Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 53
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The protein spot of interest was identified as human ubiquinol cytochrome c reductase core complex. 2DE gels were silverstained using a modified Amersham Biosciences kit.homology search [43]. A second approach uses a database searching algorithm SEQUEST [44]. This matches uninterpreted experimental MS/MS spectra with predicted fragment patterns generated in silico from sequences in protein and nucleotide databases. These techniques are highly sensitive as stated above.
A nano-electrospray ion source that has a spraying time of more than 30 minutes from only1 ll of sample, can achieve sensitivities of low femtomole range. This allows thesequencing of protein spots from silver stained 2DE gels containing down to 5 ngprotein [45].11.6BioinformaticsBioinformatics plays a central role in proteomics. At present the greatest bottleneck faced by all proteomic labs is image analysis of 2DE gels. This is a very timeconsuming process involving a certain amount of technical expertise if accurateconclusions are to be derived from the data. There are a variety of specialized software packages commercially available for quantitative analysis and databasing ofelectrophoretic separations and a range of bioinformatic tools for identifying proteins based on data from micro-chemical analyses.
In addition, special bioinformatic tools have been developed for the further characterization of proteins, ranging from the calculation of their physicochemical properties (e.g. pI, Mr) to theprediction of their potential post-translational modifications and their three-di-18518611 Proteomics, a Step Beyond Genomicsmensional structure. Most of these tools with their associated databases are available on the Internet through the World Wide Web (WWW), and can be accessedthrough the ExPASy proteomics server (http://www.expasy.ch/tools/).Once image analysis is complete and proteins have been identified and characterized it is essential that annotated and curated databases are constructed to storeall the data. These databases must allow effective interrogation by the user and,where possible, make data available to other scientists worldwide. Currently this isbest achieved using the WWW.
To maintain optimal inter-connectivity betweenthese 2DE gel protein databases and other databases of related information, it hasbeen suggested that such 2DE databases are constructed according to a set of fundamental rules [46]. Databases conforming to these rules are said to be ‘federated2-DE databases’, while many of the other databases conform to at least some ofthe rules. An index to these 2DE protein databases can be accessed via WORLD2DPAGE (http://www.expasy.ch/ch2d/2d-index.html).11.7Cardiovascular ProteomicsHeart diseases resulting in heart failure are among the leading causes of morbidity and mortality in the developed world. They can result from either systemic diseases (such as hypertensive heart disease and ischaemic heart disease) or specificheart muscle disease (such as dilated cardiomyopathy).
The present generation oftherapeutic treatments available for treating most cases of heart failure, do littlemore than ameliorate symptoms so that, in the majority of cases, the only optionis heart transplantation. The causes of cardiac dysfunction in heart failure are stilllargely unknown. However the pathogenic mechanisms underlying the diseaseprocesses are likely to involve significant alterations in myocardial gene and protein expression which will in turn determine the disease progression and outcome.
Detailed characterization of these changes should further our understanding of cardiac dysfunction in heart disease and heart failure providing new diagnostic and therapeutic markers.A relatively recent review describing alterations in the expression of contractileproteins, calcium homeostasis and signal transduction, highlights the fact thatmost molecular studies of cardiac dysfunction have been carried out on specificcellular systems [47]. However, as with many other areas of research, scientists inthis area have quickly acknowledged the potential of proteomics in being able tocharacterize differential protein expression in heart disease and heart failure.
Theuse of proteomics should provide new insights into the cellular mechanisms involved in cardiac dysfunction.11.7 Cardiovascular Proteomics11.7.1Heart 2DE Protein DatabasesTo facilitate proteomic research into heart diseases three groups have established2DE gel protein databases of human cardiac proteins (Tab. 11.1). These databasesknown as HSC-2DPAGE [48], HEART-2DPAGE [49], and HP-2DPAGE [50], are accessible through the WWW and conform to the rules for federated 2DE proteindatabases [46].
The databases contain information on several hundred cardiac proteins that have been identified by protein chemical methods. In addition, 2DE protein databases for other mammals, such as the mouse, rat [51], dog [52], pig andcow, are also under construction to support work on animal models of heart disease and heart failure.11.7.2Dilated CardiomyopathyDilated cardiomyopathy (DCM) is a disease of unknown aetiology.
It is a severedisease characterized by impaired systolic function resulting in heart failure. Sofar, proteomic investigations into human heart disease have centered aroundDCM. Known contributory factors of DCM are viral infections, cardiac-specific autoantibodies, toxic agents, genetic factors and sustained alcohol abuse. As many as100 cardiac proteins have been observed to significantly alter in their expressionin DCM, with the majority of these proteins being less abundant in the diseasedheart. This has been reported in numerous studies [51–57].
Many of these proteins have been identified using chemical methods such as mass spectrometry[53, 58–60] and have been classified into three broad functional classes:1. Cytoskeletal and myofibrillar proteins2. Proteins associated with mitochondria and energy production3. Proteins associated with stress responsesTab. 11.1 2DE heart protein databases accessible via the World Wide WebDatabaseHEART-2DPAGEWeb addressOrganhttp://userpage.chemie.fu-berlin.de/~pleiss/dh- Human heartzb.html(ventricle)Human heart (atrium)HP-2DPAGEhttp://www.mdc-berlin.de/~emu/heart/Human heart(ventricle)HSC-2DPAGEhttp://www.harefield.nthames.nhs.uk/nhli/Human heartprotein/(ventricle)Rat heart (ventricle)Dog heart (ventricle)RAT HEART-2DPAGE http://www.mpiib-berlin.mpg.de/2D-PAGE/Rat heartRAT-HEART/2d/18718811 Proteomics, a Step Beyond GenomicsThe next major challenge is to now investigate the contribution of these changesto altered cellular function underlying cardiac dysfunction.
This has already begun. For example fifty-nine isoelectric isoforms of HSP27 have been observed tobe present in human myocardium using traditional 2DE large format gels. Twelveof these protein spots in the pI range of 4.9–6.2 and mass range of 27,000–28,000Da were significantly altered in intensity in myocardium taken from patients withDCM. Ten of these protein spots were significantly changed in myocardium takenfrom patients with ischaemic heart failure [57].11.7.3Animal Models of Heart DiseaseInvestigations of human diseased tissue samples can be compromised by factorssuch as the disease stage, tissue heterogeneity, genetic variability and the patient’smedical history/therapy. It is extremely difficult to avoid any of the above complications when working with human samples.
An alternative approach is to applyproteomics to appropriate models of human disease. Animal models are an attractive alternative.There are several models of cardiac hypertrophy, heart disease and heart failurein small animals, particularly the rat. Examples of proteomic analysis of thesemodels are studies of changes in cardiac proteins in response to alcohol [61, 62]and lead [63] toxicity. Unfortunately the cardiac physiology of these small animalmodels and their normal pattern of gene expression (e.g.
isoforms of the majorcardiac contractile proteins) differ from that in larger mammals such as humans.Researchers have thus moved their investigations into higher mammals and recently two proteomic studies of heart failure in large animals have been published. One study investigated pacing-induced heat failure in the dog [64, 65],whilst the second, based in our laboratory, investigated bovine DCM [66]. Bothstudies demonstrated shared similarities with the proteome analysis of humanDCM, with the majority of changes involving reduced protein abundance in thediseased heart.Identifying altered canine and bovine proteins proved to be particularly challenging since these species are poorly represented in current genomic databases.As a result of this, new bioinformatic tools (MultiIdent) have had to be developedto facilitate cross-species protein identification [67].
For bovine DCM, the most significant change was a seven-fold increase in the enzyme ubiquitin carboxyl-terminal hydrolase (UCH) [66]. This could facilitate increased protein ubiquitination inthe diseased state, leading to proteolysis via the 26S proteosome pathway. Interestingly there is evidence to suggest that inappropriate ubiquitination of proteinscould contribute to the development of heart failure [68].More recently we have investigated whether the ubiquitin-proteosome system isperturbed in the heart of human DCM patients [69].
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