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47.1 INTRODUCTION Proteomics

Genomics, which is the study of the genetic content of a particular cell or organism, has revolutionized scientific research. The success of various genome sequencing projects, including the complete annotation of the genome sequences of plant species such as A. thaliana and rice as well as large-scale EST sequencing initiatives http://www.ncbi.nlm.nih.gov/genomes/PLANTS/PlantList.html, have provided the scientific community with a wealth of information in the areas of plant physiology, biochemistry, and morphology. The next logical endeavor for the scientific community involves the structural and functional characterization of the products of the identified genes. Those products are proteins, and the burgeoning area of scientific research involving the elucidation of their various roles is known as proteomics. The term "pro-teome" was first used in the mid-1990s to describe the total set of proteins expressed in a given cell at a given time, and the word was derived from the fact that the study of proteomics is basically the study of the protein complement of the genome [1].

Proteomics can essentially describe any research that involves (a) the detailed characterization of proteins, and (b) the characterization of the proteome, including levels of expression, PTMs, localization, and interactions with other molecules in order to obtain a global view of cellular processes at the proteome level [2]. Research in this field of study is directed toward analyzing protein dynamics within an organism, tissue, or cell and is becoming quite common in plant biology to decipher the functions of various genes. Proteomics can therefore be for the discovery of novel targets (discovery proteomics) or the characterization of the structure and function of proteins (functional proteomics).

One of the most commonly utilized techniques in "discovery proteomics" to profile the proteomes of cells and organelles consists of protein extraction and separation by 2-DGE or by LC [3-5]. 2-DGE is a PAG-based protocol that separates proteins on the basis of two distinct parameters: molecular weights, and pI [6]. It is possible for this procedure to efficiently separate more than 1000 proteins stained with silver in an individual PAG [7]. Although 2-DGE is one of the classical and most widely used methods of proteome analysis [8], there are certain inherent disadvantages to this method such as low sensitivity, difficulty in resolving proteins that are large (over 150 kDa) or small (less than 10 kDa), or proteins that have very low (less than 3) or very high (greater than 10) pI values and the inability to resolve proteins with low solubility [3, 9, 10]. LC is a column-based procedure that uses chromatographic resins to separate peptides and proteins based on differences in specific characteristics such as affinity for specific ligands, size, ionic character, or hydrophobicity. It can be used as a prefractionation technique to reduce the complexity of a protein sample or to study the protein content of specific cellular components such as membranes or organelles. LC or 2D-LC has been demonstrated to be very reproducible, and crude protein extracts can be analyzed after only a few purification steps [11]. LC-based methods have been used to characterize large numbers of plant proteins [12, 13]. Selected protein spots from 2D gels or LC-protein fractions can be digested with enzymes that cleave at specific peptide bonds and then subjected to MS analysis for qualitative and quantitative comparisons. DIGE is another proteomics research technique and is quite similar to 2-DGE; however, there are differences because this type of gel electrophoresis involves tagging two protein solutions to be analyzed with different fluorescent dyes, such as Cy2, Cy3, or Cy5, and then mixing the solutions and analyzing them on the same gel [14, 15]. This allows for the visualization of any differences in protein amounts between the analyzed samples based on the predominance of one fluorescent dye over the other. In addition to 2-DGE and LC, there are other techniques that can be used in proteomics studies including MudPIT (which has been shown to be especially effective when attempting to identify as many proteins as possible from a particular sample [16]), ICAT, iTRAQ for relative and absolute quantitation, and SILAC [9, 17-21]. Such discovery techniques are discussed in detail elsewhere in this book and are beyond the scope of this chapter.

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