Abstract
This chapter is devoted to proteomics, defined as the study of the expressed proteins in the cell. Firstly, practical ways of making measurements on these proteins is described. Then, some of the theoretical challenges such as protein identification are dealt with. The ensemble of kinases and phosphatases, known as the kinome, is covered and, generalizing, biochemical signalling.
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Notes
- 1.
Northern blotting allows detection of specific RNA sequences. RNA is fractionated by agarose gel electrophoresis, followed by transfer (blotting) to a membrane support, followed by hybridization with known DNA or RNA probes that are radioactively or fluorescently labelled to facilitate their detection. The technique can be thought of as a variant of Southern blotting, in which specific DNA sequences from a sample are probed in a similar fashion.
- 2.
If one is trying to determine whether certain genes are present in a bacterial culture (for example), the array would be coated with patches of complementary nucleic acid sequences. The DNA is extracted from the bacteria, subjected to some rudimentary purification, separated into single strands, and usually cut into fragments with restriction enzymes before pouring over the microarray.
- 3.
See Chumakov et al. (2005) for a discussion of design principles.
- 4.
Fodor et al. (1991).
- 5.
E.g. ethidium bromide, the fluorescence of which becomes about 20-fold stronger after it is intercalated into double-stranded DNA.
- 6.
An expression profile is defined as a two-column table, with conditions in the left-hand column and the corresponding (relative) amounts of expressed proteins (possibly as RNA) in the right-hand column.
- 7.
Yeakley et al. (2002) These researchers combined their fibre optic array with the technique of RNA-mediated annealing, selection and ligation (RASL), in which the mRNAs produced in a particular cell type are extracted and mixed with DNA oligomers whose sequences are complementary to those at which two RNA sections could be joined by splicing (“splice junctions”); the presence of a particular splice junction leads to binding of the DNA oligomers, which can then be multiplied, fluorescently labelled and exposed to the optical fibre array with which the sequences can be identified.
- 8.
Linderstrøm-Lang worked out a method of taking these correlations into account; his formula works practically as well as more sophisticated approaches (including explicit numerical simulation by Brownian dynamics; cf. Madura et al. 1994) and is much simpler and more convenient to calculate (see Ramsden et al. 1995 for an application example).
- 9.
See Patel and Weber (2003) for a review.
- 10.
The N-terminal of the protein is derivatized with phenylisothiocyanate to form a phenylthiocarbamate peptide, and the first amino acid is cleaved by strong acid resulting in its anilothiazolinone derivative plus the protein minus its first N-terminal amino acid. The anilothiazolinone derivative is converted to the stabler phenylthiohydantoin for subsequent high-performance liquid chromatography (HPLC) identification.
- 11.
See Bell et al. (2009) for efforts to overcome errors in mass spectrometry-based proteomics.
- 12.
See Chem. Rev. 103 (2003), issue no 2.
- 13.
Developed by Aebersold (see Gygi et al. 1999).
- 14.
As an alternative way to prepare oligopeptide receptors, the phage display technique invented by Dyax is very useful. The gene for the coat protein expressed abundantly on the surface of a bacteriophage virus is modified by adding a short sequence coding for an oligopeptide to one end. Typically, a large number (\(\sim \) \(10^9\)) of random oligonucleotides are synthesized and incorporated (one per phage) into the virus gene. The phages are then allowed to multiply by infecting a host bacterium; the random peptide is expressed in abundance on the coat of the phage along with the regular coat protein. The phage population is then exposed to an immobilized target (e.g., a protein). Any phage (a single one suffices) whose peptide interacts with the target during this screening is retained and recovered, and then multiplied ad libitum in bacteria.
- 15.
See Oh et al. (2007) for an example of this kind of approach.
- 16.
- 17.
Fearn (2015).
- 18.
See, e.g., Kolch et al. (2005).
- 19.
See Johnson and Hunter (2005) for a review of experimental methods for determining phosphorylation.
- 20.
Lapadula et al. (1992).
- 21.
And, indeed, to the neurologist—see Chap. 17.
- 22.
This limitation is imposed physicochemically; for example, there is only room for a small number of other proteins to cluster round and interact with a central one and, of course, the entire surface of the central protein is unlikely to be sensitive to the presence of other proteins; the possibilities for interaction are typically limited to a small number of specific binding sites.
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Ramsden, J. (2015). Proteomics. In: Bioinformatics. Computational Biology, vol 21. Springer, London. https://doi.org/10.1007/978-1-4471-6702-0_14
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DOI: https://doi.org/10.1007/978-1-4471-6702-0_14
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