title: Thermal stabilization of tissues and the preservation of protein phosphorylation states for two-dimensional gel electrophoresis.
abstract: 2-DE is typically capable of discriminating proteins differing by a single phosphorylation or dephosphorylation event. However, a reliable representation of protein phosphorylation states as they occur in vivo requires that both phosphatases and kinases are rapidly and completely inactivated. Thermal stabilization of mouse cerebral cortex homogenates effectively inactivated these enzymes, as evidenced by comparison with unstabilized tissues where abscissal pI shifts were a common feature in 2-D gels. Of the 588 matched proteins separated on 2-D gels comparing stabilized and unstabilized tissues, 53 proteins exhibited greater than twofold differences in spot volume (ANOVA, p<0.05). Phosphoprotein-specific staining was corroborated by the identification of 16 phosphoproteins by nano-LC MS/MS and phosphotyrosine kinase activity assay.
authors.affiliation: Harvard Catalyst, The Harvard Clinical and Translational Science Center, Laboratory for Innovative Translational Technologies, Boston, MA, USA.
authors.forename: Gary B
authors.initials: GB
authors.lastname: Smejkal
authors.forename: Chiara
authors.initials: C
authors.lastname: Rivas-Morello
authors.forename: Jae-Hyung Robert
authors.initials: JH
authors.lastname: Chang
authors.forename: Emily
authors.initials: E
authors.lastname: Freeman
authors.forename: Alexander J
authors.initials: AJ
authors.lastname: Trachtenberg
authors.forename: Alexander
authors.initials: A
authors.lastname: Lazarev
authors.forename: Alexander R
authors.initials: AR
authors.lastname: Ivanov
authors.forename: Winston P
authors.initials: WP
authors.lastname: Kuo
mesh_terms: D000595:Amino Acid Sequence; D000704:Analysis of Variance; D000818:Animals; D020134:Catalytic Domain; D002540:Cerebral Cortex / Q000737:chemistry*; D002853:Chromatography, Liquid / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D007091:Image Processing, Computer-Assisted; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D055550:Protein Stability; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D010750:Phosphoproteins; D011505:Protein-Tyrosine Kinases; D014357:Trypsin