This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. It is of great importance for the productive outcome that all proteomics technologies are integrated in a logical way leading from the identification of unique differences in protein expression (biomarkers) in a complex mixture to the identification and full characterization of a protein in question. It is inevitable that each approach has strengths and weaknesses which will be considered in designing work-flow protocols. For example, every study will begin with at least one-dimensional fractionation to reduce sample complexity before screening and/or profiling can be applied. This is very important step because of two reasons. One is to reduce usually high range of concentration of proteins (high v. low abundant) in sample. Second, mass spectrometric analyses based on MALDI-TOF mode of protein/peptide ionization are very sensitive to contamination with detergents. Another example is to use custom design affinity chromatography columns. Removal of actin from cell lysates will vastly reduce the complexity of an initial sample. Another goal is to integrate high throughput assays with in-depth analyses. From our perspective 2DE is very useful technique because of its ability to make targeted analysis of selected protein spots instead of analyzing entire sample containing proteins, which are differentially expressed mixed with those which are not. Detection of proteins labeled with different fluorescent dyes in 2DE DIGE (Cy3, green and Cy5, red) greatly enhances analytical power of this technique of profiling because it does not reduce the ability of protein identification. Sensitivity of established method of protein identification based on peptide fingerprinting using ABI4800 MALDI-TOF/TOF instrument is approximately 10 fmoles. This approach is also a high throughput one. Moreover, isoforms of proteins which are different in PTMs such as N-terminal acetylation can be separated by 2DE, because they will differ in isoelectric point, but their difference in molecular weight or physicochemical properties are too small to separate them by other means such as 1DE or liquid chromatography. ABI4800 mass spectrometer also allows peptide sequencing in TOF/TOF configuration if enough material is available in the sample already used for peptide fingerprinting. Our experience indicates that 50 fmoles of peptide mixture in one sample can be used for 3 to 4 analyses. This enable us to perform one peptide fingerprinting determination in a TOF configuration and 2 to 3 individual peptides can be subsequently sequenced. Although 2DE DIGE is a powerful profiling technique, mass spectrometry based techniques such as iTRAQ offer additional information. It is now quite obvious that one profiling approach will reveal only partial characteristics of an investigated sample. In our future experiments we will use two or more techniques. Our current effort is directed to create a comprehensive system of data analysis.
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