Identification of human myocardial proteins separated by two-dimensional electrophoresis using an effective sample preparation for mass spectrometry.
Peptide mass fingerprinting is a powerful tool for the identification of proteins separated by two-dimensional gel electrophoresis (2-DE). The identification of in-gel digested proteins by peptide mass fingerprinting was significantly improve in comparison to blot-digests by using a peptide-collecting device. This device allows the effective purification and concentration of enzymatic digests of low-intensity spots without expensive equipment and is described in detail. Sensitivity in the fmol range was demonstrated by unequivocal identification of bovine serum albumin after sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Furthermore the high performance liquid chromatography pattern of in-gel digests indicated a 2- to 3-fold higher yield of the separated peptides. Therefore, a higher amount of the peptides was available to perform N-terminal sequencing. The identification of 16 proteins from a high-resolution 2-DE gel map of human myocardium tissue has been achieved by means of this technique. Three of these proteins were associated with changes in spot intensity with dilated cardiomyopathy.
Otto
A
A
Max-Delbrück-Centrum für, Molekulare Medizin, Berlin-Buch, Germany.
Thiede
B
B
Müller
E C
EC
Scheler
C
C
Wittmann-Liebold
B
B
Jungblut
P
P
D000102:Acetyl-CoA C-Acyltransferase / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D003402:Creatine Kinase / Q000302:isolation & purification; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000302:isolation & purification; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009206:Myocardium / Q000737:chemistry*; D009211:Myoglobin / Q000302:isolation & purification; D010449:Peptide Mapping; D011506:Proteins / Q000302:isolation & purification*; D014357:Trypsin
D009211:Myoglobin; D011506:Proteins; D007521:Isocitrate Dehydrogenase; D000102:Acetyl-CoA C-Acyltransferase; D003402:Creatine Kinase; D014357:Trypsin