title: Top-down proteomics by means of Orbitrap mass spectrometry.
abstract: Top-down proteomics has become a popular approach for the analysis of intact proteins. The term "top down" has been coined for the analysis of proteins not involving any enzymatic or chemical cleavage but rather the ionization of the protein as a sound molecule and mass analysis of intact species and fragment ions thereof produced upon dissociation inside a mass spectrometer. One or several charge states of the protein are mass-isolated and subjected to dissociation (MS/MS) in the gas phase. The obtained fragment masses, predominantly from cleavages of the protein along its amino acid backbone, are directly related to the intact protein. Using bioinformatics tools the fragment masses are matched against a known protein sequence or can alternatively be used for partial or full de novo sequencing, depending on the size of the protein and the number of fragment ions obtained. Moreover, this approach provides global information about modification states of proteins including the number and types of isoforms and their stoichiometry and allows for the precise localization of modifications within the amino acid sequence. Top-down analysis of a single, purified protein can be performed by matrix-assisted laser desorption ionization or electrospray ionization upon direct infusion without online chromatographic separation, whereas top-down analysis of complex protein mixtures makes pre-fractionation combined with an efficient front-end chromatographic separation coupled online to the mass spectrometer inevitable.
authors.affiliation: Thermo Fisher Scientific, Im Steingrund 4-6, 63303, Dreieich, Germany, kai.scheffler@thermofisher.com.
authors.forename: Kai
authors.initials: K
authors.lastname: Scheffler
mesh_terms: D002853:Chromatography, Liquid; D019295:Computational Biology; D004591:Electrophoresis, Polyacrylamide Gel; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*