title: Sequencing electroblotted proteins by tandem mass spectrometry.
abstract: Electroblotting proteins separated by gel electrophoresis provides a suitable support for further manipulations and analysis of small amounts of relatively pure samples. On-membrane digestion, peptide mapping by mass spectrometry, and database searching offer sensitive and fast tools to identify the analyte. By providing sequence information, tandem mass spectrometry can go a step further, confirming the database identification, solving problems connected with post-translational modifications and sequence variations, or supplying the stretches of internal sequence necessary to synthesize an oligonucleotide probe for gene isolation. The viability of this approach was successfully evaluated using different tandem mass spectrometric techniques: metastable decomposition in a matrix-assisted laser desorption/ionization (MALDI) time-of-flight instrument with a curved-field reflectron; low energy collision-induced dissociation in a MALDI quadrupole ion trap mass spectrometer; and high energy collision-induced dissociation in a high-performance four-sector mass spectrometer with massive cluster-impact ionization.
authors.affiliation: Structural Biochemistry Center, University of Maryland Baltimore County 21228, USA.
authors.forename: D
authors.initials: D
authors.lastname: Fabris
authors.forename: M M
authors.initials: MM
authors.lastname: Vestling
authors.forename: M M
authors.initials: MM
authors.lastname: Cordero
authors.forename: V M
authors.initials: VM
authors.lastname: Doroshenko
authors.forename: R J
authors.initials: RJ
authors.lastname: Cotter
authors.forename: C
authors.initials: C
authors.lastname: Fenselau
mesh_terms: D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin
chemical_list: D010455:Peptides; D011506:Proteins; D014357:Trypsin