Src homology 2 domains enhance tyrosine phosphorylation in vivo by protecting binding sites in their target proteins from dephosphorylation.

Phosphotyrosine (pTyr)-dependent signaling is critical for many cellular processes. It is highly dynamic, as signal output depends not only on phosphorylation and dephosphorylation rates but also on the rates of binding and dissociation of effectors containing phosphotyrosine-dependent binding modules such as Src homology 2 (SH2) and phosphotyrosine-binding (PTB) domains. Previous in vitro studies suggested that binding of SH2 and PTB domains can enhance protein phosphorylation by protecting the sites bound by these domains from phosphatase-mediated dephosphorylation. To test whether this occurs in vivo, we used the binding of growth factor receptor bound 2 (GRB2) to phosphorylated epidermal growth factor receptor (EGFR) as a model system. We analyzed the effects of SH2 domain overexpression on protein tyrosine phosphorylation by quantitative Western and far-Western blotting, mass spectrometry, and computational modeling. We found that SH2 overexpression results in a significant, dose-dependent increase in EGFR tyrosine phosphorylation, particularly of sites corresponding to the binding specificity of the overexpressed SH2 domain. Computational models using experimentally determined EGFR phosphorylation and dephosphorylation rates, and pTyr-EGFR and GRB2 concentrations, recapitulated the experimental findings. Surprisingly, both modeling and biochemical analyses suggested that SH2 domain overexpression does not result in a major decrease in the number of unbound phosphorylated SH2 domain-binding sites. Our results suggest that signaling via SH2 domain binding is buffered over a relatively wide range of effector concentrations and that SH2 domain proteins with overlapping binding specificities are unlikely to compete with one another for phosphosites in vivo.

Early signaling dynamics of the epidermal growth factor receptor.

Despite extensive study of the EGF receptor (EGFR) signaling network, the immediate posttranslational changes that occur in response to growth factor stimulation remain poorly characterized; as a result, the biological mechanisms underlying signaling initiation remain obscured. To address this deficiency, we have used a mass spectrometry-based approach to measure system-wide phosphorylation changes throughout the network with 10-s resolution in the 80 s after stimulation in response to a range of eight growth factor concentrations. Significant changes were observed on proteins far downstream in the network as early as 10 s after stimulation, indicating a system capable of transmitting information quickly. Meanwhile, canonical members of the EGFR signaling network fall into clusters with distinct activation patterns. Src homology 2 domain containing transforming protein (Shc) and phosphoinositol 3-kinase (PI3K) phosphorylation levels increase rapidly, but equilibrate within 20 s, whereas proteins such as Grb2-associated binder-1 (Gab1) and SH2-containing tyrosine phosphatase (SHP2) show slower, sustained increases. Proximity ligation assays reveal that Shc and Gab1 phosphorylation patterns are representative of separate timescales for physical association with the receptor. Inhibition of phosphatases with vanadate reveals site-specific regulatory mechanisms and also uncovers primed activating components in the network, including Src family kinases, whose inhibition affects only a subset of proteins within the network. The results presented highlight the complexity of signaling initiation and provide a window into exploring mechanistic hypotheses about receptor tyrosine kinase (RTK) biology.

Computer aided manual validation of mass spectrometry-based proteomic data.

Advances in mass spectrometry-based proteomic technologies have increased the speed of analysis and the depth provided by a single analysis. Computational tools to evaluate the accuracy of peptide identifications from these high-throughput analyses have not kept pace with technological advances; currently the most common quality evaluation methods are based on statistical analysis of the likelihood of false positive identifications in large-scale data sets. While helpful, these calculations do not consider the accuracy of each identification, thus creating a precarious situation for biologists relying on the data to inform experimental design. Manual validation is the gold standard approach to confirm accuracy of database identifications, but is extremely time-intensive. To palliate the increasing time required to manually validate large proteomic datasets, we provide computer aided manual validation software (CAMV) to expedite the process. Relevant spectra are collected, catalogued, and pre-labeled, allowing users to efficiently judge the quality of each identification and summarize applicable quantitative information. CAMV significantly reduces the burden associated with manual validation and will hopefully encourage broader adoption of manual validation in mass spectrometry-based proteomics.

Measurement of Phosphorylated Peptides with Absolute Quantification.

Mass spectrometry, when coupled to on-line separation such as liquid chromatography or capillary electrophoresis, enables the identification and quantification of protein expression and post-translational modification changes under diverse conditions. To date most of the methods for mass spectrometry-based quantification have either provided relative quantification information (e.g., comparison to a selected condition) or utilized one-point calibration curves, or calibration curves in a different biological matrix. Although these quantitative methods have been used to generate insight into the differences between biological samples, additional biological insight could be gained by accurately measuring the absolute quantity of selected proteins and protein modifications. To address this challenge, we have developed the MARQUIS (Multiplex Absolute Regressed Quantification with Internal Standards) method, designed to provide absolute quantification for potentially hundreds of peptides across multiple samples in a single analysis, using a multi-point internal calibration curve derived from synthetic, isotopically distinct standard peptides.

Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases.

While the affinities and specificities of SH2 domain-phosphotyrosine interactions have been well characterized, spatio-temporal changes in phosphosite availability in response to signals, and their impact on recruitment of SH2-containing proteins in vivo, are not well understood. To address this issue, we used three complementary experimental approaches to monitor phosphorylation and SH2 binding in human A431 cells stimulated with epidermal growth factor (EGF): 1) phospho-specific mass spectrometry; 2) far-Western blotting; and 3) live cell single-molecule imaging of SH2 membrane recruitment. Far-Western and MS analyses identified both well-established and previously undocumented EGF-dependent tyrosine phosphorylation and binding events, as well as dynamic changes in binding patterns over time. In comparing SH2 binding site phosphorylation with SH2 domain membrane recruitment in living cells, we found in vivo binding to be much slower. Delayed SH2 domain recruitment correlated with clustering of SH2 domain binding sites on the membrane, consistent with membrane retention via SH2 rebinding.

MARQUIS: a multiplex method for absolute quantification of peptides and posttranslational modifications.

Absolute quantification of protein expression and posttranslational modifications by mass spectrometry has been challenging due to a variety of factors, including the potentially large dynamic range of phosphorylation response. To address these issues, we have developed MARQUIS-Multiplex Absolute Regressed Quantification with Internal Standards-a novel mass spectrometry-based approach using a combination of isobaric tags and heavy-labelled standard peptides, to construct internal standard curves for peptides derived from key nodes in signal transduction networks. We applied MARQUIS to quantify phosphorylation dynamics within the EGFR network at multiple time points following stimulation with several ligands, enabling a quantitative comparison of EGFR phosphorylation sites and demonstrating that receptor phosphorylation is qualitatively similar but quantitatively distinct for each EGFR ligand tested. MARQUIS was also applied to quantify the effect of EGFR kinase inhibition on glioblastoma patient-derived xenografts. MARQUIS is a versatile method, broadly applicable and extendable to multiple mass spectrometric platforms.

Molecular network analysis of phosphotyrosine and lipid metabolism in hepatic PTP1b deletion mice.

Metabolic syndrome describes a set of obesity-related disorders that increase diabetes, cardiovascular, and mortality risk. Studies of liver-specific protein-tyrosine phosphatase 1b (PTP1b) deletion mice (L-PTP1b(-/-)) suggest that hepatic PTP1b inhibition would mitigate metabolic-syndrome through amelioration of hepatic insulin resistance, endoplasmic-reticulum stress, and whole-body lipid metabolism. However, the altered molecular-network states underlying these phenotypes are poorly understood. We used mass spectrometry to quantify protein-phosphotyrosine network changes in L-PTP1b(-/-) mouse livers relative to control mice on normal and high-fat diets. We applied a phosphosite-set-enrichment analysis to identify known and novel pathways exhibiting PTP1b- and diet-dependent phosphotyrosine regulation. Detection of a PTP1b-dependent, but functionally uncharacterized, set of phosphosites on lipid-metabolic proteins motivated global lipidomic analyses that revealed altered polyunsaturated-fatty-acid (PUFA) and triglyceride metabolism in L-PTP1b(-/-) mice. To connect phosphosites and lipid measurements in a unified model, we developed a multivariate-regression framework, which accounts for measurement noise and systematically missing proteomics data. This analysis resulted in quantitative models that predict roles for phosphoproteins involved in oxidation-reduction in altered PUFA and triglyceride metabolism.