title: Recovery of gel-separated proteins for in-solution digestion and mass spectrometry.
abstract: A protocol for mass spectrometry of gel-separated proteins resulting in significantly increased sequence coverage and in improved possibilities for detection and identification of posttranslational modifications was developed. In relation to the standard in-gel digestion procedure, the sequence coverage using a combination of matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry was on the average increased by 30%. The method involves electroblotting of the gel-separated proteins to a poly(vinylidene difluoride) membrane. The proteins are extracted from the membrane using a solution of 1% trifluoroacetic acid in 70% acetonitrile and lyophilized. After reconstitution of the protein extract in digestion buffer, proteolytic cleavage is carried out in-solution as opposed to the standard in-gel digestion procedure. This allows recovery of large and hydrophobic peptides for mass spectrometry and reduces the risk for entrapment of proteolytic peptides in the gel matrix. The method was applied to proteins in the 30-40-kDa range with highly different structural properties. The improved ability to localize and determine protein modifications is shown for N-terminal acetylation and methylation of a histidine residue. Furthermore, the method enables fast screening of homologous protein sequences.
authors.affiliation: Department of Medical Biochemistry and Biophysics, Medical Nobel Institute, Karolinska Institutet, Stockholm, Sweden.
authors.forename: A P
authors.initials: AP
authors.lastname: Jonsson
authors.forename: Y
authors.initials: Y
authors.lastname: Aissouni
authors.forename: C
authors.initials: C
authors.lastname: Palmberg
authors.forename: P
authors.initials: P
authors.lastname: Percipalle
authors.forename: E
authors.initials: E
authors.lastname: Nordling
authors.forename: B
authors.initials: B
authors.lastname: Daneholt
authors.forename: H
authors.initials: H
authors.lastname: Jornvall
authors.forename: T
authors.initials: T
authors.lastname: Bergman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D016415:Sequence Alignment; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D011506:Proteins