title: Top-down proteomics on a high-field Fourier transform ion cyclotron resonance mass spectrometer.
abstract: Mass spectrometry is the tool of choice for sequencing peptides and determining the sites of posttranslational modifications; however, this bottom-up approach lacks in providing global information about the modification states of proteins including the number and types of isoforms and their stoichiometry. Recently, various techniques and mass spectrometers, such as high-field Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometers, have been developed to study intact proteins (top-down proteomics). While the protein molecular mass and the qualitative and quantitative information about protein isoforms can be revealed by FTICR-MS analysis, their primary structure (including the identification of modifications and their exact locations in the amino acid sequence) can directly be determined using the MS/MS capability offered by the FTICR mass spectrometer. The distinct advantage of top-down methods are that modifications can be determined for a specific protein isoform rather than for peptides belonging to one or several isoforms. In this chapter, we describe different top-down proteomic approaches enabled by high-field (7, 9.4, and 12 T) FTICR mass spectrometers, and their applicability to answer biological and biomedical questions. We also describe the use of the free flow electrophoresis (FFE) to separate proteins prior to top-down mass spectrometric characterization.
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC, USA.
authors.forename: Séverine A
authors.initials: SA
authors.lastname: Ouvry-Patat
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Torres
authors.forename: Craig A
authors.initials: CA
authors.lastname: Gelfand
authors.forename: Hung Hiang
authors.initials: HH
authors.lastname: Quek
authors.forename: Michael
authors.initials: M
authors.lastname: Easterling
authors.forename: J Paul
authors.initials: JP
authors.lastname: Speir
authors.forename: Christoph H
authors.initials: CH
authors.lastname: Borchers
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017357:Cyclotrons; D005583:Fourier Analysis; D007525:Isoelectric Focusing; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D011506:Proteins