Quantitative analysis of cell signaling and drug action via mass spectrometry-based systems level phosphoproteomics.

Protein phosphorylation is a primary form of information transfer in cell signaling pathways and plays a crucial role in regulating biological responses. Aberrant phosphorylation has been implicated in a number of diseases, and kinases and phosphatases, the cellular enzymes that control dynamic phosphorylation events, present attractive therapeutic targets. However, the innate complexity of signaling networks has presented many challenges to therapeutic target selection and successful drug development. Approaches in phosphoproteomics can contribute functional, systems-level datasets across signaling networks that can provide insight into suitable drug targets, more broadly profile compound activities, and identify key biomarkers to assess clinical outcomes. Advances in MS-based phosphoproteomics efforts now provide the ability to quantitate phosphorylation with throughput and sensitivity to sample a significant portion of the phosphoproteome in clinically relevant systems. This review will discuss recent work and examples of application data that demonstrate the utility of MS, with a particular focus on the use of quantitative phosphoproteomics and phosphotyrosine-directed signaling analyses to provide robust measurement for functional biological interpretation of drug action on signaling and phenotypic outcomes.

Profiling protein tyrosine phosphorylation: a quantitative 45-plex peptide-based immunoassay.

Cellular homeostasis and responses to stimuli are mediated by complex signaling network events dominated by changes in protein phosphorylation states. Understanding information flow in the network is essential for correlating signaling changes to cell physiology. Tyrosine phosphorylation constitutes only a small portion of all protein phosphorylation, but its importance is manifested by the significant role it plays in diseases such as cancer. A peptide-based immunoassay microarray, designed to provide site specificity, quantification, broad coverage, and accessibility, is described that profiles 45 tyrosine phosphorylation sites across 34 proteins. Epidermal growth factor-stimulated A431 cells in the absence and presence of kinase inhibitors analyzed by microarrays showed biologically validated tyrosine phosphorylation changes and unanticipated activation of other targets. The approach is scalable for increasing the breadth of content as well as for interrogating other types of protein posttranslational modifications.

Quantitative measurement of epidermal growth factor receptor-mitogen-activated protein kinase signal transduction using a nine-plex, peptide-based immunoassay.

Aberrant epidermal growth factor receptor (EGFR, ErbB1) signaling is implicated in cell transformation, motility, and invasion in a variety of cell types, and EGFR is the target of several anticancer drugs. However, the kinetics of EGFR signaling and the individual contributions of site-specific phosphorylation events remain largely unknown. A peptide-based, multiplex immunoassay approach was developed to simultaneously measure both total and phosphorylated protein in a single sample. The approach involves the proteolytic digestion of proteins prior to the isolation and quantitation of site-specific phosphorylation events within an individual protein. Quantitation of phosphorylated and total proteins, in picomolar to nanomolar concentrations, were interpolated from standard curves generated with synthetic peptides that correspond to the peptide targets used in the immunoassays. In this study, a bead-based, nine-plex immunoassay measuring total and phosphorylated protein was constructed to measure temporal, site-specific phosphorylation of key members of the EGFR pathway (ErbB1 receptor, MEK1, MEK2, ERK1, and ERK2) in A431 cells stimulated with epidermal growth factor. The effect of MEK inhibition on this pathway was determined using a known MEK kinase inhibitor, SL327. The results reported herein are the first quantitative measurements of site-specific phosphorylation events and total proteins in a single sample, at the same time representing a new paradigm for standardized protein and phosphorylation analysis using multiplexed, peptide-based, sandwich immunoassays.

The challenges of bringing autologous HSP-based vaccines to commercial reality.

Based on genetic diversity in the population, there is an expectation, born out by decades of experience, that a given drug or treatment will not be equally efficacious for all patients. While this fact cannot be avoided, with ever increasing knowledge of the drug's biological mechanism of action and the relationship between efficacy and the patient's genetic profile, more directed treatments, with greater potential for efficacy are becoming possible. For example, Herceptin, Genentech's antibody based treatment for HER2 positive metastatic breast cancer, is prescribed based on the results of a diagnostic test, the outcome of which is able to screen out patients who have no chance of responding to the treatment. At the extreme of tailoring medicines to those patients who will receive the greatest benefit, is an autologous, or patient specific approach. Oncophage, a cancer vaccine in late stage clinical trials, is designed to accommodate the unique genetic mutations underlying each patient's cancer. This chapter of the book presents the challenges involved in bringing autologous HSP-based vaccines to commercial reality based on the experiences gained to date in the clinical manufacture of Oncophage. Two guiding principles have dominated our efforts. First, only the product should be autologous. All processes should be standardized to the greatest extent possible. Second, maintaining complete segregation between patient samples at all steps of processing is paramount.