Abstract
Protein phosphorylation is a dynamic post-translational modification that plays a critical role in the regulation of a wide spectrum of biological events and cellular functions including signal transduction, gene expression, cell proliferation, and apoptosis. Determination of the sites and magnitudes of protein phosphorylation has been an essential step in the analysis of the control of many biological systems. A high throughput analysis of phosphorylation of proteins would provide a simple, logical, and useful tool for a functional dissection and prediction of biological functions and signaling pathways in association with these important molecular events. We have developed a functional proteomics technique using the ProteinChip array-based SELDI-TOF-MS analysis for high throughput profiling of phosphoproteins/phosphopeptides in human serum for the early detection and diagnosis as well as for the molecular staging of human cancer. The methodology and experimental approach consists of five steps: (1) generation of a total peptide pool of serum proteins by a global trypsin digestion; (2) rapid isolation of phosphopeptides from the total serum peptide pool by an affinity selection, purification, and enrichment using a novel automated micro-bioprocessing system with phospho-antibody-conjugated paramagnetic beads and a hybrid magnet plate; (3) high throughput phosphopeptide analysis on ProteinChip arrays by automated SELDI-TOF-MS; and (4) bioinformatics and statistical methods for data analysis. This method with appropriate modifications may be equally applicable to serine-, threonine- and tyrosine-phosphorylated proteins and for selectively isolating, profiling, and identifying phosphopeptides present in a highly complex phosphor-peptide mixture prepared from various human specimens such as cells, tissue samples, and serum and other body fluids.