Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 72
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Proteomic analysis is complicated since it is not only the changingamounts of different proteins but also the widespread post-translational processessuch as phosphorylation that alter dynamic function at the cellular level. As a result, an important motivation for functional proteomics is to find ways of concentrating on those proteins that are involved in a particular biological function of interest.15.1.3Structural ProteomicsSome proteomic approaches are mapping protein-protein interactions, determining the structure of protein complexes, and elucidating the 3-dimensional topology of proteins [9]. Currently, 2-hybrid analysis and protein array chips are beingused for screening protein binding partners.
While, automated and reproducibleprotein production, purification, crystallization and structure determination byX-ray beams from synchrotron radiation sources are allowing the high-throughputelucidation of protein structure. The deduction of protein structure is crucial forrational drug design. Co-crystallization and structural determination of hit compounds with target proteins may enable prioritization and optimization of leaddrug molecules. The information obtained from structural proteomic studies willprovide important information about the overall architecture of cells.15.2Proteomic Tools for Smooth Muscle Physiologists15.2.1Protein Separation: The 2DE TechnologyTraditionally, the “workhorse” for protein expression profiling has been two-dimensional polyacrylamide gel electrophoresis (2DE).
2DE is currently the mostgenerally applicable technique for separating and visualizing a large number ofproteins. A protein mixture can be resolved into its individual components by firstseparating proteins on the basis of their isoelectric points using ampholyte gradients in one dimension. Proteins are further resolved on the basis of their mobility(dependent upon molecular weight) through a polyacrylamide gel matrix in thesecond dimension. The initial set of 2DE experiments was able to resolve *1,000proteins from E. coli, and it was predicted that up to 5,000 distinct protein spotswould be discernable [10].
In addition, the application was able to detect post-translational modifications and amino acid mutations that altered a protein”s characteristic mobility in the 2D-gel. Protein abundance could also be quantified by using35S-methionine labeling; proteins differing in concentration by ratios of 10–4 to10–5 could be detected. Technological advances in the electrophoresis field hasgreatly improved 2DE resolution. Over 10 000 protein spots can now be resolvedon a single gel [11], although reports are normally below 2000 protein spots. The25725815 Investigations in Smooth Muscle Cell Physiologyresolution continues to be improved through the development of new isoelectric focusing media with narrower pH ranges and new gel matrices [12]. By resolving thesame protein mixture on multiple narrow range 2DEs, the total number of resolvable spots has increased.
Despite the outstanding properties and ease of use of 2Dgels systems, they did not become an integral part of protein expression profilingfor nearly two decades. 2DE technology did not reach its potential for the simplereason that methods for the rapid, routine identification of the protein spots inthe gels was not available. Three crucial developments have emerged to addressthese challenges. First, whole genome sequencing from a myriad of different organisms is defining at the gene level all the proteins that exist in that organism. This hasmeant that, instead of requiring time consuming complete Edman N-terminal sequence to identify a protein, partial information on fragments or sequence is adequate to identify the protein at the gene level within a genomic sequence database.
Second, protein sequencing by mass spectrometric methods can now supplythe needed information to identify the protein at sensitivities well below 1 pmol.And third, the development of algorithms for the identification of proteins by massspectrometric data matched to genomic databases. It is now clear that the sequencing of multiple whole genomes and the field of bioinformatics along with corresponding advances in mass spectrometric methods have drastically altered the landscape of biological research at the protein level.Although 2DE is currently the best protein analog of cDNA arrays, it does notproduce a comprehensive display of the entire cellular proteome.
At least part ofthis problem arises from the well-documented difficulty in resolving certain typesof proteins (i.e. membrane proteins, proteins with mass greater than 100 kDa,highly basic proteins, and highly acidic proteins) by 2DE [13, 14]. Many spots arelikely to contain multiple overlapping proteins that are not resolved in gels that attempt to cover most of the pI range appropriate for all cellular proteins. Oneapproach to improve resolution has been to use immobilized pH gradient (IPG)strips to cover narrower ranges of pI. Other disadvantages of 2DE include a lackof reproducibility and a lack of dynamic range. The detection of proteins that occur with low copy number remains a major limitation [11, 15].
Indeed one of themain impediments to easily observe all soluble proteins is the large variation inthe concentrations of cellular proteins with estimates placing the dynamic rangeof protein expression at between 7 to 9 orders of magnitude [11]. With limitationson the amount of total protein able to be loaded on a gel that is consistent withgood resolution of individual protein spots on analytical 2DEs, detection of lowabundance proteins remains difficult if not impossible. Currently, the total proteinload that can be applied to analytical 2DE and the chemical staining methods limit analysis to less than half of the yeast proteome [16].
This problem is even moreserious in proteomes isolated from more complex organisms such as humans.As the field of proteomics evolves rapidly away from a simple visual representationof cellular proteins on 2DE, the availability of new data and technologies has triggered many initiatives to start large-scale proteomic studies. 2DE involves a greatdeal of expertise and hands-on-time to execute, thus there is a strong need to workon replacement technologies that are easily automated and highly reproducible.15.2 Proteomic Tools for Smooth Muscle Physiologists15.2.2Mass SpectrometersMass spectrometry (MS) was originally developed in chemistry laboratories as atool for small molecule analysis and structure determination.
Mass spectrometersoriginally required small charged gaseous molecules as analytes. Proteins arelarge and polar molecules in comparison, and ionization methods were not available to introduce proteins into the MS instrumentation. Beginning in the early1980s as the development of techniques capable of presenting proteins in an ionized gaseous form to the MS instrument were perfected, MS slowly emergedfrom chemistry laboratories to become an important tool in protein biochemistryand the key technology driving the proteomics field. Electrospray ionization (ESI)[17] and matrix-assisted laser desorption ionization (MALDI) [18] are the two ionization techniques, which are most responsible for the success of mass spectrometry in proteomics.
MALDI creates ions by using a laser to excite a crystalline mixture of analyte molecules and energy desorbing matrix into the gas phase. ESIuses a potential difference between a capillary and the inlet of the mass spectrometer to cause charged droplets to be released from the tip of the capillary. As thedroplets evaporate, gas phase charged ions are desorbed from the droplets. In addition to the ionization source, a mass analyzer and an ion detector are also present in all mass spectrometers.A number of excellent and exhaustive reviews have addressed new technical developments in mass spectrometry [19, 20]. See also chapters 12 and 13. Ratherthan engage in a lengthy description of the current advances in mass spectrometry, we have chosen to highlight some significant features, which have directedour current experience with mass spectrometers.
Three different methods areused to measure the mass-to-charge ratio of analytes such as proteins, peptides,or peptide fragments. The most common mass spectrometer in use in the proteomics field is the time of flight (TOF) mass analyzer, which measures the mass/charge ratio of ions by the time it takes them to travel through a flight tube to thedetector. The other frequently used mass detection methods are quadrupole MS,the separation of analytes by quadrupole electric fields generated by metal rods,and ion-trap MS the separation of analytes by selective ejection of ions from athree-dimensional electrical-trapping field.
Both ESI and MALDI ionizations canbe used to create ions for analysis in TOF-MS. Typically, however, MALDI iscoupled with TOF-MS and ESI with quadrupole and ion-trapping MS becauseMALDI produces short bursts of ions and ESI produces a continuous beam ofions. For peptide sequencing, two steps of mass spectrometry are completed intandem (tandem mass spectrometry or MS/MS). These mass spectrometers areable to select a particular peptide ion, fragment it in a collision chamber, andthen detect the resulting fragment ions.
The actual amino acid sequence is reconstructed from the fragmentation pattern with computer software. The primary advantage of tandem mass spectrometry is the ability to select a particular peptideion from a mixture, allowing for the identification of components of complex mixtures.25926015 Investigations in Smooth Muscle Cell PhysiologyMS/MS is performed by using the same mass separation principle twice or bycombining two different separation principles. To date, the quadrupole-TOF(qTOF) has been an effective MS/MS performer. Two recent advances are theMALDI-qTOF [21] and the two section TOF separated by a high energy collisionchamber (MALDI-TOFTOF) [22] mass spectrometers.
These instruments providevery fast, automated analysis of large numbers of samples with high sensitivity,and the ability to obtain amino acid sequence from any selected peptide.15.2.3Protein Identification by Database SearchingBoth ESI-MS/MS and MALDI-MS are routinely used for protein identificationfrom 2DE although different search strategies have been developed depending onthe type of data obtained from the mass spectrometer.Peptide Mass Fingerprinting. Mass fingerprinting uses the characteristic distribution of peptide masses obtained by digesting proteins with a sequence-specificprotease such as trypsin.
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