Protein

Interest in proteomics-based technology and strategies was buoyed, in part, by the somewhat surprising finding that the human genome contained 30,000-40,000 open reading frames, a number much smaller than expected and similar to that of lower organisms. This suggested that the level of diversity and complexity in the human is partly caused by alternative mRNA splicing and posttranslational protein modifications, including such processes as phosphorylation and oxidation/reduction.

Proteomics is defined as the protein component of the human genome. The recent establishment of 10 national pro-teomics centers funded by the National Heart, Lung, and Blood Institute's Proteomics Initiative will likely provide enormous resources, reagents, and techniques to the scientific community. This program was patterned after the Programs for Genomics Applications; it is considered instrumental in the development of novel and sensitive proteomics-based technologies.

Dimensional Gel
Fig. 2. Two-dimensional gel electrophoresis image; each black spot refers to a protein. From ref. 42. Used with permission from the Max Delbruck Center for Molecular Medicine.

Many protein databases have been established for the public, including SWISS-PRO/TrEMBL Protein Knowledgebase, the Protein Information Resource, and the Protein Data Bank. The majority of the information in these sites is collated by the National Center for Biotechnology Information's Entrez-Pro-tein database.

Two-dimensional polyacrylamide gel electrophoresis has been routinely used to separate large numbers of proteins (~2000 from a total cardiac protein extract) (35-38). However, the human heart may express more than 10,000 proteins, making sufficient separation difficult. Alternative approaches for identifying these molecules include liquid chromatography and mass spectrometry. Mass spectrometry has become a favored approach for protein identification because of its high sensitivity and high-throughput capacity. In this approach, identifying proteins is done by peptide mass fingerprinting by simply comparing the peptide masses obtained by mass spectrometry of a protein digest with theoretical peptide masses generated in silico using protein and nucleotide sequence databases. This works well when the protein of interest has been previously identified, but poorly when trying to make a novel discovery.

The novel identification of amino acid sequences is typically accomplished by employing automated chemical Edman microsequencing or tandem mass spectrometry. The establishment of public protein databases, including HSC-2DPAGE (39), HEART-2DPAGE (40,41), and HP-2DPAGE (42) has proven very helpful. A representative illustration of a 2D gel is shown in Fig. 2.

Essentials of Human Physiology

Essentials of Human Physiology

This ebook provides an introductory explanation of the workings of the human body, with an effort to draw connections between the body systems and explain their interdependencies. A framework for the book is homeostasis and how the body maintains balance within each system. This is intended as a first introduction to physiology for a college-level course.

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