Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 61
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(Fig. 13.3 B). In this study one culture of yeast cells wasgrown on medium containing the natural abundance of the isotopes of nitrogen14N (99.6%) and 15N (0.4%) and another yeast culture was grown on the same medium enriched in 15N (> 96%). In these experiments stable isotope incorporation wastherefore achieved by metabolic labeling. After an appropriate growth period the proteins from both yeast samples were extracted and separated by reverse-phase highperformance liquid chromatography (RP-HPLC) and then separated by sodium-docecyl sulfate PAGE (SDS-PAGE).
In-gel digestion of spots of interest resulted in peptide fragments that were used in protein identification by peptide mass mapping.The 15N incorporation shifted mass of the peptides upwards by one mass unit perincorporated nitrogen. Peptides therefore appeared as doublets in the mass spectrum and the relative peak heights of the two signals indicated the relative abundance of the two forms.
The ratio of peak intensities was linear over two orders ofmagnitude. This study lead to the quantification of 42 highly abundant proteins"Fig. 13.3 Demonstration of stable isotope la-beling for quantitative protein profiling. A) Digested protein containing an equal amount ofthe light 14N and heavy 15N isotopes resultsin isotopic peptide pairs of equivalent ion intensities that are shifted by the mass difference of the isotope in the mass spectrometer.B) Oda et al used in vivo stable isotopic labeling in combination with 2-dimensional sepa-ration to quantify protein abundance in yeast.Cells grown on regular medium 14N (99.6%)or enriched medium 15N (> 96%) were extracted, combined, and separated by HPLC.Resulting HPLC fractions were resolved bySDS-PAGE, protein spots excised, trypsin digested, and isotopic peptides were quantifiedand identified in the mass spectrometer(MALDI).13.3 Isotopic Methodologies to Quantitative Proteomics21913 Differential Expression Proteomic Analysis Using Isotope Coded Affinity TagsQuantification and protein identification22013.4 Isotope-Coded Affinity Tags (ICATTM) for Quantitative Proteome Analysisfor two pools of yeast cells that were genetically identical but differed in their abilityto express the G1 cyclin CLN2.
The technique had a percentage error of only ± 10%,underscoring the potential for very precise quantification. Metabolic stable isotopiclabeling is not without limitations. Its major shortcoming is that it is very impractical and cost prohibitive to grow and label mammalian cells with stable isotopes. Thisis in contrast to bacterial or yeast samples which are readily metabolically labeled[56]. Furthermore, applying this approach to tissues is impractical if not impossible, even though there is a very strong interest in developing methodologies thatcan accurately and robustly compare differences in protein abundance in normaland disease tissue [57].13.4Isotope-Coded Affinity Tags (ICATTM) for Quantitative Proteome AnalysisRecently, we have developed a new class of reagent termed ICATTM [58].
These reagents are designed to incorporate stable isotope tags into proteins post isolation,by chemical reactions. The method based on these reagents is intended to measure quantitative differences in protein abundance between two samples, irrespective of the source of the sample. As shown in Fig. 13.4A, the ICAT reagent introduces stable isotope tags into proteins via the selective alkylation of the sidechains of reduced cysteines with either a deuterium-labeled heavy form (d8) or hydrogen-labeled light form (d0) of the reagent (Fig. 13.4B).
The ICAT-reagents arecomposed of three elements: a biotin affinity tag, a linker region in which the8 hydrogens or 8 deuteriums are located, and a functional group that reacts withsulfhydryl groups within proteins. In ICAT reagent based protein profiling experiments protein mixtures derived from two distinct cell populations are reducedand treated with the isotopically heavy and normal forms of sulfhydryl-specificICAT-reagent, respectively.
The labeled samples are then combined and digestedwith trypsin, and the resulting complex peptide mixtures are fractionated by ionexchange chromatography. The biotinylated (stable isotope labeled, cysteine containing peptides) in individual ICAT-labeled ion-exchange peptide fractions are selectively enriched by avidin-biotin chromatography and then subjected to microca3Fig. 13.4 The ICAT reagent method for quantitative protein profiling. A) The ICAT reagentis composed of three elements: an affinity tag(biotin), used for purification of ICAT-labeledpeptides, a linker that incorporates an isotope, and a reactive group that has specificitytoward thiol containing moieties.
The ICAT reagent exist in two forms, a light form referredto as d0 and a heavy form referred to as d8.B) The ICAT method involves extracting protein from cells in two different states, dena-turing, reducing, and reacting these proteinswith the ICAT reagent. The d0 or d8 labeledprotein samples are combined, trypsinized,and subjected to ion-exchange chromatography. The ICAT-labeled peptides are isolated bypurification over an aviding affinity matrix andsubsequently analyzed by reverse phase HPLCMS/MS. The ratio of the ion intensities of theICAT pairs is quantified which results in thequantification of protein abundance betweenthe two cell states.22122213 Differential Expression Proteomic Analysis Using Isotope Coded Affinity Tagspillary reversed-phase high performance liquid chromatography electrospray ionization tandem mass spectrometry (lHPLC-ESI-MS/MS).
Pairs of chemically identical ICAT-labeled peptides essentially co-elute from the RP column (the d8 formof the peptide elutes slightly before the d0 form of the peptide) and are recognized by the mass spectrometer. The relative ion intensities of the two differentially isotopically tagged peptides indicate their relative abundance in the sample.Concurrently, the detected peptides are selected for (CID) and the CID spectra aresearched against sequence databases for the purpose of identifying the protein fromwhich the peptide originated. The method therefore allows for the simultaneousidentification and quantification of the components of complex protein mixtures.In the intial study describing the method Gygi et al.
identified and quantified34 yeast proteins that were extracted from yeast cells grown on either galactose orethanol carbon sources. Many of the proteins identified and quantified were actually enzymes involved in carbon metabolism. For example the ADH2 protein,which is repressed by glucose and galactose sugars, was shown to be induced bymore than 200-fold in yeast cells grown on ethanol as a carbon source. These results support the known biochemical activity of the ADH2 enzyme, since this enzyme catalyzes the conversion of ethanol into acetaldehyde, which is ultimatelyfed back into the tricarboxylic and glyoxylate cycles for energy utilization [59].
Themeasured ICAT peptide signal intensity ratios were very robust as reflected in thelow percent error values (< 12%) for ratios of a standard ICAT-labeled peptide mixture in vitro. The ICAT method has a number of advantages. First, the methodprovides quantitative protein profiles of complex samples without the need forprior protein separation by gel electrophoresis or other methods. Second, allclasses of proteins with the exception of proteins devoid of cysteine are amenableto the ICAT method.
The detection of low abundance proteins and hydrophobicmembrane proteins has already been demonstrated [40, 60]. Third, the ICAT labeling strategy is applicable to the extraction of proteins from any biological source,including cells, tissues, or bodily fluids under any number of physiological ornon-physiological conditions. Fourth, the alkylation of cysteines by the ICAT reagent is very specific and is not compromised by the presence of non-ionic detergents (NP-40, Triton-X 100) and strong denaturing agents (SDS, urea, guanidineHCl) [58, 60, 61].
This is critical for the analysis of hydrophobic or very insolubleprotein complex. Fifth, the complexity of peptide mixtures is greatly reduced bytargeting only those peptides that contain a reactive cysteine residue. This purification step is critical because it reduces the number of peptides that have to bemass spectrometrically analyzed and processed. This is important because thenumber of peptides eluting from the reverse phase chromatograph has to be lowenough to not overwhelm the duty cycle of the mass spectrometer operating inMS/MS mode. Lastly, the ICAT method can be applied to any type of front-endbiochemical, immunological, or physical fractionation scheme for the analysis ofprotein complexes and or low abundance proteins. However, the ICAT method isnot without limitations.
First, the ICAT label is relatively large and the fragmentation pattern induced after CID can complicate the interpretation of the fragmention spectra that are used for protein identification via sequence database search-13.5 Biological Applications of the ICAT Methoding. Second, the ICAT method only works for those proteins that contain at leastone cysteine residue. Even though the number of proteins that are cysteine-free isquite small in yeast and other eukaryotic species, some microbial species have amuch higher fraction of cysteine-free proteins (*8%). The development of newICAT reagents with specificities different from SH groups will alleviate this problem.
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