A3_Systems Proteomics

Project Summary:

Phosphorylation is essential for regulating signal transduction events in general. The hepatocyte growth factor, transforming growth factor, and other signal transduction pathways important for hepatocyte regeneration, in particular, are also tightly controlled by phosphorylation. To understand these events in detail we dissect the phosphoproteome of hepatocytes using our advanced mass spectrometric techniques. We employ hybrid linear ion trap Fourier Transform mass spectrometers (LTQ-FT and LTQ-Orbitrap) with fast sequencing speed, high sensitivity and resolution, and mass accuracy better than one part per million. With the latest developments in phosphopeptide enrichment, we routinely detect over 3000 phosphorylation sites in mammalian cell lines. These phosphorylation sites can also be quantified by using stable isotope labeling with amino acid in cell culture (SILAC) - for example in response to stimulus. We plan to fully label mouse liver cell line Hepa1-6 by SILAC and then "spike-in" with primary hepatocytes. Both the proteome and the phosphoproteome of mouse liver cells can be quantified to a depth of several thousand proteins and sites. We will measure the response of hepatocytes to the several growth factors, both separately and in combination. This will result in indispensable data for systems modeling of hepatocyte regeneration.