Proteomics Cartoon_modified

Adapted from Science 2001 291: 1221

 

I. Integrated proteomic techniques for molecular signaling

The long term goal of this project is to develop novel proteomics strategies valuable for understanding the role of phosphorylation in regulating cell proliferation on the molecular level. We have been focusing on the development and optimization of effective and efficient methodologies and strategies for the analysis of phosphoproteomes. The project currently includes two major directions:

(i) Novel reagents based on soluble polymers for the isolation of phosphopeptides with high efficiency. We have designed biochemical reagents, termed soluble polymer-based metal ion affinity capture (PolyMAC) with different immobilized metal ions (i.e., PolyMAC-Ti and PolyMAC-Fe), to provide a homogeneous environment for more efficient isolation of phosphopeptides. The approach has been used on Syk-dependent signaling as our model system and to establish protocols that will ultimately provide a powerful method to dissect any signaling pathway regulated by protein-tyrosine phosphorylation.

(ii) Develop strategies for the identification of specific tyrosine-kinase substrates and their phosphorylation sites. Using Syk as our model kinase, we are developing a tactic based on quantitative proteomics for the identification of novel substrates that will be generally applicable to virtually any kinase.

Role of Tyr-290 in the location and activity of Syk.

Role of Tyr-290 in the location and activity of Syk.


II. Soluble nanopolymers for targeted proteomics in vitro and in living cells

Engagement of the B cell receptor for antigen (BCR) leads to immune responses through a cascade of intracellular signaling events.

Engagement of the B cell receptor for antigen (BCR) leads to immune responses through a cascade of intracellular signaling events.

High-throughput drug discovery methods typically focus on protein targets which are screened in vitro against existing compounds for high specificity and affinity. This strategy, however, could result in unexpected or undetected off-target effects, leading to high abrasion rates in the later stages of drug development. Ideally, unbiased identification of proteins and associated complexes that bind to a drug or drug candidate would provide direct evaluation and therefore would be more appealing, allowing for valuable insight into target cellular functions. We have combined drug delivery with proteomic analyses to identify drug targets in vitro and in living cells. The new strategy based on multi-functionalized soluble nanopolymers demonstrates that the dendrimer-based nanomedicine has a great potential to successfully probe the drug target proteins in living cells. It highlights chemical and technological approaches that seek to increase the quality of information obtained from high throughput experiments. The combination of mass spectrometry and functionalized dendrimers provides an unprecedented opportunity for sensitive, fast identification of proteins of interest in the most physiologically relevant environment.


III. Novel phosphorylation assays based on multi-functionalized soluble nanopolymers

Graphic illustration (top) and proposed schematic structure (bottom) of the pIMAGO molecule. The structure includes the dendrimer foundation functionalized with phosphonate-bound titanium ions and biotin groups.

Graphic illustration (top) and proposed schematic structure (bottom) of the pIMAGO molecule. The structure includes the dendrimer foundation functionalized with phosphonate-bound titanium ions and biotin groups.

Assessing the phosphorylation status of an individual protein or classes of proteins, qualitatively or quantitatively, has become a routine but extremely important step in a majority of research labs in life sciences. Current methods for phosphorylation analyses include the use of phospho-specific antibodies, 32P radioactive labeling, and mass spectrometry. A simple and reliable phosphorylation assay method is still missing for routine detection of phosphorylation in complex and typically heterogeneous biological samples. We have devised a novel technology for the highly efficient assay of protein phosphorylation in high throughput format without the use of phospho-specific antibodies or radioisotopes. The technique is based on a water-soluble, nanosize polymer, termed pIMAGO that is multi-functionalized with titanium (IV) ions for specific binding to phosphoproteins, and with biotin groups that allow for enzyme-linked spectrometric detection. As low as 100 pg of phosphoprotein can be measured quantitatively with the pIMAGO chemiluminescence assay. The pIMAGO assay was applied to an in vitro kinase assay, kinase inhibitor screening, and measurement of endogenous phosphorylation events. The technique provides a universal, quantitative method for global phosphorylation analysis with high sensitivity and specificity.