Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 60
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KGaA, WeinheimISBN: 3-527-30596-321413 Differential Expression Proteomic Analysis Using Isotope Coded Affinity Tagspresenting two different cellular states. We will also discuss how differential expression proteomic analysis via ICAT reagent labeling can lead to the identification of critical regulators within defined signaling pathways, and highlight its applications in biology and medicine.13.2Traditional Methodologies for Measuring Protein AbundanceOver the past 25 years two-dimensional polyacrylamide-gel electrophoresis (2DE)has been the cornerstone proteomic technology. The method is capable of separating complex protein mixtures that are composed of thousands of proteins intotheir individual components and of indicating the quantity of each detected feature [18–21].
During 2DE, proteins are extracted from cells or tissues are separated in the isoelectric focusing (IEF) first-dimension based upon their overall intrinsic charge. Each protein will migrate to an equilibrium position in the gel thatis referred to as its isoelectric point (pI). IEF is typically performed in polyacrylamide gels that can span narrow or broad range of pI ranges (Fig. 13.1). Once focused, the proteins are then separated in the second dimension by their size. Thisis achieved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). The gels are subsequently fixed and proteins are being visualized usingtraditional staining methods (e.g.
silver-stain and coomasie blue) or more recently,fluorescent stains [22–32]. The staining intensity indicates the quantity of each detected spot. Spots containing the stained proteins are excised from the gel, trypsin-digested and the resulting peptides are subjected to MS analysis for proteinidentification [33–36]. The 2DE strategy has been the method of choice for identifying proteins and for measuring differences in protein abundance between twoexperimental samples (e.g. treated cells versus untreated cells) by detecting differences in the staining intensities of spots with identical coordinates present in twoor more gel patterns.
Conceptually, 2DE is relatively simple. However, in practicethe strategy has been plagued with technical difficulties that have prevented itfrom being a very robust quantitative and comprehensive tool for quantitative proteome profiling. For example, a high level of skill and expertise are required togenerate nearly identical 2D gel patterns with identical samples. Therefore it hasbeen extremely difficult to compare the protein spot intensities between two gelsrun under the same exact conditions, particularly if the gels were run in differentlaboratories.
Second, it is widely known that 2DE does not resolve complex protein mixtures very well, especially proteins that are very acidic, very basic, of higher molecular weight, or very hydrophobic [37–41]. Despite recent advances in IEFtechnology (e.g. narrow range gels) and pre-fractionations schemes that reducesample complexity, these methods commonly lead to increases in the amount ofwork to be done and are also plagued with reproducibility problems [42–44]. Athird limitation to using 2DE for quantitating protein abundance is that low abundance proteins go routinely undetected [40]. This is a direct reflection of the lowloading capacities inherent to 2D gels [40].
Exceeding the loading capacity of theFig. 13.1 Diagram demonstrating the 2D-PAGE methodand its application to quantitative protein profiling. Proteins extracted from cells or tissues are applied to an isoelectric focusing gel strip that separates the proteins according to their isoelectric point. Once focused, the gelstrip is applied to a SDS-PAGE gel where the proteins areseparated in the second dimension according their size.The gels are fixed and stained with coomasie blue or silverstain.
The pattern and quantity of the protein spots arerecorded with image analysis software and protein spotsof interest are cut out and analyzed by mass spectrometryto facilitate protein identification.13.2 Traditional Methodologies for Measuring Protein Abundance21521613 Differential Expression Proteomic Analysis Using Isotope Coded Affinity Tagsgel will result in poorer gel resolution, which complicates the identification andquantitation of individual proteins within the gel [40].
Fourth, it cannot be assumed that a protein spot contains a single protein species. Frequently two ormore proteins co-migrate to the same gel coordinates, making precise proteinquantification difficult. Lastly, one of the most significant shortcomings to using2DE as a quantify proteomic tool is related to the staining methods and imageanalysis software tools that are used to quantitate the signal intensities of resolvedprotein spots in the 2D gels [45, 46]. Silver and coomasie staining are the mostcommon staining techniques used for visualizing proteins in 2D gels.
Neithermethod is optimal for quantitative analysis because they display a very limited dynamic range for protein quantification and limited sensitivity [47–50]. The imageanalysis software tools available for measuring the spot intensities can be compromised by problems associated with accurately defining spot boundaries in the gel,difficulties in normalizing staining between gels, variation in spot intensities between gels and correctly matching a large number of protein spots between gels[45].
New developments in protein detection have tried to make the 2DE methoda more viable methodology for quantitative proteomic studies. For example, newfluorescent dyes called propyl-Cy3 and methyl-Cy5 can be covalently bound to proteins and therefore be used for protein detection and quantification (Fig. 13.2)[32]. Two protein samples can be individually labeled with either the Cy3 or Cy5dye and subsequently combined and run together in one 2D-gel.
The ratio of relative signal intensities emitted by the Cy3 and Cy5 dyes for each protein spot willreflect the relative protein abundance of that protein between the two samples.This technology is very similar to the dye technology that commonly used incDNA microarray analysis [2].
However, labeling proteins with these fluorescentdyes is not without limitations. Covalent modification of the protein may cause alterations in the proteins mobility and solubility during 2DE. Photobleaching, aswell as differences in the number of functional groups available for modificationwith the fluorophore may affect the overall sensitivity of the analysis [51]. Despitethese limitations there have been numerous reports of the successful applicationof 2DE the analysis of clinical or biological samples in the literature.
A study thatcompared differences in the secreted proteins between normal and diseased patients with bladder squamous cell carcinomas found a urinary marker that wassubsequently identified as the protein psoriasin [52]. It was suggested that thisprotein could be used as a marker to track the progression of patients with bladder squamous cell carcinomas. Another study that compared proteins from normal human luminal and myoepithelial breast cancer cells resulted in the identification of 51 proteins out of a total of 170 protein spots that were 2-fold differentially regulated [52]. Generally, 2DE technology has been most successful whenused as a differential-display technology to find differences in expression of readily soluble and relatively abundant proteins.Fig.
13.2 Diagram demonstrating protein quantification viafluorescent dyes in combination with 2D-PAGE. Proteins extracted from cells or tissues are incubated with a fluorescent dye (e.g. Cy3, Cy5) that reacts with functional groups(e.g. amines, carboxyls) in the protein to form covalentlylinked protein-dye conjugates. The dye-protein conjugatesare combined and subjected to 2D-PAGE analysis. The fluorescence intensity ratios of the protein spots are measuredusing image analysis software tools.
Proteins of interestedare identified as described in Fig. 13.1.13.2 Traditional Methodologies for Measuring Protein Abundance21721813 Differential Expression Proteomic Analysis Using Isotope Coded Affinity Tags13.3Isotopic Methodologies to Quantitative ProteomicsMass spectrometry (MS) based approaches without the involvement of gel electrophoresis have gained momentum as methods for large-scale protein identificationover the last few years [53, 54]. Unfortunately, the intensity of a peptide signal inthe mass spectrometer cannot be easily correlated to the amount of analyte presentin the sample.
To achieve accurate quantification by MS, suitable external or internalstandards need to be employed. The optimal internal standard for a particular peptide is the isotopically labeled form of that peptide. Therefore, the newest methodsfor quantitative proteome analysis are based upon the venerable technique of stableisotope labeling [55]. This method involves incorporation of a stable isotope ( e.g. 2H,13C, 15N) in one of the two samples (Fig. 13.3 A), and the concurrent analysis of thecombined samples by mass spectrometry.
The ratio of signal intensities for the isotopically heavy and normal form of a peptide indicates the ratio of abundance of thetwo peptides in the two samples being compared, as it is assumed that the peptidepairs from these samples contain the same physiochemical properties and behavesimilarly under all conceivable isolation and separation steps. This strategy therefore allows measurements in protein abundance between two samples if one ofthe protein samples is isotopically different from a second reference sample. Oneof the first applications of this strategy to quantitate protein expression in vivo wasperformed by Oda et al.
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