Grace Y. J. Chen

Cell-based proteome profiling of potential dasatinib targets by use of affinity-based probes.

Protein kinases (PKs) play an important role in the development and progression of cancer by regulating cell growth, survival, invasion, metastasis, and angiogenesis. Dasatinib (BMS-354825), a dual Src/Abl inhibitor, is a promising therapeutic agent with oral bioavailability. It has been used for the treatment of imatinib-resistant chronic myelogenous leukemia (CML). Most kinase inhibitors, including Dasatinib, inhibit multiple cellular targets and do not possess exquisite cellular specificity. Recent efforts in kinase research thus focus on the development of large-scale, proteome-wide chemical profiling methods capable of rapid identification of potential cellular (on- and off-) targets of kinase inhibitors. Most existing approaches, however, are still problematic and in many cases not compatible with live-cell studies. In this work, we have successfully developed a cell-permeable kinase probe (DA-2) capable of proteome-wide profiling of potential cellular targets of Dasatinib. In this way, highly regulated, compartmentalized kinase-drug interactions were maintained. By comparing results obtained from different proteomic setups (live cells, cell lysates, and immobilized affinity matrix), we found DA-2 was able to identify significantly more putative kinase targets. In addition to Abl and Src family tyrosine kinases, a number of previously unknown Dasatinib targets have been identified, including several serine/threonine kinases (PCTK3, STK25, eIF-2A, PIM-3, PKA C-α, and PKN2). They were further validated by pull-down/immunoblotting experiments as well as kinase inhibition assays. Further studies are needed to better understand the exact relevance of Dasatinib and its pharmacological effects in relation to these newly identified cellular targets. The approach developed herein should be amenable to the study of many of the existing reversible drugs/drug candidates.

Developing site-Specific immobilization strategies of peptides in a microarray

In peptide-based microarrays, most existing methods do not allow for site-specific immobilization of peptides on the glass surface. We have developed two new approaches for site-specific immobilization of kinase substrates onto glass slides: (1) slides were functionalized with avidin for attachment of biotinylated peptides; and (2) slides were functionalized with thioester for attachment of N-terminally cysteine-containing peptides via a native chemical ligation reaction.

Antibody-based fluorescence detection of kinase activity on a peptide array

Peptide-based microarrays allow for high-throughput identification of protein kinase substrates. However, current methods of detecting kinase activity require the use of radioisotopes. We have developed a novel fluorescence-based approach for quantitative detection of peptide phosphorylation on chip using fluorescently-labeled anti-phosphoserine and anti-phosphotyrosine antibodies. This method is sensitive, specific and extremely fast, presenting obvious advantages and may find wider uses in high-throughput kinase screenings.

A sensitive two-photon probe to selectively detect monoamine oxidase B activity in Parkinson’s disease models

The unusually high MAO-B activity consistently observed in Parkinsons disease (PD) patients has been proposed as a biomarker; however, this has not been realized due to the lack of probes suitable for MAO-B-specific detection in live cells/tissues. Here we report the first two-photon, small molecule fluorogenic probe (U1) that enables highly sensitive/specific and real-time imaging of endogenous MAO-B activities across biological samples. We also used U1 to confirm the reported inverse relationship between parkin and MAO-B in PD models. With no apparent toxicity, U1 may be used to monitor MAO-B activities in small animals during disease development. In clinical samples, we find elevated MAO-B activities only in B lymphocytes (not in fibroblasts), hinting that MAO-B activity in peripheral blood cells might be an accessible biomarker for rapid detection of PD. Our results provide important starting points for using small molecule imaging techniques to explore MAO-B at the organism level.

Developing a Strategy for Activity-Based Detection of Enzymes in a Protein Microarray

An emerging area in proteomics is the development of arraybased, high-throughput screening techniques for discovery of novel protein functions and interactions. 2] Since the seminal work by MacBeath et al. , who demonstrated the feasibility of immobilizing proteins on a glass slide while retaining their native biological functions, significant advances have been made. These advances further expand the potential of this technology by increasing the number of spotted molecules, improving protein immobilization and surface chemistry, and other modifications. However, few reports have thus far addressed the downstream and fundamentally critical issue of detection, identification, and characterization of proteins in a microarray. Most existing strategies for the detection of TMhit∫ molecules in a microarray rely on strong, noncovalent binding between the proteins and their natural ligands. These strategies can only be used to identify potential receptors, antigens, and proteininteracting proteins effectively in an array format, which excludes key groups of proteins such as various classes of enzymes. Enzymes are critical to the vital functioning of any living system and play a fundamental role in all cellular processes and metabolic transformations. Earlier work on enzymes relied on the use of substrate-based peptide arrays to detect the enzymatic activity of a kinase in solution, which limits the strategy primarily to a TMone slide, one enzyme∫ format. More recently, a method has been developed to detect more than 100 different kinases inside microwells made from glass slides. However, this strategy is similar to the traditional microplatebased methods and is thus not compatible with the fluorescence detection methods used in slide-based microarray technologies, nor is it easily adapted for the study of other types of enzymes. In order to fully realize the enormous potential of protein microarrays, there is a need to develop not only strategies that cater for the measurement of protein binding on a glass slide, but also techniques that allow for determination of the activity and function of the immobilized proteins. We report here the first microarray strategy that allows high-throughput, activity-based detection of enzymes immobilized on a glass slide, and its potential application for rapid screenings of enzyme inhibitors. Our approach takes advantage of fluorescently labeled, mechanism-based suicide inhibitors of enzymes (Figure 1), which have mostly been used as agents for routine biochemical studies of proteins, protein modification and engineering, and

Combinatorial peptide microarrays for the rapid determination of kinase specificity.

We report a rapid method for profiling of kinases using a strategy that couples the merits of combinatorics (in rapid diversity generation) with the throughput attainable using microarrays (in parallel screening). Alanine-scanning, deletion and positional-scanning peptide libraries of a kinase substrate were synthesized and site-specifically arrayed onto glass slides. The phosphorylation pattern of target sequences detected using fluorescently-labeled antiphosphoamino acid antibodies revealed the substrate preference of the kinase through its activity profile.

Recent Advances in Microarray Technologies for Proteomics

Proteins are fundamental components of all living systems and critical drivers of biological functions. The large-scale study of proteins, their structures and functions, is defined as proteomics. This systems-wide analysis leads to a more comprehensive view of the intricate signaling transduction pathways that proteins engage in and improves the overall understanding of the complex processes supporting the living systems. Over the last two decades, the development of high-throughput analytical tools, such as microarray technologies, capable of rapidly analyzing thousands of protein-functioning and protein-interacting events, has fueled the growth of this important field. Herein, we review the most recent advancements in microarray technologies, with a special focus on peptide microarray, small molecule microarray, and protein microarray. These technologies have become prominent players in proteomics and have made significant changes to the landscape of life science and biomedical research. We will elaborate on their performance, advantages, challenges, and future directions.

Array-based technologies and their applications in proteomics.

Latest microarray-based technologies, including small molecule-, peptide-, protein- and cell-based arrays, and their applications in the field of proteomics are reviewed.

Activity-based fluorescent probes that target phosphatases

We have successfully designed and synthesized two fluorescently-labeled, activity-based probes, Probe 1 and Probe 2, which were shown to label protein tyrosine phosphatases specifically, as well as other types of phosphatases. The probes were not reactive towards the other non-phosphatase enzymes tested. These probes may find potential applications in large-scale proteomic experiments whereby subclasses of proteins may be selectively identified.

One- and Two-Photon Live Cell Imaging Using a Mutant SNAP-Tag Protein and Its FRET Substrate Pairs

A small molecule-assisted protein labeling strategy based on a mutant SNAP-Tag (mSNAP) and its FRET substrate pairs has been developed. Both one- and two-photon fluorescence microscopic experiments were successfully demonstrated in living cells.