Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 82
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Complex isolation is commonlydone by one of two methods: immunoprecipitation [4, 8, 10–13] or recombinantprotein-based affinity pull down [2, 3, 8, 12, 13]. With both of these techniques,the protein of interest is the bait with which one identifies the associated proteins(for a technical review of immunoprecipitation, please see reference 33). Briefly,immunoprecipitation requires that antibodies to the protein of interest exist, andmust be controlled with IgG-based parallel experiments. Accordingly, affinity pulldown experiments (e.g.
GST-based pull down experiments) require the development of recombinant proteins and must be controlled with null proteins (i.e. byconducting parallel pull down experiments with the selection epitope, GST). Confirmation of results with both of these methods provides strong support for observed protein interactions, as will be discussed in greater detail below.29729817 Proteomic Characterization of Signaling ComplexesRegardless of which method is employed, proteins are then separated by one- ortwo-dimensional electrophoresis.
Proteomic studies have classically relied heavily onthe marriage of two-dimensional electrophoresis with mass spectrometry to identifyproteins and post-translational modification. However, recent studies have illustrated the utility of standard large format one-dimensional SDS-PAGE for the identification of proteins of high molecular weight and of high and low isoelectric points[10], which have been traditionally problematic with a two-dimensional electrophoretic approach [34]. Furthermore, the advent of powerful and somewhat more affordable tandem mass spectrometric instruments and quantitative mass spectrometryapproaches empowers the characterization of post-translational modification andprotein expression at the level of the mass spectrometer [35–37]. An increasinglypopular approach circumvents electrophoresis and directly subjects isolated proteincomplexes to mass spectrometric analysis [37, 38].
This method is particularly appealing in that it obviates concerns of protein resolution on a gel. We have foundthat all three of these methods (one- and two-dimensional electrophoresis followedby mass spectrometry, and direct subjection of isolated complexes to mass spectrometry following solution digest) yield novel data regarding the components of protein complexes [10]. As a result, signaling complex analysis is most thoroughly accomplished with an approach that combines these three methods.Following separation, protein identification is then made by mass spectrometry.In the case of the two-dimensional gel, discernable gel features are excised andtrypsinized in the traditional manner prior to mass spectrometric analysis.
If aone-dimensional gel is used, protein spots from the entire continuum of the gel,as opposed to solely from densely stained regions of the gel, are excised, digestedand analyzed. This is done because previous studies have indicated that the detection limit of the mass spectrometer exceeds that of the gel stain (e.g. coomassie,silver, or SYPRO) [10, 39], indicating that unstained regions of the gel may alsocontain members of the isolated subproteome, and therefore require analysis.This highlights another advantage of one- verses two-dimensional electrophoresis,in that many unstained regions of the two-dimensional gel presumably also contain proteins, but for reasons of practicality, are much more difficult to analyzethen are unstained regions of a one-dimensional gel.
Peptide mass fingerprintingis still a widely used technique to make protein identification [8, 36, 40], but onethat is being increasingly replaced by more powerful tandem mass spectrometryapproaches [2, 3, 10, 38]. These instruments allow for high resolution and massaccuracy and can be operated in data-dependent LC/MS/MS mode (such as aquadrupole TOF hybrid instrument, like the Q-Tof2), consequently increasing theconfidence of database search results. In addition, tandem mass spectrometry instruments are far superior for the identification of post-translational modification.Functional Characterization of the Subproteome.
Numerous studies employing variations on the above described theme have been published in the past few years[2–4, 8, 10]. These include studies focused on a single molecule and its interacting proteins [4, 8, 10] and others that take a more large scale approach to examinemultiple proteins and their associated complexes [2, 3]. Regardless of the17.2 Subproteomic Analysis of PKCe SignalingFig. 17.2 Task specific modules as a meansfor signal transduction. In this model, distinctPKCe modules would exist at various subcellular compartments, containing different proteincomponents, and all orchestrating differentsignaling tasks essential to the myocyte’s resistance to ischemia.approach, it is becoming increasingly clear that ancillary validation methods arerequired to not only confirm the members of a subproteome, but moreover, to understand the role that these proteins have in the genesis of a phenotype. In thiskey final step of the proteomic platform described herein, the role of the proteincomplex to engender a phenotype is discerned.First, all proteins that are detected via mass spectrometry are confirmed asmembers of the subproteome of interest by co-immunoprecipitation followed byelectrophoresis and western blotting.
If antibodies to any of the identified proteinsare not available, other methods of confirmation include recombinant proteinbased interaction assays [8, 12, 13, 20]. The advent of antibody- and protein-basedmicroarrays presents a powerful high-throughput method to screen for changes inprotein expression and protein-protein interactions [41, 42]. This approach willlikely allow for automation of these confirmatory experiments in the near future.Next, one must discriminate proteins that form direct interactions with the protein of interest (i.e. binding partners) from those that associate indirectly with thetarget of complex isolation (Fig. 17.3). It is often overlooked in studies that employ immunoprecipitation and/or affinity pull down techniques that these methods can in fact isolate proteins that do not directly interact with the target of isolation.
Nonetheless, these proteins may still be important members of the subproteome in question. Thus, after confirmation with immunoprecipitation and immunoblotting from cell lysates, the determination of whether direct protein-protein interactions are formed between each molecule in the complex and the pro-29930017 Proteomic Characterization of Signaling ComplexesFig. 17.3 Detection of direct and indirect protein-protein interactions. Whether the protein X complex isisolated via immunoprecipitation or affinity pull down,this complex may contain proteins that directly (protein Y) and indirectly (protein Z) associate with protein X. Thus, ancillary interaction studies are necessary to validate the nature of protein interactions within a multi-protein complex, as discussed in the text.tein of interest is done in vitro either with recombinant proteins or by methodssuch as in vitro transcription/translation [8, 12, 13].
Commercial kits are available(e.g. Promega TnT) to expedite this process, and the interactions can be detectedeither by western blotting or through the use of radioactively-labeled amino acids/proteins. These processes are of paramount importance as recapitulation of protein interactions and detection with additional methods (i.e. other than mass spectrometry) greatly reduces the likelihood that the observed findings are artifactual.The differences in complex formation that occur at distinct subcellular locationsare studied using confocal microscopy and subcellular fractionation.
As was addressed in the foregoing examples, the need to examine sub-cellular specific signal transduction events underlying a given phenotype cannot be over-emphasized.Often times, minute alterations in protein expression and/or activity at the levelof a specific organelle are attenuated when the entire cellular component is analyzed [12, 13]. Thus, to insure a lucid characterization of protein function, organellar differences must be taken into consideration through subcellular fractionation(Fig. 17.2).Lastly, the functional roles of the proteins within the complex are determined.This is the final step that links the proteomic data to a physiological phenotype.Accordingly, this step involves established biochemical and physiological assays tocharacterize the individual components of the subproteome [9].
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