Photocleavable peptide-oligonucleotide conjugates for protein kinase assays by MALDI-TOF MS.

Robust methods for highly parallel, quantitative analysis of cellular protein tyrosine kinase activities may provide tools critically needed to decipher oncogenic signaling, discover new targeted drugs, diagnose cancer and monitor patients. Here, we describe proof-of-principle for a novel protein kinase assay with the potential to help overcome these challenges. MALDI-TOF mass spectrometry provides an ideal tool for label-free multiplexed analysis of peptide phosphorylation, but is poorly matched to homogeneous assays and complex samples. Thus, we conjugated a common oligonucleotide tag to multiple peptide substrates, offering efficient capture from solution-phase kinase reactions by annealing to the complementary sequence tethered to PEG-passivated superparamagnetic microparticles. To enable reversible conjugation, we developed a novel bifunctional cross-linker allowing simple and efficient preparation of photocleavable peptide-oligonucleotide conjugates. After washing away contaminants and following photorelease, MALDI-TOF analysis yielded relative phosphorylation of each peptide with high sensitivity and specificity. Validating the hybridization-mediated multiplexed kinase assay, when three peptide substrate-oligonucleotide conjugates were mixed with the tyrosine kinase c-Abl and ATP, we readily observed their differential phosphorylation yet measured a common IC(50) for the Abl kinase inhibitor imatinib. This new assay enables analysis of protein kinase activities in a multiplexed format amenable to screening inhibitors against multiple kinases in parallel, an important capability for drug discovery and predictive diagnostics.

Annotator: postprocessing software for generating function-based signatures from quantitative mass spectrometry.

Mass spectrometry is used to investigate global changes in protein abundance in cell lysates. Increasingly powerful methods of data collection have emerged over the past decade, but this has left researchers with the task of sifting through mountains of data for biologically significant results. Often, the end result is a list of proteins with no obvious quantitative relationships to define the larger context of changes in cell behavior. Researchers are often forced to perform a manual analysis from this list or to fall back on a range of disparate tools, which can hinder the communication of results and their reproducibility. To address these methodological problems, we developed Annotator, an application that filters validated mass spectrometry data and applies a battery of standardized heuristic and statistical tests to determine significance. To address systems-level interpretations, we incorporated UniProt and Gene Ontology keywords as statistical units of analysis, yielding quantitative information about changes in abundance for an entire functional category. This provides a consistent and quantitative method for formulating conclusions about cellular behavior, independent of network models or standard enrichment analyses. Annotator allows for "bottom-up" annotations that are based on experimental data and not inferred by comparison to external or hypothetical models. Annotator was developed as an independent postprocessing platform that runs on all common operating systems, thereby providing a useful tool for establishing the inherently dynamic nature of functional annotations, which depend on results from ongoing proteomic experiments. Annotator is available for download at http://people.cs.uchicago.edu/∼tyler/annotator/annotator_desktop_0.1.tar.gz .

A magnetic bead-based protein kinase assay with dual detection techniques.

A novel magnetic bead-based protein kinase assay was developed using MALDI-TOF mass spectrometry (MALDI-TOF MS) and immunochemifluorescence as two independent detection techniques. Abltide substrate was immobilized onto magnetic beads via noncovalent biotin-streptavidin interactions. This noncovalent immobilization strategy facilitated peptide release and allowed MALDI-TOF MS analysis of substrate phosphorylation. The use of magnetic beads provided rapid sample handling and allowed secondary analysis by immunochemifluorescence to determine the degree of substrate phosphorylation. This dual detection technique was used to evaluate the inhibition of c-Abl kinase by imatinib and dasatinib. For each inhibitor, IC50 (half-maximal inhibitory concentration) values determined by these two different detection methods were consistent and close to values reported in the literature. The high-throughput potential of this new approach to kinase assays was preliminarily demonstrated by screening a chemical library consisting of 31 compounds against c-Abl kinase using a 96-well plate. In this proof-of-principle experiment, both MALDI-TOF MS and immunochemifluorescence were able to compare inhibitor potencies with consistent values. Dual detection may significantly enhance the reliability of chemical library screening and identify false positives and negatives. Formatted for 96-well plates and with high-throughput potential, this dual detection kinase assay may provide a rapid, reliable, and inexpensive route to the discovery of small-molecule drug leads.

Peptide reporters of kinase activity in whole cell lysates.

Kinase assays are used to screen for small-molecule inhibitors that may show promise as targeted pharmaceutical therapies. Using cell lysates instead of purified kinases provides a more accurate estimate of inhibitor sensitivity and selectivity in a biological setting. This review summarizes the range of homogeneous (solution-phase) and heterogeneous (solid-supported) formats available for using peptide substrates to monitor kinase activities in cell lysates. With a focus on heterogeneous kinase assays, the peptide substrate Abltide is used as a model to optimize presentation geometries and the modular arrangement of short sequences for kinase recognition. We present results from peptides immobilized on two- and three-dimensional surfaces such as hydrogels on 96-well plates and glass slides, and fluorescent Luminex beads. We discuss methods to increase assay sensitivity using chemifluorescent ELISAs, antibody-based recognition, and label-free mass spectrometry. Monitoring the activity of specific kinases in cell lysates presents challenges that can be overcome by manipulating peptide substrates to optimize assay conditions. In particular, signal-to-background ratios were improved by (1) adding long branched hydrophilic linkers between the substrate and the surface, (2) changing the orientation of peptides relative to the surface, and (3) including peptide ligands in cis or in trans to recruit kinases to the surface. By improving the accessibility of immobilized peptide substrates to kinases in solution, the apparent rate of phosphorylation increased and assays were more sensitive to changes in endogenous kinase activities. These strategies can be generalized to improve the reactivity of most peptide substrates used in heterogeneous kinase assays with cell lysates.

A bead-based activity screen for small-molecule inhibitors of signal transduction in chronic myelogenous leukemia cells.

Chronic myelogenous leukemia is characterized by the presence of the chimeric BCR-ABL gene, which is expressed as the constitutively active Bcr-Abl kinase. Although kinase activity is directly responsible for the clinical phenotype, current diagnostic and prognostic methods focus on a genetic classification system in which molecularly distinct subcategories are used to predict patient responses to small-molecule inhibitors of the Bcr-Abl kinase. Point mutations in the kinase domain are a central factor regulating inhibitor resistance; however, compensatory signaling caused by the activation of unrelated kinases can influence inhibitor efficacy. Kinase activity profiling can be used as a complementary approach to genetic screening and allows direct screening of small-molecule inhibitors. We developed a quantitative assay to monitor tyrosine kinase activities and inhibitor sensitivities in a model of chronic myelogenous leukemia using peptide reporters covalently immobilized on Luminex beads. Kinase activity is quantified by nonlinear regression from well-specific internal standard curves. Using optimized synthetic substrates and peptides derived from native substrates as probes, we measured kinase inhibition in cell lysates by the signal transduction inhibitors imatinib and dasatinib. Taking advantage of a convenient 96-well plate format, this assay also allows a straightforward and quantitative analysis of the differential effects of ATP and inhibitors on kinase activity. This method for analyzing a focused signaling network benefits from rigorous statistical analysis and short processing times, thereby offering a powerful tool for drug discovery and clinical testing.

The high- and low-affinity receptor binding sites of growth hormone are allosterically coupled.

Growth hormone regulates its biological properties via a sequential hormone-induced receptor homodimerization mechanism. Using a mutagenesis-scanning analysis of 81 single and 32 pairwise double mutations, we show that the hormone's two spatially distal receptor binding sites (Site1 and Site2) are allosterically coupled. These allosteric effects are focused among a relatively few residues centered around the interaction between Asp-116 of the hormone and Trp-169 of the receptor in Site2. A rearrangement of this interaction triggered by mutations in Site1 produces both a major conformation and energetic reorganization of Site2, surprisingly without a reduction in overall binding affinity. Additionally, the data suggest a change in the conformational dynamics of several groups in Site2 that appear to be important in defining the Site2 interaction. Changes in binding energy of the affected Site2 residues usually range in magnitude from 3- to 60-fold, but in one case are as large as 10(4).

Site2 binding energetics of the regulatory step of growth hormone-induced receptor homodimerization.

Receptor signaling in the growth hormone (GH)-growth hormone receptor (GHR) system is controlled through a sequential two-step hormone-induced dimerization of two copies of the extracellular domain (ECD) of the receptor. The regulatory step of this process is the binding of the second ECD (ECD2) to the stable preassociated 1 : 1 GH/ECD1 complex on the cell surface. To determine the energetics that governs this step, the binding kinetics of 38 single- and double-alanine mutants in the hGH Site2 contact with ECD2 were measured by using trimolecular surface plasmon resonance (TM-SPR). We find that the Site2 interface of hGH does not have a distinct binding hot-spot region, and the most important residues are not spatially clustered, but rather are distributed over the whole binding surface. In addition, it was determined through analysis of a set of pairwise double alanine mutations that there is a significant degree of negative cooperativity among Site2 residues. Residues that show little effect or even improved binding on substitution with alanine, when paired with D116A-hGH, display significant negative cooperativity. Because most of these pairwise mutated residues are spatially separated by >or=10 A, this indicates that the Site2 binding interface of the hGH-hGHR ternary complex displays both structural and energetic malleability.