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- Delfina C Domínguez, PhD⇑
- Rosana Lopes, PhD
- M Lorraine Torres, MS
- Address for correspondence: Delfina C Domínguez PhD, University of Texas at El Paso, Clinical Laboratory Science, College of Health Sciences.1101 N. Campbell Street. El Paso TX, 79902-4238. (915) 747-7238, (915) 747-7207 (fax). Delfina{at}utep.edu.
Recognize the different tools applied to proteomics analysis.
Differentiate the procedures utilized in proteomics studies starting from protein sample preparation to protein identification methods.
Identify the steps needed for analysis of protein expression by software packages.
Distinguish the methods employed for qualitative and quantitative protein identification.
Extract
Proteomics techniques are essential tools for protein detection and characterization. Besides several advances in the proteomics field, the two-dimensional electrophoresis (2-DE) technique is the most important method for protein separation. The combination of 2-DE technique, new advances in mass spectrometry and bioinformatics promises to unveil protein function and pathological mechanisms of disease.
After geneticists sequenced the human genome and learned of the poor correlation between mRNA and protein numbers, special attention was given to the gene products, the proteins. From approximately 25,000 predicted genes in human beings,1 it is expected that there will be more than 500,000 protein products as a result of splicing and protein modifications.2,3 Proteomics analyses utilize several techniques for protein separation, detection, and identification. For separation of simple and complex protein mixtures, two-dimensional electrophoresis (2-DE) is the technique of choice. Several staining procedures can be applied to 2-DE gels for protein detection. Mass spectrometry is considered the best method for protein identification.
Sample preparation A wide variety of samples can be utilized to identify and characterize proteins in proteomic studies. Some of these samples include: samples fractionated from an organism (eukaryote or prokaryote), tissue, cell lysates, and physiological fluids.2,4 A careful research design should be planned to acquire a meaningful representation of the proteins of interest. It is essential to ensure that proteins are soluble in order to obtain an accurate protein analysis. Protein solubilization is achieved utilizing chaotrops (urea), reducing agents (dithiothreitol), detergents (CHAPS, Triton-X), buffers (Tris), and <0.2% ampholytes. In addition,…
ABREVIATIONS: 2-DE = two-dimensional electrophoresis; CHAPS = (3-[ (3-Cholamidopropyl)-Dimethylammonio]-1-Propane Sulfonate); ESI - MS= electrospray ionization mass spectrometry; ICAT = isotope-code affinity tag; IPG = immobilized pH gradient; LC-MS = liquid chromatography mass spectrometry; MALDI = matrix assisted laser desorption ionization; MALDI-TOF = matrix assisted laser desorption ionization time-of-flight; mRNA = messenger ribonucleic acid; pI = isoelectric point; SELDI-MS = surface-enhanced laser desorption/ionization time-of-flight.
Recognize the different tools applied to proteomics analysis.
Differentiate the procedures utilized in proteomics studies starting from protein sample preparation to protein identification methods.
Identify the steps needed for analysis of protein expression by software packages.
Distinguish the methods employed for qualitative and quantitative protein identification.
- © Copyright 2007 American Society for Clinical Laboratory Science Inc. All rights reserved.
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