Bioinformatics for Omics Data Methods and Protocols
Presenting an area of research that intersects with and integrates diverse disciplines, including molecular biology, applied informatics, and statistics, among others, Bioinformatics for Omics Data: Methods and Protocols collects contributions from expert
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1. Introduction High-throughput Omics techniques are dominated by a rather limited set of analytical methods. For transcriptomics, these techniques are microarrays and next-generation sequencing. In proteomics (and to a large extent also in metabolomics), mass spectrometry, coupled to various separation techniques, is the key analytical tool. A typical proteomics sample contains – depending on its origin – hundreds to tens of thousands of different proteins. Hence, it is necessary to reduce the complexity of the sample by separation techniques and then to conduct one or more mass spectrometric analyses. Common separation techniques are highperformance liquid chromatography (HPLC), capillary electrophoresis (CE), and two-dimensional gel electrophoresis (2D PAGE). Techniques like HPLC or CE have certain advantages over 2D PAGE for high-throughput analysis because they can be Bernd Mayer (ed.), Bioinformatics for Omics Data: Methods and Protocols, Methods in Molecular Biology, vol. 719, DOI 10.1007/978-1-61779-027-0_15, © Springer Science+Business Media, LLC 2011
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readily automated. HPLC, on the other hand, has difficulties separating intact proteins, so, in general, a shotgun proteomics approach is applied: proteins are enzymatically digested into peptides, which are then easily separated using HPLC. Peptides elute off the HPLC column at different retention times due to their interaction with the stationary phase of the column. The eluting peptides are then typically spotted onto a MALDI target (LC-MALDI-MS) or continuously injected into a mass spectrometer using electrospray ionization (LC-ESI-MS). In matrix-assisted laser desorption/ionization (MALDI)-MS, the ionization of the peptides is achieved by evaporation of the analyte together with a matrix. The analyte is spotted onto a metal target using an organic matrix. Matrix and analyte are dried on the target, and this solid is then evaporated using laser shots. The matrix is chosen to have an absorption maximum at the laser wavelength. It is thus rapidly heated by the laser shots and evaporates into the gas phase together with the analyte. The matrix then transfers protons to the analyte in a gentle ionization process, which is very well suited to peptides. While LC-MALDI-MS requires the spotting and handling of targets, LC-ESI-MS directly couples the HPLC column to the mass spectrometer. Electrospray Ionization (ESI) ionizes the analytes at atmospheric pressure by applying a strong electrostatic field at a very fine tip (typically 3–6 kV). The analyte is dissolved in the mobile phase and is dispersed into very fine, highly charged droplets. The fine spray then enters the mass spectrometer, where the solvent evaporates and, during this process, ionizes the analyte. Inside the mass spectrometer, the mass-to-charge ratio of the ionized analyte is measured using a mass analyzer. There are various types of mass analyzers like quadrupole analyzers, quadrupole ion trap analyzers, time-of-flight analyzers, ion cyclotron resonance and Fourier tr
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