Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 80
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The supernatant, containing outer membrane (OM)and intermembrane space (IMS), was centrifuged for 30 min at 100,000 g to separate OM (pellet) and IMS (supernatant). The mitoplast pellet was resuspended in500 ll of MC buffer (300 mM sucrose, 1 mM EGTA, 1 mM Na3VO4, 20 mMMOPS, pH 7.4) and sonicated on ice in 5 cycles of 20 s bursts and 40 s rest intervals with output setting at 8–10 Watts. The sonicated mitoplast preparation wascentrifuged for 10 min at 10,000 g to remove any remaining intact mitoplasts ormitochondria, followed by centrifugation at 100,000 g for 30 min to separate innermembrane (IM) and matrix (MTX).28929016 Phosphoproteins in Heart Mitochondria16.3.5Preparation of Samples for 2D PAGE and MSTo prepare p46 for identification by mass spectrometry, 14 mg of metrizamide-purified mitochondria were obtained from two untreated hearts.
Two aliquots of mitochondria (7 mg each) were subjected to phosphorylation as above, except one ofthe aliquots was phosphorylated with non-radioactive “cold” ATP. Following the reaction, the mitochondria of both reactions were subjected to suborganellar fractionation as described above to obtain the IM. IM was chosen as the source foridentification of p46 because of its small volume for subsequent 2D gel analysisand the reasonable abundance of p46 (Fig. 16.5).
“Hot” and “cold” IM were resuspended in buffer containing 8 M urea and 20 mM Tris-HCl (pH 7.4) and resolvedby 2D gel electrophoresis under identical conditions using the Pharmacia IPGphor IEF system. Both gels were Coomassie-blue stained and dried. The gel containing the radiolabeled sample was exposed to X-ray film to localize p46. Usingposition markers, the autoradiogram was superimposed on the gel. Three closelyspaced radioactive spots were found to precisely overlie three Coomassie bluestained spots. These spots were located in a relatively clean portion of the gel withfew other spots nearby, making their recognition straightforward.
The same threespots on the non-radioactive gel were visually identified, confirmed by overlay ofthe autoradiograph, and excised for mass spectrometry analysis.16.3.6In-gel Digest of p46Tryptic in-gel digest of all three spots was performed as described previously [15].Unseparated tryptic peptide mixtures were diluted with 50% acetonitrile-5% trifluoroacetic acid to a final volume of 15 ll.16.3.7MALDI AnalysisMALDI analysis was performed as described previously [15] using a Voyager DE-StrMALDI-TOF instrument (Applied Biosystems, Framingham MA) equipped with anitrogen laser (337 nm), operated in delayed-extraction [16] and reflectron mode[17]. Mass spectra were calibrated internally on the trypsin autolysis peptides.16.3.8Peptide Sequencing by MS/MS AnalysisFor MS/MS analysis, the crude peptide mixture obtained after in-gel digest waspurified over a C18 reversed-phase Zip-Tip® (Millipore; Bedford, MA).
The purified sample was supplied into a nanospray needle (Protana, Odense; Denmark)and analyzed on a Q-Star quadrupole time-of-flight instrument (Sciex, Toronto;Canada) in nanospray mode. The ion spray voltage was set to 1,100 V. For MS/MSexperiments the collision energy Q0 was set to 50.16.4 References16.4References12345678He, H., M. Chen, N. K.
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Thus, to understand organ phenotype inhealth and disease, one must understand the nature of these subcellular complexes. As a result, the study of disease is more than ever becoming the study ofprotein interactions.The field of proteomics is poised to accept this challenge. In addition to biomarker discovery and expression profiling, a powerful application of proteomics is tofacilitate the understanding of how changes in protein interactions engender disease.
This is typified by proteomic investigations aimed to characterize protein interactions in specific disease states [4, 8, 14] and by more recent studies designedto map entire cellular networks in lower eukaryotes [2, 3]. Incumbent with thesestudies is the need for functional validation of proteomic data. In other words, ifone wants to understand the relevance of observed protein interactions, one mustunderstand whether these interactions occur in vivo, and if so, specifically howthey contribute to the organism’s phenotype.17.1Protein Kinase C Signaling ComplexesCardiac tissue possesses an extremely limited (if any) ability to regenerate, and asa result, cell death in the heart is a particularly perilous occurrence [15].
Considerable research in the past has accordingly been aimed at preventing myocardial celldeath, and more recently, with understanding the minute molecular changes responsible for myocyte death and survival. This is chiefly relevant with regard tocell death due to ischemia, or myocardial infarction, the most serious consequence of heart disease. Significant evidence supports a central role for the serine/threonine kinase protein kinase C epsilon (PKCe) in the protective signaltransduction mechanism that prevents cell death due to ischemia [8, 16–21]. Importantly, recent evidence from proteomic studies suggests that PKCe forms mulProteomic and Genomic Analysis of Cardiovascular Disease.Edited by Jennifer E.
van Eyk, Michael J. DunnCopyright © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN: 3-527-30596-329417 Proteomic Characterization of Signaling Complexesti-protein complexes with various different proteins to regulate key subcellularprocesses [8, 10, 11].
Interestingly, these processes appear to include metabolismand protein synthesis (both at the level of transcription and translation) [10]. Accordingly, this chapter aims to 1) discuss examples of task-targeted proteomics; 2)develop the concept of functional validation of proteomic data in the context ofcurrent research in the field; and 3) specifically discuss the use of proteomics tocharacterize PKCe complexes in the heart.17.1.1Metabolism-targeted ProteomicsThere is a scarcity of studies reporting large scale proteomic characterizations ofmetabolic systems, but a few seminal examples are briefly discussed. It should benoted that these studies were chosen, as were those in the transcription and translation section below, both for the quality of the study and for the uniqueness ofthe proteomic approach used by the authors to conduct their investigation.Seow and colleagues used a mammalian cell line in culture to study the effectsof resource-induced metabolic shift [22]. Cells were incubated in a low nutrientmedium (cells in normal media served as controls) – a protocol known to inducethe aforementioned metabolic shift by decreasing the rate of lactate produced relative to glucose consumed.
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