Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 62
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These will also give the ICAT method a broader application to those fields(e.g. neurobiology, cardiovascular biology) where small peptides that are criticalfor signaling pathways have been difficult to quantitatively analyze to date [62–64].Since the original publication of the ICAT method there have been several studies that have applied a similar strategy to isolating and quantifying proteins viaisotopic labeling [65–68]. These reports either label proteins in vivo similar to theapproach of Oda et al., or capture proteins post-isolation via their reactive cysteines.
These studies were successful in measuring protein abundance throughthe quantification of isotopic peptide pairs in different mass spectrometers (e.g.ion-trap, fourier transform ion cyclotron resonance (FTICR) mass spectrometers).13.5Biological Applications of the ICAT MethodThe ICAT method was initially used to characterize protein expression differencesin yeast cells grown on either galactose or ethanol [58]. More recently, the methodhas also been successfully applied to mammalian systems.
Han et al. reported thequantitative protein profiling of differentiation-induced microsomal proteins fromhuman myeloid leukemia (HL-60) cells. The report identified and quantified 491proteins isolated from the microsomal fractions of naive and 12-phorbol 13-myristate acetate (PMA) treated HL-60 cells. Of the 491 proteins *11% were knowncell surface antigens, receptors, and membrane proteins. A large class of previously uncharacterized proteins containing transmembrane domains were identified and quantified as well. This was an important study for several reasons. Itsuccessfully identified and quantified membrane and membrane associated proteins from the microsomal fraction of mammalian cells, a class of proteins thathas been difficult to analyze by more traditional methods.
Proteins located in orassociated with the plasma membrane have very important physiological roles incells. These proteins encompass a number of critical homeostatic and regulatoryfunctions including ion channels, pores, pumps, and transmembrane receptorsthat can sense the environment and transit signals within or between other cells.Acquiring a better understanding of their localization and abundance during specific signals may help define their role and potential function within the cell.These proteins also contain considerable diagnostic and therapeutic value becauseof their accessibility [69]. Traditionally, the analysis of membrane proteins hasbeen extremely difficult because of low solubility of many of these proteins incommonly used solvents [70]. A crude microsomal preparation strategy was usedto enrich for membrane and membrane associated proteins (Fig.
13.5) [71]. Someof the observed results also suggested new hypotheses on the mechanisms of22322413 Differential Expression Proteomic Analysis Using Isotope Coded Affinity Tags13.5 Biological Applications of the ICAT MethodPMA induced cell differentiation. A 20-fold reduction in squalene synthase (SQS)protein in PMA stimulated microsomal fractions in comparison to unstimulatedmicrosomal fractions and the concurrent change in abundance in the microsomalfraction of a number of proteins that depend upon prenylation for membrane association (e.g.
Rac1 and RhoG) is consistent with a critical role of SQS in the distribution of farnesyl pyrophosphate (FPP) utilization, a critical component of theprenylation process [72]. SQS is located at a metabolic branch point in the mevalonate pathway for controlling the distribution of the FPP pools for use in eitherthe cholesterol/sterol biosynthetic pathway or for the production of nonsteroidalproducts such as precursors of prenylation reactions [73]. Proteolysis-induced release of SQS from membranes could therefore lead to the utilization of FPP incholesterol and sterol biosynthesis and activation of SQS could result in the depletion of FPP for use in the prenylation pathway, explaining the observed PMA-induced downregulation of prenylated protein products in the microsomal fraction[74].
The use of the ICAT method alone, like any discovery science method, is notby itself sufficient to explain an entire biological process like PMA-inducedchanges in HL-60. However, the ICAT method can reveal new proteins that mayparticipate in a particular process or pathway that can lead to new hypotheses thatcan be tested by hypothesis-driven research methods. This iterative cycle of applying discovery science and hypothesis-driven approaches can be extended until amore complete understanding of the system comes to form.
This study also demonstrated the utility and the application of the ICAT method to a complex mammalian system and illustrated the need for extensive pre-fractionation methods ifcomplex samples are being studied.We have also used the ICAT method to examine the role of the hormone androgen upon prostate cancer cells. It is well known that androgens are critical for thedevelopment and maintenance of normal and cancerous prostate [76, 77].
Wehave been interested in defining the complete subset of proteins that are responsive to the hormone androgen, with the expectation that this may help us identifythe proteins that are involved in androgen receptor signal transduction. This isclinically important since these proteins may act at critical regulatory junctures inthe cell and may give us some clues on how prostate cells transit from androgendependent to androgen-independent cell growth during prostate cancer [78].
Wehave been systematically analyzing the cytosolic, membrane, and nuclear proteincomponents of prostate cancer cells and their respective responses to the hormone androgen. For example, we have isolated nuclear proteins from androgen3Fig.
13.5 Strategy for extracting and analyzing membrane proteins via ICAT. Membraneand membrane associated proteins frommammalian cells were isolated using differential centrifugation. Whole cells were incubatedin a hypotonic lysis buffer and dounce homogenized to form a microsomal homogenate.This homogenate was spun at low speeds(3K) to remove unbroken cells and nuclei.The supernatant was subjected to a highspeed centrifugation step (100,000 g) and theresulting microsomal pellet was solubilized ina mixture of urea and SDS. These solubilizedproteins were subjected to the ICAT method.22522613 Differential Expression Proteomic Analysis Using Isotope Coded Affinity Tags13.6 Conclusions and Future Directionsstarved and stimulated LNCaP prostate cancer epithelia cells and subjected themto ICAT analysis (Fig.
13.6 A). We have identified and quantified > 1,000 proteinslocated within nuclei or associated with the nuclear membrane of LNCaP cells.These included discrete classes of nuclear proteins associated with nuclear proteintransport, architecture, and transcription. Interestingly, we were successful inidentifying and quantifying androgen receptor protein expression levels by theICAT method. ICAT analysis revealed that androgen receptor levels increased by3-fold in response to androgen starvation. Western-blots on the nuclear proteinsisolated from androgen starved and stimulated LNCaP cells confirmed the resultsobtained by the ICAT method (Fig. 13.6 B), and this conclusively demonstratedthat androgen receptor levels increase in the nuclear compartment of LNCaP prostate epithelial cells when starved of the androgen hormone.
We are in the processof following up many of the biological observations that are apparent from thislarge proteomic dataset and are hopeful that these discoveries will help increaseour understanding of androgen signaling in prostate cancer.The ICAT method can resemble a large-scale quantitative western-blot (Fig.13.7). If used appropriately, the method can provide new insights into proteinabundance, localization, and turnover. Application of the ICAT method will growand undoubtedly lead to new and exciting biological discoveries.13.6Conclusions and Future DirectionsOne of the primary goals of proteomics research is to build functional and physical maps of all the proteins expressed by a cell and understand how these proteins function with each other to form biological networks on a global scale. Thisscenario has already been taken place at the level of RNA through the applicationof cDNA microarray technology [2].
Using suitable algorithms, cDNA microarraytechnology has revealed that many concurrently regulated genes participate in thesame biochemical pathway and that cellular states can be ascribed to distinct andunique transcriptional profiles [2]. This phenomenon has yet to be documented atthe level of proteins, even though there have been hints that a similar processmay be occurring at the protein level as well [79]. Recent advances in the field ofproteomics now give scientist the opportunity to address this question more directly.
Technologies that can rapidly identify proteins and accurately measure pro3Fig. 13.6 Strategy for extracting and analyzing nuclear proteins via ICAT. A) Nuclear proteins were extracted using the Dignam method. In short, nuclear pellets isolated using traditional differential centrifugation were incubated in a high-salt (420 mM) solution thatextracts most nuclear proteins except the histones.
The nuclear proteins were dialyzed toreduce the salt concentrations and subjectedto the ICAT method. B) Extracted nuclear proteins (30 lg/lane) from androgen starved/stimulated (10 nM R1881, 72 hours) LNCaPprostate epithelia were subjected to SDSPAGE and blotted with a monoclonal antibody to the androgen receptor.22722813 Differential Expression Proteomic Analysis Using Isotope Coded Affinity Tags13.6 Conclusions and Future Directionstein expression levels between two cellular states on a global scale will lead theway for this type of analysis. One method that is expected to provide such data isthe application of the ICAT reagent method in combination with mass spectrometry [58]. The ICAT reagent technology makes proteomics quantitative, because iteffectively measures relative changes in protein abundance between two cellularstates.
Since proteins are the biological effectors in the cell there is great interestin accurately measuring protein levels in response to a given stimuli. Differentialprotein profiling can be used to understand differences in protein expression between normal and disease tissue.
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