The control of enzyme activity by reversible phosphorylation and the role of protein kinases in signal transduction illustrate well the pivotal role of protein phosphorylation in cell regulation. There are numerous examples of the role of protein kinases and phosphatases in disease, and protein kinases in particular are

From: Cancer Drug Discovery and Development: Protein Tyrosine Kinases: From Inhibitors to Useful Drugs Edited by: D. Fabbro and F. McCormick © Humana Press Inc., Totowa, NJ

now established as druggable therapeutic targets (1). Phosphoproteomics includes the large-scale determination of protein phosphorylation in cells and tissue, sometimes referred to as phosphoprofiling. Phosphoprofiling is a global approach that can be used to characterize biological states including therapeutic responses. It is augmented by another proteomic method, protein kinase (and protein phosphatase) interaction mapping, which together have the potential—yet to be fully realized—to provide comprehensive pictures of cellular states. A particular utility for these methods will be in drug discovery and development.

The amount of protein phosphorylation in a cell is vast. Approximately one-third of mammalian proteins are phosphorylated (2). Therefore the identification of those protein modifications associated with a given pathway, cellular process, or cellular response of interest is an important goal in phosphoproteomics. A noteworthy feature of protein kinases as drug targets is the fact that their activity—and hence modulation by drugs—is frequently reflected not only in their own modification by phosphorylation (e.g., autophosphorylation), but also by the phosphorylation of their substrates, and by their protein-protein interactions. These features are the primary data of phosphoproteomics. Differential phosphoproteomics— that is, the identification of changes in protein phosphorylation against a background of largely unchanging cellular phosphorylation—is a logical but challenging approach to illuminate phosphorylated proteins of particular interest. For example, differential phosphoproteomics may reveal drug-induced changes in phosphorylation as a means to understand drug mechanisms and toxicity in pre-clinical models and monitor patients in clinical studies. Hence phosphoproteomics will logically find its first pharmaceutical applications in the area of protein kinase-directed therapeutics. Moreover, because phosphorylation is a universal mechanism of cell regulation, phosphoproteomics holds promise of becoming a generally applicable tool in drug discovery and development.

Phosphoprofiling is based on an ability to isolate phosphorylated peptides from an enzymatic digest of whole-cell or tissue protein extractions by using immobilized metal affinity chromatography (IMAC), followed by the identification of phosphopeptides and their corresponding proteins by mass spectrometry (3). Such samples contain many thousands of phosphopeptides and their identification and characterization by mass spectrometry and data analysis have benefited from the development of new mass spectrometry instrumentation and software solutions, as described below. Imbedded in the data are two basic pieces of information: the sequence context of the phosphorylation sites, and, in a differential experiment, a ratio of the abundance of a given peptide in two samples. The former can be used to identify the protein from which the peptide was derived by searching protein databases, whereas the latter can be used to quantify the peptide, at least in a relative or comparative sense. In addition, the total data set itself is likely to contain information in the form of patterns (e.g., drug or disease signatures) that reflect the physiological state of the cell.

The importance of "global"—in a total cellular context—attempts at protein phosphorylation analysis predate current proteomics trends and terminology. For example, early practitioners of two-dimensional gel electrophoretic analysis of 32P metabolically labeled cell-derived protein extracts appreciated the potential information content inherent to cellular phosphorylation patterns (4). Although greatly facilitating our understanding of signal transduction and oncogene function, these methods did not readily provide insight into the identity of phosphorylated proteins or sites of phosphorylation. The introduction of protein phospho-specific antibodies and other tools such as protein phosphotyrosine-binding SH2 domains allowed the identification, by nongenetic means, of the substrates and interacting proteins in kinase-mediated signal transduction (5,6). Although various sequence-specific antibodies are available and being put to use in array format to characterize disease states (7), a limitation exists in that the antibody reagents available for these applications are based on the limited set of known phosphorylations discovered largely in experimental systems that may not reflect precisely those that may occur in animals. A goal for phosphoproteomics is therefore the global analysis of protein phosphorylation in tissue that will provide both detail at the level of individual protein regulation, and phosphoprofile patterns that reflect cellular states as a means to recognize and understand developmental and disease progression, and therapeutic responses, just to name a few.

In this chapter, we briefly review advances in measuring protein interactions of protein kinases, and follow with a more focused review of new approaches for global protein phosphorylation mapping by application of advanced proteomics methods based largely on mass spectrometry-based analytical methods. A significant bioinformatics infrastructure and tools for acquiring and analyzing mass spectrometry-based phosphopeptide data are required to support phosphopro-teomics, but are not the subject of this chapter.

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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