Chemical biology

Some forms of chemical biology attempt to answer biological questions by directly probing living systems at the chemical level. In contrast to research using biochemistry, genetics, or molecular biology, where mutagenesis can provide a new version of the organism, cell, or biomolecule of interest, chemical biology probes systems in vitro and in vivo with small molecules that have been designed for a specific purpose or identified on the basis of biochemical or cell-based screening (see chemical genetics). Chemical biology is one of several interdisciplinary sciences that tend to differ from older, reductionist fields and whose goals are to achieve a description of scientific holism. Chemical biology has scientific, historical and philosophical roots in medicinal chemistry, supramolecular chemistry, bioorganic chemistry, pharmacology, genetics, biochemistry, and metabolic engineering. Proteomics investigates the proteome, the set of expressed proteins at a given time under defined conditions. As a discipline, proteomics has moved past rapid protein identification and has developed into a biological assay for quantitative analysis of complex protein samples by comparing protein changes in differently perturbed systems. Current goals in proteomics include determining protein sequences, abundance and any post-translational modifications. Also of interest are protein–protein interactions, cellular distribution of proteins and understanding protein activity. Another important aspect of proteomics is the advancement of technology to achieve these goals. Protein levels, modifications, locations, and interactions are complex and dynamic properties. With this complexity in mind, experiments need to be carefully designed to answer specific questions especially in the face of the massive amounts of data that are generated by these analyses. The most valuable information comes from proteins that are expressed differently in a system being studied. These proteins can be compared relative to each other using quantitative proteomics, which allows a protein to be labeled with a mass tag. Proteomic technologies must be sensitive and robust; for these reasons, the mass spectrometer has been the workhorse of protein analysis. The high precision of mass spectrometry can distinguish between closely related species and species of interest can be isolated and fragmented within the instrument. Its applications to protein analysis was only possible in the late 1980s with the development of protein and peptide ionization with minimal fragmentation. These breakthroughs were ESI and MALDI. Mass spectrometry technologies are modular and can be chosen or optimized to the system of interest. Chemical biologists are poised to impact proteomics through the development of techniques, probes and assays with synthetic chemistry for the characterization of protein samples of high complexity. These approaches include the development of enrichment strategies, chemical affinity tags and probes. Samples for Proteomics contain a myriad of peptide sequences, the sequence of interest may be highly represented or of low abundance. However, for successful MS analysis the peptide should be enriched within the sample. Reduction of sample complexity is achieved through selective enrichment using affinity chromatography techniques. This involves targeting a peptide with a distinguishing feature like a biotin label or a post translational modification. Methods have been developed that include the use of antibodies, lectins to capture glycoproteins, immobilized metal ions to capture phosphorylated peptides and suicide enzyme substrates to capture specific enzymes. Here, chemical biologists can develop reagents to interact with substrates, specifically and tightly, to profile a targeted functional group on a proteome scale. Development of new enrichment strategies is needed in areas like non-ser/thr/tyr phosphorylation sites and other post translational modifications. Other methods of decomplexing samples relies on upstream chromatographic separations. Chemical synthesis of affinity tags has been crucial to the maturation of quantitative proteomics. iTRAQ, Tandem mass tags (TMT) and Isotope-coded affinity tag (ICAT) are protein mass-tags that consist of a covalently attaching group, a mass (isobaric or isotopic) encoded linker and a handle for isolation. Varying mass-tags bind to different proteins as a sort of footprint such that when analyzing cells of differing perturbations, the levels of each protein can be compared relatively after enrichment by the introduced handle. Other methods include SILAC and heavy isotope labeling. These methods have been adapted to identify complexing proteins by labeling a bait protein, pulling it down and analyzing the proteins it has complexed. Another method creates an internal tag by introducing novel amino acids that are genetically encoded in prokaryotic and eukaryotic organisms. These modifications create a new level of control and can facilitate photocrosslinking to probe protein–protein interactions. In addition, keto, acetylene, azide, thioester, boronate, and dehydroalanine- containing amino acids can be used to selectively introduce tags, and novel chemical functional groups into proteins. To investigate enzymatic activity as opposed to total protein, activity-based reagents have been developed to label the enzymatically active form of proteins (see Activity-based proteomics). For example, serine hydrolase- and cysteine protease-inhibitors have been converted to suicide inhibitors. This strategy enhances the ability to selectively analyze low abundance constituents through direct targeting. Structures that mimic these inhibitors could be introduced with modifications that will aid proteomic analysis- like an identification handle or mass tag. Enzyme activity can also be monitored through converted substrate. This strategy relies on using synthetic substrate conjugates that contain moieties that are acted upon by specific enzymes. The product conjugates are then captured by an affinity reagent and analyzed. The measured concentration of product conjugate allow the determination of the enzyme velocity. Other factors such as temperature, enzyme concentration and substrate concentration can be visualized. Identification of enzyme substrates (of which there may be hundreds or thousands, many of which unknown) is a problem of significant difficulty in proteomics and is vital to the understanding of signal transduction pathways in cells; techniques for labelling cellular substrates of enzymes is an area chemical biologists can address. A method that has been developed uses 'analog-sensitive' kinases to label substrates using an unnatural ATP analog, facilitating visualization and identification through a unique handle.