Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 79
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The presence of multiply phosphorylated EF-Tumt in vivo confirms that thisis not merely an artifact of in vitro labeling.16.2Perspective and CommentsThe recounting of our experience with EF-Tumt allows us to offer several observations and speculations that may be applied to other systems.16.2.1Analysis of Post-translational ModificationsWhile most strategies in proteomics deal with changes in protein levels, posttranslational modifications often represent pathological consequences at the molecular level without changes in abundance and, therefore represent a critical phenomenon to study. The present report elucidates an approach to studying onesuch post-translational modification, phosphorylation, which is an importantevent in a variety of signaling cascades initiated in the heart in response to physiological and pathological stimuli.
Comparison of phosphorylation profiles in genetically modified mice may aid in identifying relevant mitochondrial targets ofprotein kinases, particularly those already suspected to translocate to the mitochondria, such as PKCe [5, 6].Comparison of phosphorylation profiles in different conditions, including drugtreatments, receptor stimulation or blockade, etc., may lead to identification of relevant targets of signal transduction cascades.
Furthermore, it may be possible todevise similar schemes to detect the subset of proteins modified by ubiquitinylation, lipid acylation, and so forth, and to compare differential patterns.16.2.2Detection of Phosphoproteins by Comparison of Two Conditions Can Be a Toolto Select Proteins of Potential Interest for Proteomic AnalysisBy comparing mitochondria phosphorylated by cytosol from control and ischemichearts, we were able to select only those proteins whose phosphorylation statechanged in response to ischemia. A similar approach could be applied to othersettings, such as drug treatment or receptor stimulation, in order to examine16.2 Perspective and Commentsshort-term posttranslational changes. Another example is illustrated in the comparison between the CalTG and control mice heart mitochondria.16.2.3Selection of an Organelle Simplifies the AnalysisWe would not have been able to distinguish changes in the whole cell lysate, andinspection of cytosol after in vitro phosphorylation revealed too many bands to detect changes.
However, relatively few proteins in the mitochondria underwentphosphorylation. Therefore the approach of identifying mitochondrial phosphoproteins that were targets of cytosolic signaling greatly simplified the analysis. Inaddition to organelles and cytoskeleton, it is possible to recover signaling scaffoldsby immunoprecipitating one component under conditions that preserve the integrity of the entire complex. This provides some exciting functional relevance, asdiscussed in this book in the chapter by Peipei Ping.The organellar approach could also be used to identify changes in the proteinpopulation of the outer mitochondrial membrane.
Mitochondria from hearts subjected to two different treatments can be isolated and subjected to biotinylationunder gentle conditions in which outer membrane proteins are selectively labeled[7]. Comparison of the 2-D spot patterns (after detection by streptavidin-peroxidase) may reveal the appearance of new spots in the experimental condition. Thebiotin provides a convenient handle for partial purification of the labeled proteins,and the two protein mixtures can next be analyzed with the use of isotope codedaffinity tags [8].16.2.4Further Simplification of the Mixture of Proteins to be Analyzed is DesirableThe tools of cell biology (e.g., sub-organellar fractionation) and biochemistry(chromatography) should be employed whenever possible.
Mitochondria contain> 1,000 different polypeptides of widely varying abundance. While detection of themost abundant proteins on 2D gels is straightforward, one is likely to miss manyof the less abundant proteins. In addition membrane proteins are problematicdue to the limiting factor of solubilization. Therefore it is useful to enrich for thesubset of proteins one is most interested in.16.2.5Selection of a Protein Separation Strategy is EssentialWhile 2-D gels are one widely used strategy to separate complex mixtures of proteins for mass spectrometry, other approaches exist and are discussed elsewherein this textbook. One approach that shows great promise is that developed byYates and colleagues, utilizing multidimensional liquid chromatography coupledto tandem mass spectrometry [9]. In this approach, a complex mixture of proteinsis subjected to tryptic digestion or cyanogen bromide hydrolysis, then loaded onto28728816 Phosphoproteins in Heart Mitochondriaa microcapillary column packed with a C18 reverse-phase matrix and with a cation exchange material.
Peptides are eluted through the column with an automated program using an acetonitrile gradient and a step gradient of ammoniumacetate. The eluted peptides are injected directly into the mass spectrometer ananalyzed by the SEQUEST algorithm [10].16.3Specific Methods Employed16.3.1Isolated Perfused HeartMale New Zealand White rabbits (2.0–2.5 kg) were anesthetized and a midsternalthoracotomy was performed. The heart was rapidly excised and mounted onto aLangendorff heart perfusion apparatus using a protocol adapted from Tsuchida etal. [11]. The heart was perfused at a constant pressure of 60 mmHg with KrebsRinger buffer consisting of (in mmol/L) NaCl 118, KCl 4.75, KH2PO4 1.18,MgSO4 · 7 H2O 1.18, CaCl2 · 2 H2O 2.5, NaHCO3 25, and glucose 11.
The perfusate was bubbled with a mixture of 95% O2 and 5% CO2 at 37 8C. Perfused heartswere stabilized for 15 min, then subjected to global ischemia for 30 min by turning off the perfusion system. After 30 min of ischemia the perfusion system wasrestarted, and the hearts were reperfused for up to 90 min. Ischemic preconditioning was induced by three 5 min cycles of no-flow ischemia and reperfusion immediately preceding the regular ischemia and reperfusion. The efficacy of these interventions was verified by measurement of creatine kinase release (Sigma Chemicals, St.
Louis, MO) and infarct size measurement using triphenyl tetrazoliumstaining [12]. All animal procedures were approved by the Animal Care and UseCommittee of The Scripps Research Institute.16.3.2Isolation of Cytosol and MitochondriaUpon completion of global ischemia, the heart was removed from the cannulaand the ventricles were minced in 20 ml per heart of ice-cold MSE buffer (225mM mannitol, 75 mM sucrose, 1 mM EGTA, 1 mM Na3VO4, 20 mM HEPESKOH, pH 7.4). The heart was further polytron homogenized for 5 s at maximalpower output by PowerGen 125 (Fisher Scientific) equipped with a 10 mm diameter rotor knife.
The homogenate was centrifuged for 10 min at 600 g, 4 8C. Thepellet was discarded and the supernatant was centrifuged for 10 min at 10,000 gto pellet mitochondria and lysosomes. The postmitochondrial supernatant generally contains about 15 mg protein/ml. This supernatant was further centrifugedfor 30 min at 100,000 g to obtain particulate-free cytosol (S100). The 10,000 g mitochondrial pellet from the previous centrifugation was resuspended in 10 ml ofMSE buffer and centrifuged for 10 min at 8,000 g.
This wash step was repeated16.3 Specific Methods Employedonce. The final pellet was resuspended in 3 ml of MSE buffer and was further purified by hybrid Percoll/metrizamide discontinuous gradient purification consisting of 5 ml of 6% Percoll, 2 ml of 17% metrizamide and 2 ml of 35% metrizamide, all prepared in 0.25 M sucrose and set up in 13 ml tubes [13]. Three ml ofthe sample were overlaid on top of the gradient and centrifuged for 20 min at50,000 g, 4 8C, using a Beckman SW41 rotor. The mitochondrial fraction at the interface between 17% and 35% metrizamide was collected and diluted at least 10fold with MSE buffer, followed by centrifugation for 10 min at 10,000 g to removemetrizamide.
The pellet was resuspended in 20 ml of MSE and centrifuged again.The final pellet was resuspended in 3 ml of MSE and aliquots were stored at –80 8C. Protein concentration was determined using the Bradford assay, and for allexperiments, equal amounts of mitochondrial protein were loaded on the gels. Cytosol concentrations were adjusted to be equal in all conditions before incubatingwith mitochondria.16.3.3In Vitro Phosphorylation ReactionsFor phosphorylation reactions, 100 lg of purified mitochondria were incubated inMSE supplemented with 25 mM HEPES-KOH, pH 7.5, 10 mM magnesium acetate, 10 lM ATP (cold) and 10 lCi [c-32P]ATP for 30 min at 30 8C with or without250 lg of cytosol.
The reaction mixture was subsequently centrifuged for 5 min at10,000 g. The mitochondrial pellet was resuspended in 500 ll MC buffer and waswashed twice. The mitochondrial proteins were resolved on a 12% polyacrylamidegel, transferred to nitrocellulose, and detected by autoradiography.16.3.4Submitochondrial FractionationWe used a modification of the method of Comte and Gautheron to fractionate mitochondria [14]. Freshly isolated purified mitochondria were pelleted by centrifugation for 5 min at 10,000 g. The mitochondrial pellet was resuspended in 10 mMKH2PO4, pH 7.4 and incubated on ice for 20 min for hypotonic swelling. The mitochondria were centrifuged for 15 min at 10,000 g, 4 8C to pellet mitoplasts (inner membrane and matrix).
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