Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 81
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The cells were then lysed and analyzed using two-dimensional electrophoresis and matrix-assisted laser desorption ionization time-offlight mass spectrometry (MALDI-TOF MS). Protein spots that were not readilyidentified with peptide mass fingerprinting were then subjected to electrospray ionization and MS/MS [22]. The investigators found that multiple proteins, someidentifiable directly from the gel patterns and others identified by mass spectrometry, that had altered their expression and/or post-translational modification toallow the cell to metabolically accommodate the decreased nutrient environment[22].
This is a good example of a uniquely customized study that uses classicalproteomic techniques to ask broad spectrum questions about cellular metabolism.In a truly innovative study that samples both genomic and proteomic approaches, Ideker and colleagues investigated genetically altered yeast strains to understand global changes responsible for the metabolism of galactose [23]. Thisapproach utilized extensive nucleotide microarray technology to understand thechanges in mRNA levels that occurred in response to genetically induced galactose metabolism pathway perturbations.
Importantly, the investigators employedstate-of-the-art isotope coded affinity tag technology and tandem mass spectrometry to characterize proteomically which of these mRNA changes was matched by aprotein change [23]. This step was extremely important, as the correlation between mRNA and protein levels continues to be a contentious issue regarding theinterfacing of genomic and proteomic data. They found that there was a considerable correlation between mRNA and protein levels in this model, but that a significant number of proteins appeared to change independent of their mRNA counterparts, suggesting posttranscriptional modification. Lastly, the investigators alsodeveloped a model to integrate the observed protein changes with previously pub-17.1 Protein Kinase C Signaling Complexeslished changes associated with galactose metabolism, and to develop a visual representation of the galactose utilization network as it relates to other cellular processes [23].
By combining advanced genomic and proteomic tools in a novel manner, this study provides detailed information about the protein infrastructure responsible for a catabolic process, galactose metabolism.17.1.2Transcription-targeted ProteomicsProtein abundance is reliant upon dynamic interplay between transcription, translation, protein degradation, and other processes, and is therefore the study of noone organelle or specific family of molecules. Nonetheless, various reports in thepast few years have examined sub-organelle proteomes [14, 24, 25] (e.g. the nucleolus) and protein complexes [1, 6] (e.g.
nucleoporin) within the nucleus that relate to the control of this organelle’s function. In addition, various studies havetargeted other organelles such as the golgi apparatus [26], the ribosome [27], andthe endoplasmic reticulum [28] which are known to be key regulators, along withthe nucleus, of protein synthesis. This section will review two distinct proteomicapproaches that share the common theme of characterizing transcriptional control.RNA polymerase II is a critical enzyme for transcription and is well known tobe regulated by a battery of transcription factors. Despite this knowledge, the nature of the interactions between the different members of a key regulator of RNApolymerase II, the Mediator complex, was unclear. Liu and colleagues used epitope-tagged-containing yeast strains to isolate Mediator complexes for analysis [6].Affinity chromatography and gel filtration were used to isolate and separate thecomplexes, and mass spectrometry and immunoblotting were then employed toidentify proteins within the isolated complexes.
The investigators found that thecomplex existed primarily in two forms of markedly different molecular weight,and subsequently went on to characterize the protein components of each form ofthe complex. This type of approach is particularly useful if some of the components of a protein complex are known or suspected, such that they can be identified by western blotting, whilst a simultaneous non-biased analysis of all proteincomponents (via mass spectrometry) allows for the detection of previously unknown members of a complex.In contrast to focusing on a specific protein of interest that regulates transcription, Predic and colleagues investigated gene expression changes in response toagonist stimulation through a proteomic approach [29].
Fibroblasts were treatedwith endothelin-1 and protein expression was monitored by pulsing cells withradioactively labeled amino acids. This labeling technique facilitated detection ofchanges in extremely low abundance proteins on the gel, which is generally notpossible with conventional stain-based labeling methods.
This technique allowedfor the visualization of proteins with abundance as low as 10 copies per cell.Furthermore, in that only newly synthesized proteins were labeled, the approachallowed for “gene expression” to be monitored by protein level, eliminating quanti-29529617 Proteomic Characterization of Signaling Complexestative discrepancies between mRNA and protein. The investigators used thismethod to detect expression changes on the two dimensional gel and then identified the altered proteins by standard MALDI-TOF MS. This method also allowedfor time-dependent characterization of protein expression changes following thetreatment with endothelin-1, thus adding additional detail to the fibroblast regulatory profile provided by the study [29].Having highlighted a number of distinct approaches that have been used byothers to examine metabolic changes and the control of protein synthesis, thechapter will henceforth focus on the comprehensive proteomic platform developedby our laboratory to characterize molecular signaling tasks in the heart.17.2Subproteomic Analysis of PKCe Signaling17.2.1IntroductionMechanisms to reduce the deleterious effects of ischemia and to prevent myocardialcell death are of particular clinical importance to prevent heart attack-induced mortality [15, 17, 30].
Intense research over the past decade has suggested that the serine/threonine kinase protein kinase C epsilon (PKCe) is a critical signaling kinasethat, once activated, coordinates a protective response within the heart to preventcell death [8, 16, 17]. Indeed, activation of PKCe in the heart is sufficient to significantly reduce myocardial infarct size due to coronary artery occlusion [31]. Importantly, multiple investigations have divulged a collection of molecules that are activated in the heart in a PKCe-dependent fashion, some of which also appear to benecessary for cardiac protection [8, 13, 16, 17, 20].
In other words, while it is clearthat PKCe plays a necessary role in protection of the ischemic myocardium, it alsoseems apparent that there are other molecular players involved in the responseorchestrated by PKCe. Thus, in the subproteome constituted by all molecules necessary to protect the myocyte against ischemic cell death, PKCe appears to be a centralregulator. Accordingly, the following questions were precipitant from these findings:Does such a subproteome defined by PKCe and its associated proteins exist? And ifso, how does one define such a subproteome in vivo?To answer these questions, our laboratory has devised a comprehensive proteomic platform that allows for the functional characterization of the PKCe signalingsubproteome in the normal and in the protected heart.
For these studies, it was ofparamount importance that the platform be versatile enough to directly link theobserved protein interactions to the genesis of a cardiac protective phenotype invivo. This approach, as diagramed in Fig. 17.1, allows for an unbiased, yet focused, investigation of all molecules participating in a given biological process,and thereby provides a detailed blueprint of the entire signaling network [8, 10,32].
This method can be easily modified to examine signaling complexes in othercell types and disease states.17.2 Subproteomic Analysis of PKCe SignalingFUNCTIONAL PROTEOMIC PLATFORMFig. 17.1 Functional proteomic platform for the characterization of protein complexes. Pleasesee text for explanation.17.2.2Functional Proteomic PlatformIsolation, Separation, and Identification of the Subproteome. In order to effectivelycharacterize the proteins that associate with a protein of interest, one must firstisolate this subproteome from the cell lysate. This process can be preceded bysubcellular fractionation [11, 12], a method that has been described elsewhere [12]will not be discussed in detail in this chapter.
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