Abstract
Phosphorylation is one of the most common forms of protein modification. The most frequent targets for protein phosphorylation in eukaryotes are serine and threonine residues, although tyrosine residues also undergo phosphorylation. Many of the currently applied methods for the detection and localization of protein phosphorylation sites are mass spectrometry-based and are biased against the analysis of tyrosine-phosphorylated residues because of the stability and low reactivity of phosphotyrosines. To overcome this lack of sensitive methods for the detection of phosphotyrosine-containing peptides, we have recently developed a method that is not affected by the more predominant threonine or serine phosphorylation within cells. It is based on the specific detection of immonium ion of phosphotyrosine at 216.043 Da and does not require prior knowledge of the protein sequence. In this report, we describe the first application of this new method in a proteomic strategy. Using anti-phosphotyrosine antibodies for immunoprecipitation and one-dimensional gel electrophoresis, we have identified 10 proteins in the epidermal growth factor receptor signaling pathway, of which 8 have been shown previously to be involved in epidermal growth factor signaling. Most importantly, in addition to several known tyrosine phosphorylation sites, we have identified five novel sites on SHIP-2, Hrs, Cbl, STAM, and STAM2, most of which were not predicted to be phosphorylated. Because of its sensitivity and selectivity, this approach will be useful in proteomic approaches to study tyrosine phosphorylation in a number of signal transduction pathways.
Highlights
Phosphorylation is one of the most common forms of protein modification
One commonly used electrospray method is based on the neutral loss of HPO3 (80 Da) or H3PO4 (98 Da) moieties [4]. This technique is quite useful for phosphorylated serine or threonine residues, it is less suitable for phosphotyrosines because the phosphate group on tyrosine residues is more stable and does not detach
After washing the column several times, the bound proteins were eluted with phenylphosphate, which mimics phosphotyrosine residues and competitively binds to the anti-phosphotyrosine antibody
Summary
Is the selective detection of a characteristic negatively charged fragment ion at m/z Ϫ79 (PO3Ϫ) for the identification of any phosphorylated species in peptide mixtures [5,6,7]. We describe the application of a novel tandem mass spectrometry (MS/MS)-based technique that relies on specific detection of tyrosine-phosphorylated peptides [8, 9] This method, termed phosphotyrosine-specific immonium ion (PSI) scanning, is based on detection of the specific immonium ion of phosphotyrosine at 216.043 Da as a characteristic “reporter” ion for precursor tyrosine-phosphorylated species in complex peptide mixtures. A number of tyrosine phosphorylation sites on these molecules were identified; five of them have never been described in the literature despite the fact that most of these proteins have been intensively studied by several groups None of these novel tyrosine-phosphorylated residues conform to the known consensus motifs for SH2 or phosphotyrosine binding domains. PSI scanning can be used in high-throughput identification of in vivo tyrosine phosphorylation sites on proteins, which is an important step in the elucidation of regulatory networks within a cell
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
