Intregrated analysis of the human cardiac transcriptome, proteome and phosphoproteome

Abstract

Altered expression of different classes of genes has been shown to differentiate between failing and nonfailing human hearts. However, characterization of proteins and the post-translational modifications that regulate their functions is required for understanding both the physiology of cardiac muscle and the mechanisms leading to pathological states associated with cardiac diseases. We present in this paper, an analysis of the human cardiac transcriptome, proteome and phosphoproteome. Data from two sources (i) experiments performed in our laboratory and (ii) bioinformatics searches of public databases (SWISS-PROT, NCBI, Cardiac Gene Expression Knowledge Base, Gene Ontology Consortium and Affymetrix) are reported in a relational database that allows user-designed specific queries. Microarray experiments were performed with Affymetrix Hu95Av2. Cardiac proteins were digested with trypsin. An 11 step cation exchange procedure produced fractions for analysis in separate reversed phase high-performance liquid chromatography-tandem mass spectrometry (MS/MS) experiments. Immobilized metal affinity chromatography was used to select the phosphopeptides from the same tryptic peptide mixture. They were then further investigated by MS/MS. Gel-free approaches were used to detect 267 proteins and 47 phosphopeptides. Our human cardiac database contains 447 entries. We propose the use of this platform, built with data derived from nonfailing hearts, as a template for initiating the effort to characterize the human cardiac proteome and its associated post-translational modifications.