Large-Scale Protein Phosphorylation Profiling by High Performance Phosphoproteomics

Yasushi Ishihama

Institute for Advanced Biosciences, Keio University

ABSTRACT

Recent advances in mass spectrometry coupled with miniaturized on-line liquid chromatography allow us identifying and quantifying thousands of proteins with high throughput and have been applied to analyzing post-translational modifications (PTM) of proteins. Among them, protein phosphorylation is one of the most attractive targets of proteomics because it is the most widespread PTM regulating a lot of cell functions. Very rapid progresses in phosphopeptide enrichment approaches in last couple of years have been contributed to the recent large-scale phosphoproteome studies, where thousands of phosphorylated sites of proteins were identified in combination with pre-fractionation methods or other phospho-enrichment methods. In general, these studies required a lot of starting materials and several days or weeks for the analysis. Then, as the number of LC-MS runs increases, the false positive identification would also increase unless proper identification criteria are employed. In order to avoid these problems, we intend to develop a 'high performance' phosphoproteomics platform where only a single and  unbiased phosphopeptide enrichment method is employed directly for the cell lysate without any pre-fractionation to minimize the starting material as well as the total analysis time. Recently, we developed a phosphopeptide enrichment approach based on aliphatic hydroxy acid-modified metal oxide chromatography (HAMMOC), where the chemo-affinity of metal oxides to phosphopeptides is enhanced by the addition of aliphatic hydroxy acids. This HAMMOC/nanoLC-MS approach accomplished the identification of thousands of phosphorylation sites per single LC-MS run from whole cell lysates. Here I will present our recent advances in the HAMMOC approach as well as the applications to the phosphorylation profiling of various organisms such as human, plants, bacteria and animals. In addition, several examples for quantitative cancer signaling regulated by molecular-targeting drugs will be shown.