An integrated approach to proteome analysis: identification of proteins associated with cardiac hypertrophy.
Hypertrophy of cardiac myocytes is a primary response of the heart to overload, and is an independent predictor of heart failure and death. Distinct cellular phenotypes are associated with hypertrophy resulting from different causes. These phenotypes have been described by others at the molecular level by analysis of gene transcription patterns. An alternative approach is the analysis of large-scale protein expression patterns (the proteome) by two-dimensional polyacrylamide gel electrophoresis. Realization of this goal requires the ability to rigorously analyze complex 2D gel images, efficiently digest individual gel isolated proteins (especially those expressed at low levels), and analyze the resulting peptides with high sensitivity for rapid database searches. We have undertaken to improve the technology and experimental approaches to these challenges in order to effectively study a cell culture model for cardiac hypertrophy. The 2D gel patterns for cell lysates from multiple samples of cardiac myocytes with or without phenylephrine-induced hypertrophy were analyzed and spots which changed in abundance with statistical significance were located. Eleven such spots were identified using improved procedures for in-gel digestion of silver-stained proteins and high-sensitivity mass spectrometry. The incorporation of low levels of sodium dodecyl sulfate into the digestion buffer improved peptide recovery. The combination of matrix-assisted laser desorption mass spectrometry for initial measurements and capillary liquid chromatography-ion trap mass spectrometry for peptide sequence determination yielded efficient protein identification. The integration of 2D gel image analysis and routine identification of proteins present in gels at the subpicomole level represents a general model for proteome studies relating genomic sequence with protein expression patterns.
Arnott
D
D
Protein Chemistry Department, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA.
O'Connell
K L
KL
King
K L
KL
Stults
J T
JT
D000595:Amino Acid Sequence; D000818:Animals; D006332:Cardiomegaly / Q000139:chemically induced / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D016208:Databases, Factual; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008969:Molecular Sequence Data; D009206:Myocardium / Q000737:chemistry*; D010449:Peptide Mapping; D010656:Phenylephrine / Q000633:toxicity; D011506:Proteins / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011506:Proteins; D010656:Phenylephrine