Structural and Functional Proteomics

As the amino acid sequence of a protein is encoded in the nucleotide sequence of a gene, it has become possible to determine the amino acid sequences or primary structures of large numbers of proteins by deciphering the nucleotide sequences of an organism deposited in the gene databank. 

2 Determining the Primary Structure of Proteins by Different Methods 

It is important to determine the total amino acid composition of a peptide before the amino acid sequence analysis is undertaken. This helps to establish the final order of amino acids in the peptide. The understanding of amino acid composition also helps to resolve the situation of whether the peptide contains two adjacent glycine (mol Wt = 57 daltons) residues or just one aspargine (mol. Wt = 114 daltons). This dilemma is based on the molecular weight data alone. 

The total amino acid composition is determined by a complete hydrolysis of the peptide with 6 M HC1 at a high temperature. The hydrolysate is run on a paper electrophoresis to separate the amino acids. The different amino acids are visualized by staining with a dye called dinitrophenol. All amino acids produce yellow spots except proline, which appears blue. The intensity of color determines the amount of each amino acid in a particular spot. The different amino acid spots are identified by comparing their mobility with the known amino acids used as standard during electrophoresis. 

Therefore, the sequence of these 10 amino acids in the peptide can be deduced as lys glu thr ala ala ala lys phen glu arg. 

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Naylor, S. and Kumar, R. (2003) Emerging role of mass spectrometry in structural and functional proteomics. Adv. Protein Chem., 65, 217 -8. 

Proteomics, on the other hand, is the application of molecular biology, biochemistry, and genetics to study the structures and functions of proteins expressed by cells. Unhke the genome, which is reasonably static, the proteome changes constantly in response to intra- and extracellular signals. As proteins are vital to our cells and the biological pathways, an in-depth understanding of proteomics would help to elucidate the processes of disease and to devise means to counteract errant cells and processes. 

The real value in the genome sequence is to find out the regions of the genome that encode proteins. Proteomics, the study of the structures and functions of proteins, further enhances our understanding of proteins and their functions, leading to insights on how they are affected in normal and disease conditions. Exhibit A2.3 shows some of the medical conditions due to genetic problems. 

The protease-activated receptors (PARs), a subclass of GPCRs that function in the coagulation cascade, suggest that a comprehensive survey of the GPCR portion of the proteome provides information about the structure and function of this receptor class. The PAR factor II (thrombin) receptor-like 2 (F2RL2) is inactive in the cascade until proteolytic cleavage of its extracellular amino terminus. A Phe240Ser variant that is located in the second intracellular loop, found at a frequency of approx. 8%, disrupts receptor activation by proteolysis. 

The marriage of HPLC to mass spectrometry (MS), now developed into a mature instrumentation, continues to greatly impact many of the separation sciences, especially in pharmaceutical analysis where it has been used in new drug discovery [23,24] and in drug metabolite identification [25-27]. HPLC-MS has also made an impact on lipid research, providing a convenient approach to the analysis of phospholipids and fatty acids [28,29]. It has also greatly benefited the field of proteomics [30-34], especially analysis of protein structure and function. 

The genome of an organism provides a blue print for its structural and functional attributes. Genome functions through the synthesis, regulation, and activity of proteins. A dynamic and well balanced network of DNA-protein, RNA-protein, and protein-protein interactions (PPIs) maintain the cellular system as a complex but cohesive unit (1). Proteomics serves as a medium to unravel protein functions and ultimately to understand a living system or a disease condition (2-5). Over the years, classical genetic... 

Before the era of proteomics, structure and function were characterized, often in... 

Structural proteomics aims the determination of three-dimensional protein structures in order to better understand the relationship between protein sequence, structure, and function. NMR and x-ray crystallography have been significant methods and indispensable tools to determine the structure of macromolecules, especially proteins. Many biotechnology companies have been using these two techniques for enlightening protein structure (Table 5.5). 

New precision technology, based on protein structures and function makes it possible for clinicians to detect cancer earlier than ever and provide individualized treatment. Following the discovery of new proteins and gene maps, cancer researchers believed that proteomics is a revolutionary approach to detect cancer and other major illnesses (e.g., HIV) during their early phases and to tailor individualized therapy. 

With the completion of genomic DNA sequencing for various species, attention has shifted towards the structural and functional characteristics of proteins. However, rapid progress in proteomics requires the following (a) availability of many proteins, and (b) availability of sufficient amounts of proteins in naturally folded states. To meet these requirements, various existing protein production technologies have been adopted for genome-wide protein translation. 

The resulting system, with or without automation, was attractive for the systematic study of protein structure and function in the context of modem proteomics. 

Bioinformatics uses computers to create and maintain large electronic databases on genomes, protein sequences, and proteomes. With the help of protein prediction software, the computer analysis of genome sequences is producing thousands of new proteins of unknown structure and function. These proteins are called hypothetical proteins because they are predicted from the gene sequence. To know if they really exist would require that they be isolated, purified, and subjected to X-ray crystallography or... 

In contrast, Functional Proteomics concerns the manner in which proteins interact and, in turn, how these interactions determine function, both normal and abnormal. This approach is less reductionist than Expression Proteomics as proteins are studied in the context of their complex cellular interactions. Finally, Structural Proteomics is concerned with the primary through tertiary structure of proteins, and modifications therein, largely determined by x-ray and NMR analysis of protein crystals (20). In this chapter we will concern ourselves only with Expression and Functional Proteomics. 

PROTEOMIC ANALYSIS USING INTERNET RESOURCES STRUCTURE AND FUNCTION... 

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