Development of a new method for absolute protein quantification on 2‐D gels

Abstract

With the development of systems biology projects aimed at modeling the cell, accurate and absolute measurements of cellular protein concentrations are increasingly important. However, methods for absolute quantification at the proteomic level remain rare. Using the yeast Saccharomyces cerevisiae, we propose a new method based on the radioactive labeling with an (35)S compound and 2-D PAGE. The principle is simple: cells are grown for more than four generations in the presence of a unique sulfur source labeled at a defined specific radioactivity, ensuring that more than 90% of the proteins are labeled at the same specific radioactivity as the sulfur source. After separation of (35)S-labeled proteins on 2-D gels, each protein is counted. The amount of each protein present in the gel is then calculated, from which is deduced the amount of each protein per cell. The method, limited to soluble and abundant proteins visible on 2-D gels, is simple, precise and reproducible and does not require an internal standard. We use it to compare the amounts of proteins in two growth conditions: 100 microM sulfate or 500 microM methionine. Up to now, we only had transcriptional data on the expression of these proteins in both conditions.