine KS, Aebersold RH, Zhang J (2004) Analysis of alpha-synuclein-associated proteins by quantitative proteomics. J Biol Chem 279: 39155–39164 Author’s addresses: F. Tribl, Department of Clinical Neurochemistry, Clinic and Polyclinic for Psychiatry & Psychotherapy, Bayerische Julius-Maximilians-Universit€at W€urzburg, F€uchsleinstraße 15, 97080 W€urzburg, Germany, Current affiliation: Medizinisches ProteomCenter, Ruhr-Universit€at Bochum, Universit€atsstraße 150, 44801 Bochum, Germany, e-mail: florian.tribl@ rub.de