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 expressio n patterns (the proteome) by two-dimensional polyacrylamide gel electr ophoresis. Realization of this goal requires the ability to rigorously analyze complex 2D gel images, efficiently digest individual gel isol ated proteins (especially those expressed at low levels), and analyze the resulting peptides with high sensitivity for rapid database search es. We have undertaken to improve the technology and experimental appr oaches to these challenges in order to effectively study a cell cultur e model for cardiac hypertrophy. The 2D gel patterns for cell lysates from multiple samples of cardiac myocytes with or without phenylephrin e-induced hypertrophy were analyzed and spots which changed in abundan ce with statistical significance were located. Eleven such spots were identified using improved procedures for in-gel digestion of silver-st ained proteins and high-sensitivity mass spectrometry. The incorporati on of low levels of sodium dodecyl sulfate into the digestion buffer i mproved peptide recovery. The combination of matrix-assisted laser des orption mass spectrometry for initial measurements and capillary liqui d chromatography-ion trap mass spectrometry for peptide sequence deter mination yielded efficient protein identification. The integration of 2D gel image analysis and routine identification of proteins present i n gels at the subpicomole level represents a general model for proteom e studies relating genomic sequence with protein expression patterns. (C) 1998 Academic Press.