Protein Identification and Analysis

Prokaryotic cells express hundreds to thousands of proteins while higher eukaryotes express thousands to tens of thousands of proteins at any given time. If these proteins are to be individually identified and characterized, they must be efficiently fractionated. One-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has typically been use to study protein mixtures of 100 proteins. Onedimensional electrophoresis is useful because nearly all proteins are soluble in SDS, molecules ranging from approximately 10,000 to 300,000 molecular weight can be resolved, and extremely basic or acidic proteins can be visualized. The major disadvantage to one-dimensional gels is that they are not suitable for complex mixtures such as proteins from whole cell lysates. 

Because the pi of a protein is based on its amino acid sequence, this technique has good resolving power. The resolution can be adjusted further by changing the range of the pH gradient. The use of immobilized pH gradient (IPG) strips has enabled reproducible micropreparative fractionation of protein samples, which is not consistently possible when ampholytes are used in the first dimension (Gorg et al., 2000). 

The second step in 2D electrophoresis is to separate proteins based on molecular weight using SDS-PAGE. Individual proteins are then visualized by Coomassie or silver staining techniques or by autoradiography. Because 2D gel electrophoresis separate proteins based on independent physical characteristics, it is a powerful means to resolve complex mixtures proteins (Fig. 2.1). Modem large-gel formats are reproducible and are the most common method for protein separation in proteomic studies. 

Another limitation of 2D gels is that membrane proteins are underrepresented. Because membrane proteins account for approximately 30% of total proteins (Wallin and Von Heijne, 1998), this is a serious problem for characterization of the proteome. The relative lack of membrane proteins resolvable on 2D gels can be attributed to thee main factors (i) they are not abundant, and therefore are difficult to detect by standard staining techniques, (ii) they often possess alkaline pi values, which make them difficult to resolve on the pH gradients most often used for isolelectric focusing, and (iii) the most important reason for under representation may be that membrane proteins are poorly soluble in the aqueous media used for isoelectric focusing (Santoni et al., 2000). Membrane proteins are designed to be soluble in lipid bilayers and are therefore difficult to solubilize in water-based solutions. 

Reducing complexity Protein fractionation prior to electrophoresis 

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Liu, H. J., Berger, S. J., Chakrahorty, A. B., Plumh, R. S., Cohen, S. A. (2002). Multidimensional chromatography coupled to electrospray ionization time-of-fhght mass spectrometry as an alternative to two-dimensional gels for the identification and analysis of complex mixtures of intact proteins. J. Chromatogr. B 782(1-2), 267-289. 

Field, H.I., Fenyo, D., Beavis, R.C. (2002). RADARS, a bioinformatics solution that automates proteome mass spectral analysis, optimises protein identification, and archives data in a relational database. Proteomics 2, 36 17. 

This chapter has presented several comprehensive 2DLC approaches combining a first-dimension IEX separation and a second-dimension RP separation for the analysis of complex protein mixtures typical in proteomics studies. Online ESI-TOF/MS detection provided sensitive detection and valuable qualitative information (MW) for proteins eluting from the MDLC system. Coordinated fraction collection and subsequent MS analysis of peptides produced by proteolysis of the fractions provided in-depth information on protein identification and a mechanism... 

A modified version of 2DE and gel image analysis, with silver staining, autoradiography, and protein identification and measurement of peptide mass, uses matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) as a rapid and sensitive technique for identifying peptides. MALDI-TOF-MS applies well to protein detection in biological fluids.56 A second advantage of this technique is... 

H. I. Field, D. Fenyo, and R. C. Beavis. RADARS, a Bioinformatics Solution that Automates Proteome Mass Spectral Analysis, Optimises Protein Identification, and Archives Data in a Relational Database. Proteomics, 2, no. 1 (2002) 36-47. 

ExPASy Proteomics tools (http //expasy.org/tools/), tools and online programs for protein identification and characterization, similarity searches, pattern and profile searches, posttranslational modification prediction, topology prediction, primary structure analysis, or secondary and tertiary structure prediction. 

The most prominent field of applications for microchip—MS concerns identification and analysis of large molecules in the field of proteomics according to the reduced separation time compared to conventional approaches such as gel-based methods for protein analysis. High-throughput analyses, with lower contamination and disposability, are other features of microfabricated devices that allow the fast screening of proteomic samples in the clinical field. Applications also include the analysis of low-molecular-weight compounds such as peptides or pharmaceutical samples. 

Proteome analysis in general involves two stages protein separation and subsequent identification and analysis. Multidimensional separations are required in order to result in an adequate resolution of complex protein or peptide... 

Rasmussen, M., Jacobsson, M., and Bjorck, L. (2003). Genome-based identification and analysis of collagen-related structural motifs in bacterial and viral proteins. J. Biol. Chem. 278, 32313-32316. 

D. R. Goodlett, Mass spectrometry-based methods for protein identification and phosphorylation site analysis, in ... 

The comprehensive molecular biology server, ExPASy (Expert Protein Analysis System), provides Proteomic tools (http //www.expasy.ch/tools) that can be accessed directly or from the ExPASy home page by selecting Identification and Characterization under Tools and Software Packages. The Protein identification and characterization tools of the Proteomics tools (Figure 11.4) provide facilities to ... 

Figure 11,4. ExPASy Proteomic tools. ExPASy server provides various tools for proteomic analysis which can be accessed from ExPASy Proteomic tools. These tools (locals or hyperlinks) include Protein identification and characterization, Translation from DNA sequences to protein sequences. Similarity searches, Pattern and profile searches, Post-translational modification prediction, Primary structure analysis, Secondary structure prediction, Tertiary structure inference, Transmembrane region detection, and Sequence alignment.

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A crucial aspect of studying gene function is identification of the protein formed by the gene in question determination of protein function is a traditional aspect of biology. The science of protein identification and their systematic, large-scale analysis is proteomics. Protein profiles can be correlated with specific effects of disease and with alterations of function. The importance of the subject is well illustrated by the recent creation of a journal entitled Proteomics. ... 

Gradually, over the past twenty years, mass spectrometers were interfaced with a number of protein chemistry assays to generate detectors providing superior information. With the increased performance and versatility of the instrumentation dedicated to the life sciences, new analytical strategies for peptide and protein identification and characterization have emerged in which MS and bioinformatic tools are key players. MS has an enormous impact on the capability for structural analysis of bio-molecules, thanks to the ability to create gas phase ions of the peptides and proteins to be analyzed. Peptides and proteins are often charged and polar, making... 

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