Proteomics methods

Many diseases are characterized by the expression of specific proteins1 in some cases, malignant cells yield unique protein profiles when total cellular protein extracts are analyzed by proteomic methods such as two-dimensional gel electrophoresis or matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS).2 High-throughput proteomic studies may be useful to differentiate normal cells from cancer cells, to identify and define the use of biomarkers for specific cancers, and to characterize the clinical course of disease. Proteomics can also be used to isolate and characterize potential drug targets and to evaluate the efficacy of treatments. 

When fresh or frozen tissue is used for proteomic analyses, the results cannot be related directly to the clinical course of diseases in a timely manner. Instead, researchers frequently reduce the number of interesting proteins to a manageable number and then attempt to use immunohistochemistry to understand the implications of proteomic changes in archival formalin-fixed, paraffin-embedded (FFPE) tissue for which the clinical course has been established.3 Unfortunately, immunohistochemistry is a semiquantitative pro-teomic method, and the choice of interesting proteins must occur without advance knowledge of the clinical course of the disease or the response to therapy. If routinely fixed and embedded archival tissues could be used for standard proteomic methods such as 2-D gel electrophoresis and mass spectrometry (MS), these powerful techniques could be used to both qualitatively and quantitatively analyze large numbers of tissues for which the clinical course has been established. However, analysis of archival FFPE tissues by... 

High-throughput proteomic methods hold great promise for the discovery of novel protein biomarkers that can be translated into practical interventions for the diagnosis, treatment, and prevention of disease. These approaches may also facilitate the development of therapeutic agents that are targeted to specific molecular alterations in diseases such as cancer. In many cases, malignant cells yield unique protein profiles when total protein extracts from such cells are analyzed by 2-D gel electrophoresis or mass spectrometry (MS) methods. Such proteomic studies have the potential to provide an important complement to the analysis of DNA and mRNA extracts from these tissues.1... 

DNLM 1. Drug Design. 2. Genomics-methods. 3. Oligonucleotide Array Sequence Analysis-methods. 4. Proteomics-methods. QV 744 M434a 2004] I. Title. 

Amacher, D.E., Alder, R Herath, A. and Townsend, R.R. (2005) Use of proteomic methods to identify serum biomarkers associated with rat liver toxicity or hypertrophy. Clinical Chemistry, 51 (10), 1796-1803. 

The term proteome means the total protein complement of a genome, and pro-teomics means the analysis for proteome. The combination of two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) is a proteomic method of high-throughput analysis of protein expression. By using this 2-DE and MS, proteomic studies have identified many proteins that may be involved in the pathogenic mechanism of cancers. These studies analyzed cancer cell lines, as well as cancer tissues or serum from patients. 

Whether we discuss silk, proteins embedded in membranes, or soluble complexes of cytosolic proteins, we must ask questions about interactions. A first step is to identify interactions720-730 among proteins either in vitro or in living cells.731 Proteomic methods, which include the yeast two-hybrid method (Box 29-F), are widely used for this purpose. It is possible to identify large sets of interacting proteins, to identify disease states, to observe effects of drugs, and to compare metabolism among species. 

More importantly insoluble proteins, such as membrane-bound and nuclear proteins, are under-represented within 2DE analysis. However, membrane proteins can be identified by MS following a IDE separation. Unfortunately, hydrophobicity is the most common reason for poor representation of abundant proteins and seems set to be with us for quite some time (Santoni et al., 2000 Rabilloud, 2002). Emerging proteomic methods without the use of 2DE are being developed, such as isotope-coded affinity tagging (ICAT) and multi-dimensional protein identification technology (MuDPIT). 

At the time of writing this book, publications regarding applications of genomics and proteomics in biocatalysis are still exceedingly rare. However, one particular pertinent example is discussed below, and many more examples are certain to be published in the next few years, and the field of metabolic engineering, which has already achieved a number of successes and is discussed in the next section, is very likely to use more and more genomics and proteomics methods to achieve its future goals. 

Abstract Proteomics methods, such as activity-based protein profiling, can be used to connect proteins to biology and disease. Some proteins found through unbiased methods are not well characterized, which makes it difficult to ascertain the role of these proteins. Metabolomics approaches are useful in characterizing proteins that regulate or bind metabolites. Here, we provide examples of the development and use of metabolomics approaches to elucidate protein-metabolite interactions. 

At present, there are advanced difference gel electrophoresis (DOGE) Systems and 2-D fluorescence difference gel electrophoresis (2-D DIGE) which enable the analyst to use simultaneously modern (more precise) methods of fluorescent analysis with 2-D electrophoresis (using internal patterns), aided by a fully integrated bioinformatics system. Such systems allow more complete differential protein analysis, while the application of internal standards eliminates differentiation between the intervals, thus ensuring that even the smallest differences will be detected irrespective of the multitude of components. This guarantees reproducibility of results and their statistical reliability. Such assays are one of the platforms employed in the research based on the proteomics method. 

Assays performed with proteomics methods combined with immunoblotting are believed to play an important role in recognition of epitopes responsible for causing allergenic reactions (Beyer et al 2002). 

Lawrie LC, Curran S. Laser capture microdissection and colorectal cancer proteomics. Methods Mol Biol 2005 293 245-253. 

Functional Proteomics Methods and Protocols, edited by Julie D. Thompson, Christine Schaeffer-Reiss, and Marius Ueffing, 2008 483. Recombinant Proteins From Plants Methods and Protocols, edited by Loic Faye and Veromque Gomord, 2008... 

The proteomic analysis of the brain has certain limitations that are related either to the sample and/or analytical approach. In the analysis of the brain, many factors may be involved, such as differences among individuals, differences in age and sex, possible other diseases, treatment with medicines, as well as technical factors, disease-unrelated factors, such as postmortem time, improper treatment of the samples, etc., all of which can affect a clear discrimination between healthy and diseased states of interest. The technical limitations involve inefficient detection of low-abundance gene products, hydrophobic proteins (they do not enter the IPG strips), and acidic, basic, high-, and low-molecular mass proteins. All these protein classes are underrepresented in 2-D gels (Lubec et al., 2003 Fountoulakis, 2004). A combination of proteomics methods with protein separation, enriching techniques, and alternative methodologies for detection will improve the detection of additional differences between AD and control brains. Such differences may be essential in the discovery of early disease markers and therapeutic approaches. 

In these proteomics methods, the separation process is split in two phases (e.g. in Bell et al. 2001). The first phase is a protein separation by denaturing zone electrophoresis, that is in the presence of denaturing detergents, most often sodium dodecyl sulfate (SDS). The second phase is carried out by chromatography on the peptides produced by digestion of the separated proteins. This has no impact on the sample preparation itself, which just needs to be compatible with the initial zone electrophoresis. 

Ong, S.E., Foster, L.J. and Mann, M. (2003) Mass spectrometric-based approaches in quantitative proteomics. Methods 29, 124-130. 

Comprehensive studies on the synaptic proteome have been rare. Not until quite recently has the mass spectrometric technical momentum developed for detecting and documenting a comprehensive and coherent map of synaptic proteins, currently numbering approximately 1000 unique proteins (Grant 2006). This momentum has been driven by an urgent need in the neuroscientific community for molecular markers of neuropsychiatric and neurodegenerative disorders, as well as a desire to understand the molecular mechanisms underlying synaptic neurotransmission and plasticity. Table 1 presents a compact list of recent proteomic efforts where protein fractions were derived from synapses, and the proteomic methods used to study them. These studies are described further in the text that follows. 

Table I. Protein fractions derived from synapses and the proteomic methods used to study them...

Due to the limitations of prediction, there is a need for projects that will collect data on subcellular location for a variety of conditions. These projects determine protein location rather than predict it. Although these projects are useful in their own right, they also serve as a way to expand the database for the prediction of protein location. One powerful approach to determination is to isolate specific organelles or stmctures and then identify their components by expression proteomics methods (Bmnet et al. 2003). This provides information rapidly for large numbers of proteins but has limited ability to distinguish different domains within an organelle. This chapter will focus on a complementary approach, automated determination of subcellular location using fluorescence microscopy. 

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