Identification and characterization of heat shock protein 27 protein species in human myocardial two-dimensional electrophoresis patterns.
Immunostaining of heat shock protein 27 (Hsp27) protein species on two-dimensional electrophoresis (2-DE) gels with enhanced sensitivity yields 59 spots reacting with anti-Hsp27 antibodies. Recombinant Hsp27 exists in 2-DE as two major protein species which comigrate in the human myocardial pattern with Hsp27 spots C754 and D899 as defined in the heart high-performance 2-DE database (http://www.mdc-berlin.de/emu/heart/). Preparative electrophoresis of human myocardial proteins and analysis of the enriched mass range 20-30 kDa by 2-DE revealed eight protein spots (C438, C582, C658, C697, C754, C595, C750) from the human myocardial database and a new spot not previously detected on silver-stained gels. These spots were identified as Hsp27 protein species by enzymatic in-gel-digestion and analysis by matrix assisted laser desorption-ionization (MALDI) peptide mass fingerprinting and, in part, MALDI-post source decay sequencing of single fragments. Possible post-translational modifications were investigated: immunostaining tests with anti-phospho-serine/-threonine/-tyrosine antibodies, although positive for other myocardial proteins, were negative for presumed Hsp27 protein species; likewise, periodate-glycostaining assays and biotinylation screening did not detect modifications in the investigated Hsp27 protein species.
Scheler
C
C
Technische Universität Berlin, Institut für Biochemie, Germany.
Müller
E C
EC
Stahl
J
J
Müller-Werdan
U
U
Salnikow
J
J
Jungblut
P
P
D000595:Amino Acid Sequence; D015180:Electrophoresis, Gel, Two-Dimensional; D006360:Heat-Shock Proteins / Q000032:analysis* / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D005810:Multigene Family; D009206:Myocardium / Q000737:chemistry*; D011994:Recombinant Proteins / Q000032:analysis; D012394:Rosaniline Dyes; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D006360:Heat-Shock Proteins; D007202:Indicators and Reagents; D011994:Recombinant Proteins; D012394:Rosaniline Dyes; C048139:Coomassie blue