Proteomics in brain research: potentials and limitations
Introduction
Proteomics is the science and methodology of the study of the proteome, i.e. all proteins expressed in a cell or tissue, rather than proteins one by one. The advent of proteomics was a major step forward, comparable to the introduction of molecular biological methods in the past. It fits within the concept that the determination of protein rather than RNA levels has major advantages as it is the proteins that carry out functions. There is a long and unpredictable way from RNA to protein, a fact known to all scientists working with gene hunting methods, like differential display and subtractive hybridization. Huge subtractive libraries have been generated, but when the differentially expressed mRNAs were studied at the protein level, only a small percentage of aberrant transcriptomes could be verified. This does not mean, of course, that proteomics is fully replacing these techniques, but proteomics is a very valuable tool for protein hunting, i.e. comparing cell and tissue proteomes under physiological and pathophysiological conditions. The trend, however, seems to be that once the protein is determined, the transcriptional level is examined to find the underlying mechanism for the increase or decrease of a certain gene product. Moreover, information about the presence of isoforms and post-translational modifications can be obtained by proteomics.
In the following, we give only an outline, an example of a proteomic method that has been used in many studies, and we are aware that much valuable work is not mentioned and many important studies are not cited. The review is written to enable the neuroscientist to catch the spirit of proteomics. After introducing the methodology, we present typical human brain protein maps, identify the protein classes, recommend applications, discuss the shortcomings and limitations of proteomics, and, finally, draw a conclusion.
Section snippets
Typical analytical protocols for brain proteomics
Proteomics consists mainly of two steps: (1) separation of proteins usually by two-dimensional gel electrophoresis and (2) protein analysis and identification, mainly by mass spectrometry. Of course, other protein identification methods, like amino acid composition analysis, N-terminal sequencing, or immunochemistry, as well as column chromatography can be used (Fountoulakis, 2001), or other biochemical techniques can be applied for protein enrichment (Fountoulakis and Takács, 2002).
A critical
Generation of a human brain protein map
To illustrate results of proteomics, we provide a practical example of an application by showing typical brain protein maps from normal human frontal cortex after subfractionation into mitochondrial, microsomal and cytosolic fractions (Fig. 1, Fig. 2, Fig. 3, respectively). In Table 1, we provide data for protein identification assignment, including peptide matches, probability of assignment of a random identity and theoretical pI and molecular weight values. Analysis of spots from the three
Cytoskeleton proteins
Cytoskeleton proteins (CP) are not only serving as scaffolding structures but also play a major role in transport and signalling (dos Remedios and Thomas, 2001). Reports about the separation and identification of CP have been published for several organ systems (Srinivasan et al., 2001, Kovarova et al., 2000, Steiner et al., 2000), including the brain (Weitzdoerfer et al., 2001, Weitzdoerfer et al., 2002, Lubec et al., 2001, Gulesserian et al., 2002), and for several cellular compartments.
Potential applications
There is a wide range of proteomics applications in neuroscience, and with the rapid development in the area, many more can be expected in the near future. First of all, proteomics can be used for protein screening in brain tissue, which may be extended to the generation of protein maps. It also can be applied for the determination of isoforms and post-translational modifications. Protein spots can be excised from the gels and further examined for post-translational modifications, i.e.
Limitations
A series of shortcomings and limitations must be indicated to prevent raising hopes and expectations too high for this methodology.
Conclusion and perspectives
This review was written to give some insight into proteomics and to catch the spirit of proteomics rather than to list the many technologies that are available or are in progress, and, therefore, many important contributions have not been respected. Proteomics is a rapidly developing area and will not only successfully complement genomics but hold centre stage. Determination of proteins is much closer to function than that of RNAs. This technology enabling high throughput and automation (
Acknowledgements
G.L. is highly indebted to the Red Bull Company, Salzburg, Austria, for support of the proteome project. We are highly indebted to Mrs. Claudia Mostoegl for excellent secretarial work.
References (98)
- et al.
Human and mouse proteomic databases: novel resources in the protein universe
FEBS Lett.
(1998) - et al.
Hydrolysis and amino acid composition analysis of proteins
J. Chromatogr.
(1998) - et al.
Identification of proteins by matrix-assisted laser desorption ionization–mass spectrometry following in-gel digestion in low-salt, nonvolatile buffer and simplified peptide recovery
Anal. Biochem.
(1997) - et al.
Enrichment and proteomic analysis of low-abundance bacterial proteins
Methods Enzymol.
(2002) - et al.
Enrichment and purification of proteins of Haemophilus influenzae by chromatofocusing
J. Chromatogr.
(1998) - et al.
Enrichment of low-abundance proteins of Haemophilus influenzae by hydrophobic interaction chromatography
J. Chromatogr.
(1999) - et al.
Postmortem changes in the level of brain proteins
Exp. Neurol.
(2001) - et al.
Immunochemical and immunohistochemical study of carbonic anhydrase II in adult rat cerebellum: a marker for oligodendrocytes
Neuroscience
(1980) - et al.
Aberrant expression of centractin and capping proteins, integral constituents of the dynactin complex, in fetal Down syndrome brain
Biochem. Biophys. Res. Commun.
(2002) - et al.
16-BAC/SDS–PAGE: a two-dimensional gel electrophoresis system suitable for the separation of integral membrane proteins
Anal. Biochem.
(1996)
A simple method for displaying the hydropathic character of a protein
J. Mol. Biol.
Profiling changes in gene expression during differentiation and maturation of monocyte-derived dendritic cells using both oligonucleotide microarrays and proteomics
J. Biol. Chem.
Identification of novel MAP kinase pathway signalling targets by functional proteomics and mass spectrometry
Mol. Cell
Mass spectrometry-based methods for phosphorylation site mapping of hyperphosphorylated proteins applied to Net1, a regulator of exit from mitosis in yeast
Mol. Cell Proteomics
Manifold reduction of moesin in fetal Down syndrome brain
Biochem. Biophys. Res. Commun.
Two-dimensional electrophoresis of membrane proteins using immobilized pH gradients
Anal. Biochem.
Proteomics analysis of cellular response to oxidative stress. Evidence for in vivo overoxidation of peroxiredoxins at their active site
J. Biol. Chem.
Brain t-complex polypeptide 1 (TCP-1) related to its natural substrate beta1 tubulin is decreased in Alzheimer’s disease
Life Sci.
Cancer proteomics: from signalling networks to tumor markers
Trends Biotechnol.
Decreased brain levels of 2′,3′-cyclic nucleotide-3′-phosphodiesterase in Down syndrome and Alzheimer’s disease
Neurobiol. Aging
Reduction of actin-related protein complex 2/3 in fetal Down syndrome brain
Biochem. Biophys. Res. Commun.
Deranged expression of molecular chaperones in brains of patients with Alzheimer’s disease
Biochem. Biophys. Res. Commun.
Proteomic profiling of mechanistically distinct enzyme classes using a common chemotype
Nat. Biotechnol.
Neurofilament proteins NF-L, NF-M and NF-H in brain of patients with Down syndrome and Alzheimer’s disease
Amino Acids
Proteomic evaluation of intermediary metabolism enzyme proteins in fetal Down’s syndrome cerebral cortex
Proteomics
Analysis of protein sequences and protein complexes by matrix-assisted laser desorption/ionization mass spectrometry
Proteomics
Stability of the human papillomavirus type 18 E2 protein is regulated by a proteasome degradation pathway through its amino-terminal transactivation domain
J. Virol.
Reliable automatic protein identification from matrix-assisted laser desorption/ionization mass spectrometric peptide fingerprints
Electrophoresis
Human umbilical cord blood cells can be induced to express markers for neurons and glia
Cell Transplant.
Oncogenic activation of c-Myb by carboxyl-terminal truncation leads to decreased proteolysis by the ubiquitin-26S proteasome pathway
Oncogene
High throughput two-dimensional blue-native electrophoresis: a tool for functional proteomics of mitochondria and signalling complexes
Proteomics
New zwitterionic detergents improve the analysis of membrane proteins by two-dimensional electrophoresis
Electrophoresis
Copper/zinc superoxide dismutase is phosphorylated and modulated specifically by granulocyte-colony stimulating factor in myeloid cells
Proteomics
Translational control of the proteome: relevance to cancer
Proteomics
Mechanism-related changes in the gene transcription and protein synthesis patterns of Haemophilus influenzae after treatment with transcriptional and translational inhibitors
Proteomics
Organic solvent extraction as a versatile provedure to identify hydrophobic chloroplast membrane proteins
Electrophoresis
Proteomics: current technologies and applications in neurological disorders and toxicology
Amino Acids
Proteomic analysis of the cell envelope fraction of Escherichia coli
Amino Acids
Effect of strong detergents and chaotropes on the protein detection in two-dimensional gels
Electrophoresis
Large-scale identification of proteins of Haemophilus influenzae by amino acid composition analysis
Electrophoresis
Two-dimensional map of Haemophilus influenzae following protein enrichment by Heparin chromatography
Electrophoresis
Reference map of the low-molecular-mass proteins of Haemophilus influenzae
Electrophoresis
Identification by redox proteomics of glutathionylated proteins in oxidatively stressed human T lymphocytes
Proc. Natl. Acad. Sci. U.S.A.
Protein identification methods in proteomics
Electrophoresis
Decreased levels of synaptosomal-associated protein 25 in the brain of patients with Down syndrome and Alzheimer’s disease
Electrophoresis
Superoxide dismutase SOD1, encoded on chromosome 21, but not SOD2 is overexpressed in brains of patients with Down syndrome
J. Investig. Med.
Proteomic characterization of early-stage differentiation of mouse embryonic stem cells into neural cells induced by all-trans retinoic acid in vitro
Electrophoresis
Cited by (153)
Biomarkers for CNS Injury and Regeneration
2015, Neural RegenerationUncovering Neurodegenerative Protein Modifications via Proteomic Profiling
2015, International Review of NeurobiologyCitation Excerpt :We then used iTRAQ technology together with a multiplexing strategy which detected the fragmented ions of mass tags that generated a reporter ion in one of four different channels on the tandem mass spectra (Ong & Mann, 2005; Ross et al., 2004; Fig. 2). Use of quantitative proteomics to profile the human brain proteome presents clear advantages, since this discovery-driven approach can generate unforeseen insight into disease pathology which is likely to be overlooked by classical hypothesis-driven research (Lubec, Krapfenbauer, & Fountoulakis, 2003). The discovery-driven approach also has the advantage of being unbiased by prior assumptions, thus allowing the analysis and comparison between experimental conditions and data from thousands of individual peptides and proteins.
Cellular and molecular neurotoxicology: Basic principles
2009, Clinical Neurotoxicology: Syndromes, Substances, EnvironmentsOmics in precision medicine
2023, Oncology: Genomics, Precision Medicine and Therapeutic Targets