title: Application of free-flow IEF to identify protein candidates changing under microgravity conditions.
abstract: Using antibody-related methods, we recently found that human thyroid cells express various proteins differently depending on whether they are cultured under normal gravity (1g) or simulated microgravity (s-microg). In this study, we performed proteome analysis in order to identify more gravity-sensitive thyroid proteins. Cells cultured under 1g or s-microg conditions were sonicated. Proteins released into the supernatant and those remaining in the cell fragments were fractionated by free-flow IEF. The fractions obtained were further separated by SDS-gel electrophoresis. Selected gel pieces were excised and their proteins were determined by MS. A total of 235 different proteins were found. Out of 235 proteins, 37 appeared to be first identifications in human thyroid cells. Comparing SDS gel lanes of equally numbered free-flow IEF fractions revealed similar patterns with a number of identical bands if proteins of a distinct cell line had been applied, irrespective of whether the cells had been cultured under 1g or s-microg. Most of the identical band pairs contained identical proteins. However, the concentrations of some types of proteins were different within the two pieces of gel. Proteins that concentrated differently in such pieces of gel are considered as candidates for further investigations of gravitational sensitivity.
authors.affiliation: FU-Berlin, Division of Biology, Chemistry, Pharmacy, Berlin, Germany.
authors.forename: Jessica
authors.initials: J
authors.lastname: Pietsch
authors.forename: Richard
authors.initials: R
authors.lastname: Kussian
authors.forename: Albert
authors.initials: A
authors.lastname: Sickmann
authors.forename: Johann
authors.initials: J
authors.lastname: Bauer
authors.forename: Gerhard
authors.initials: G
authors.lastname: Weber
authors.forename: Mikkel
authors.initials: M
authors.lastname: Nissum
authors.forename: Kriss
authors.initials: K
authors.lastname: Westphal
authors.forename: Marcel
authors.initials: M
authors.lastname: Egli
authors.forename: Jirka
authors.initials: J
authors.lastname: Grosse
authors.forename: Johann
authors.initials: J
authors.lastname: Schönberger
authors.forename: Robert
authors.initials: R
authors.lastname: Wildgruber
authors.forename: Manfred
authors.initials: M
authors.lastname: Infanger
authors.forename: Daniela
authors.initials: D
authors.lastname: Grimm
mesh_terms: D015153:Blotting, Western; D002457:Cell Extracts; D045744:Cell Line, Tumor; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods*; D013058:Mass Spectrometry; D010455:Peptides / Q000032:analysis; D020543:Proteome / Q000032:analysis*; D016622:Silver Staining; D013010:Sonication; D014893:Weightlessness
chemical_list: D002457:Cell Extracts; D010455:Peptides; D020543:Proteome