Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 91
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For themyofilament proteins, MLC-1 and MLC-2 stain black while Tm has a green toneand actin and desmin stain a golden red; diffractive scattering of light by the silver grains is responsible for this effect [57].Proteins that are problematic for current staining techniques can be visualizedby immunodetection. In our experience, western blot analysis is required for thedetection and quantification of the individual Tn subunits. This, however, complicates matters because of inherent problems with antibody specificity.
For example,an antibody may not be able to detect all isoforms or a specific PTM (Fig. 19.6).We have characterized over 25 anti-TnI antibodies which are differentially sensitive to either isoform specificity or PTMs, including degradation and phosphorylation. The anti-TnT antibody JLT-12 (Sigma) also cross-reacts to the cytosolic protein, GAPDH. This cross-reactivity caused confusion about whether cTnT is degraded in globally ischemic rat hearts [58]. Therefore, extreme care must be takenwhen interpreting immunoblots.19.3.4Alternative Methods of Protein SeparationChromatography encompasses several techniques that can be used alone or incombination.
It includes ion exchange, size exclusion, reversed-phase and affinitychromatography (conventional and high-performance liquid chromatography(HPLC)), capillary IEF, and capillary zone electrophoresis. Affinity-based separation may be achieved by conventional column chromatography, immunoprecipitation, or by the newly developed “protein chip” method (e.g., SELDI; for review, see[59]).
These methods can be used alone or in various combinations and, in somecases, can be carried out under either native or denaturing conditions. The advantage of isolating proteins in their native state is that one can exploit protein-protein interactions and use the isolated proteins in biochemical assays. The caveat isthat one has to ensure that no modification(s) is introduced by processing.We separated the myofilament-enriched fraction (obtained using IN Sequence)using reversed-phase HPLC which separates proteins based on hydrophobicity[53]. In proteomic analysis, reversed-phase HPLC has been used mainly to separate peptides, obtained from proteolytic (in-gel or whole proteome) digestion,prior to their online injection into an electrospray mass spectrometer, althoughother methods for peptide fragment methods are being developed (see [60] for review).
We have used reversed-phase HPLC to separate proteins rather than the digested protein fragments. Optimization of protein peak resolution and peak number was obtained by altering flow, gradient rate, and the organic modifiers, isopropanol and acetronitrile. The subproteome was successfully separated and alterations of cTnT and MHC were observed in hearts in failure. One must rememberthat the goal of proteomic analysis is to observe as much of the proteome or subproteome as possible.
Using a combination of 2-DE analysis and HPLC (or any19.4 Conclusionother protein separation method) will increase one’s ability to completely characterize the entire proteome.19.3.5The Next Step – Clinical Applications of Proteomics: The Development of BiomarkersSome of the earliest benefits that will emerge from proteomics of skeletal and/orcardiac muscle will be in the area of biomarkers (also termed diagnostic markers).In particular, myofilament proteins detected in the serum following release fromthe diseased cell are potential biomarkers.
Currently, cTnI is widely used for thediagnosis and management of several myocardial diseases (e.g., myocardial infraction and angina; see [61]). The current method of detection (ELISA) does not specifically differentiate between the different forms of cTnI associated with disease.Recently, we modified 1D SDS-PAGE to optimize the separation of serum proteins (western blot-direct serum analysis, WB-DSA; [49]).
Normally, the high abundance proteins (i.e., albumin and IgG) in serum will distort the migration ofother proteins. WB-DSA minimizes this distortion, allowing detection by westernblot of low quantities of proteins with masses below 60 kDa. Using WB-DSA, wehave observed proteolytic fragments of cTnI in serum from patients with AMI[49].
We have also observed that patients with AMI may not necessarily possessthe same forms of cTnI [49]. This also applies to patients with stable and unstableangina and who have low levels of cTnI in the serum [62]. These findings, if confirmed, suggest that future biomarkers may involve not just detection of a particular protein but also quantification of the various forms of proteins previously associated with a specific disease process or stage.We have also applied WB-DSA to the detection of the various isoforms of sTnI inserum, based on the assumption that skeletal muscle will, like the heart, when sufficiently injured, release cellular proteins, including sTnI, into the blood [51]. Wehave preliminary evidence that the forms (i.e., intact fast vs intact slow vs degradedforms) of sTnI detected in serum vary and therefore, may reflect different degrees ortypes of injury to fast vs slow skeletal muscle fibers.
Thus, the amount of sTnI maynot be as important as what forms are present. In addition, whether or not cTnI ispresent (and in which forms) should provide insights into the relative contributionsof the skeletal (e.g., respiratory) and cardiac components to many diseases.19.4ConclusionOur understanding of the molecular basis of muscle dysfunction is just beginning. The interplay between the myofilament proteins is extraordinarily complexand still needs to be defined in health irrespective of the many protein alterationsthat develop during disease/injury revealed by proteomics. Each protein alterationwill have specific effects on contraction, an effect complicated by the many known(and unknown) alterations coexisting in each disease state.
Moreover, these33333419 Myofilament Proteomicschanges in the proteome will be further complicated by many factors (e.g., medications, diet, genetics, gender, age, exercise, and hormones) which influence disease progression. This complexity, in turn, will be confounded by the experimental model and the proteomic approach used. This will undoubtedly create a complex array of possibilities which we are currently ill equipped to handle.
Even so,we anticipate many diagnostic and pharmacological targets will become evident,some of which should result in improved treatments of many diseases.19.5AcknowledgementsWe thank Kent Arrell and Michelle Quick; their expertise in 2-DE analysis of skeletal and cardiac muscle was critical to the results depicted in this chapter. We alsothank Ralf Labugger for his insights which contributed greatly to the successfulanalysis of troponin in samples of tissue and serum.JAS was supported by an Ontario Graduate Student Award (Science and Technology), funds from the Block Term Grant to Queen’s University from the Ontario Thoracic Society, and the School of Graduate Studies and Research, Queen’s University.This research was funded by grants to SI (Canadian Institutes of Health Research,the Ontario Thoracic Society, Block Term Grants to Queen’s University from the Ontario Thoracic Society, and the William M.
Spear Endowment Fund) and to JVE (Canadian Institutes of Health Research, the Ontario Heart and Stroke Foundation, theCanadian Foundation for Innovation, and the National Institutes of Health (USA)).JVE is a Canadian Heart and Stroke Foundation Career Investigator.19.6ReferencesAliverti, A., P. T. Macklem.
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