Time-resolved Mass Spectrometry of Tyrosine Phosphorylation Sites in the Epidermal Growth Factor Receptor Signaling Network Reveals Dynamic Modules

Abstract

Ligand binding to cell surface receptors initiates a cascade of signaling events regulated by dynamic phosphorylation events on a multitude of pathway proteins. Quantitative features, including intensity, timing, and duration of phosphorylation of particular residues, may play a role in determining cellular response, but experimental data required for analysis of these features have not previously been available. To understand the dynamic operation of signaling cascades, we have developed a method enabling the simultaneous quantification of tyrosine phosphorylation of specific residues on dozens of key proteins in a time-resolved manner, downstream of epidermal growth factor receptor (EGFR) activation. Tryptic peptides from four different EGFR stimulation time points were labeled with four isoforms of the iTRAQ reagent to enable downstream quantification. After mixing of the labeled samples, tyrosine-phosphorylated peptides were immunoprecipitated with an anti-phosphotyrosine antibody and further enriched by IMAC before LC/MS/MS analysis. Database searching and manual confirmation of peptide phosphorylation site assignments led to the identification of 78 tyrosine phosphorylation sites on 58 proteins from a single analysis. Replicate analyses of a separate biological sample provided both validation of this first data set and identification of 26 additional tyrosine phosphorylation sites and 18 additional proteins. iTRAQ fragment ion ratios provided time course phosphorylation profiles for each site. The data set of quantitative temporal phosphorylation profiles was further characterized by self-organizing maps, which resulted in identification of several cohorts of tyrosine residues exhibiting self-similar temporal phosphorylation profiles, operationally defining dynamic modules in the EGFR signaling network consistent with particular cellular processes. The presence of novel proteins and associated tyrosine phosphorylation sites within these modules indicates additional components of this network and potentially localizes the topological action of these proteins. Additional analysis and modeling of the data generated in this study are likely to yield more sophisticated models of receptor tyrosine kinaseinitiated signal transduction, trafficking, and regulation.

In-depth Analyses of Kinase-dependent Tyrosine Phosphoproteomes Based on Metal Ion-functionalized Soluble Nanopolymers

“…S9. Although there have been reports that tyrosine phosphopeptides can be analyzed with single stage affinity purification based on anti-Tyr(P) antibodies (7,34), including recent large scale phosphotyrosine profiling (34,35), tandem affinity approaches combining antiTyr(P) antibodies and metal oxide improve the selectivity and confidence in the identification of tyrosine phosphopeptides (5,8). We carried out parallel experiments to compare three methods using anti-tyrosine phosphopeptide PT66 alone, PT66 ϩ TiO 2 , and PT66 ϩ PolyMAC purifications.…”

“…Currently, there are three major strategies for the isolation of phosphopeptides: antibody-based affinity capture, chemical derivatization of phosphoamino acids, and metal ion-based affinity capture. Antibody-based methods are used mainly for the selective isolation of phosphotyrosinecontaining proteins or peptides (5)(6)(7)(8). Chemical derivatization methods begin with the ␤-elimination of phosphates from phosphoserine and phosphothreonine (9) or the formation of phosphoramidates by reactions with amines (10) to selectively immobilize phosphopeptides.…”

Protein Phosphorylation and Expression Profiling by Yin-Yang Multidimensional Liquid Chromatography (Yin-Yang MDLC) Mass Spectrometry

“…Some high-affinity chemical derivation methods [15][16][17][18] have emerged, but suffer from side reactions, marginal yields, and low specificities. Immunopurification [19][20][21][22] and affinity chromatography including immobilized metal affinity chromatography (IMAC) and titanium dioxide (TiO2) are frequently used techniques for the enrichment of phosphorylated proteins and peptides, [23][24][25][26][27][28] but also suffer from unsatisfactory specificity or sensitivity. Recently, strong-cation exchange (SCX) chromatography using salt gradient has been applied for phosphopeptide enrichment by collecting fractions from an SCX column followed by reverse-phase chromatography and mass spectrometry.…”

A multiplexed proteomics approach to differentiate neurite outgrowth patterns

“…The peptide quantification is based on the relative peak areas of the four reporter ions, m/z 114.1, 115.1, 116.1 and 117.1, produced following tandem mass spectrometry fragmentation of iTRAQ-labeled peptides (DeSouza et al, 2005). This technology has been successfully applied to elucidate biochemical pathways involved in reperfusion stress (Hirsch et al, in press), to discover cancer markers (DeSouza et al, 2005), to characterize dynamic phosphorylation events in the epidermal growth factor receptor signaling network (Zhang et al, 2005) and to evaluate diurnal peptide variation in parotid saliva (Hardt et al, 2005). …”

“…It has been used for cancer classification, signal transduction pathway studies as well as pharmacological differentiation of drug treatments (Golub et al, 1999;Ideker et al, 2001). Although commonly used in cDNA microarray studies, more recent applications of SOM to proteomics studies allow one to analyze datasets with multiplex trend data points such as time course and dose response (Zhang et al, 2005).…”