Quantitative phospho-proteomics based on soluble nanopolymers.

Abstract

Phospho-proteomics, the global analysis of protein phosphorylation, holds great promise for the discovery of cell signaling events that link changes in dynamics of protein phosphorylation to the progression of various diseases, particularly cancer and diabetes. Mass spectrometry has become a powerful tool for identification and global profiling of protein phosphorylation. However, even with continuously improving sensitivity of mass spectrometers, sub-stoichiometric nature of phosphorylation poses enormous challenges for phosphoprotein identification and, particularly, mapping phosphosites. Therefore, a successful mass spectrometry-based phospho-proteomic experiment depends largely on an effective method of phosphopeptide enrichment.We present in this chapter two robust methods based on soluble nanopolymers functionalized for phosphopeptide enrichment. The first method describes the formation of reversible phosphoramidate bonds between amines on the nanopolymer and phosphate groups on peptides, thus enabling selective isolation of phosphopeptides using a molecule size-based filtering device. The second technique is based on the selective chelation of phosphopeptides to zirconia or titania functionalized nanopolymer, which can be isolated from the complex peptide mixture by binding the nanopolymer to solid-phase support through efficient hydrazide chemistry. Combined with stable isotope labeling approaches, both strategies provide reproducible and efficient meanings for quantitative phospho-proteomics.