Purdue Research Foundation

Stowers Institute for Medical Research

Institute for Systems Biology

Tymora Analytical Operations, LLC

Weiguo Andy Tao

Leroy E Hood

Linheng Li

Xi C He

Qiang Tian

Tong Yin

Ying Zhang

Anton Ilyuk

Linna Wang

Anton B Iliuk

Changdeng Hu

Anton B Ilyuk

Hillary Andaluz

Lingfel Zeng

I-Hsuan Chen

Li Pan

Li Yang

Mayank Srivastava

A method is disclosed to capture and purify extracellular vesicles from biofluids via lipid affinity-based capture. The EVTRAP (Extracellular Vesicles Total Recovery And Purification) method enables capture of EVs onto modified beads. The EVTRAP method results in fast and reproducible capture and isolation of EVs with greater than 90% recovery yields.

Methods for affinity-based non-antibody capture and purification of extracellular vesicles

Disclosed herein is a chemo-proteomic probe for labelling and monitoring a live microbe interacting with a host cell and for qualitative and quantitative analyses of those proteins involved during a microbe infects the host cell. This probe comprises a functional group for conjugating to a surface protein of a live microbe under a physiological condition; a photo-reactive group for covalent cross-linking to an interacting cell protein of a host; and a tag for isolating the cross-linked complex of the surface protein of said live microbe and the interacting protein of a host cell for qualitative and quantitative proteomics analyses. The probe may further comprise a visualization tag. This technology takes advantage of the high throughput feature of mass spectrometry analysis and combines it with a uniquely designed chemistry to achieve high efficient isolation and analysis of host cell proteins interacting with a pathogen at different stages of an infection.

Methods and reagents to label bacteria and virus and identify their interacting proteins

The state of protein phosphorylation and glycosylation can be key determinants of cellular physiology such as early stage cancer, but the development of phosphoproteins and/or glycoproteins in biofluids for disease diagnosis remains elusive. Here we demonstrate, for the first time, a strategy to isolate and identify phosphoproteins/glycoproteins in extracellular vesicles (EVs) from human plasma as potential markers to differentiate disease from healthy states. We identified close to 10,000 unique phosphopeptides in EVs by isolating from small volume of plasma samples. Using label-free quantitative phosphoproteomics, we identified 144 phosphoproteins in plasma EVs that are significantly higher in patients diagnosed with breast cancer than in healthy controls. Several novel biomarkers were validated in individual patients using Paralleled Reaction Monitoring for targeted quantitation. Similarly a group of glycoproteins in plasma EVs are identified. The study demonstrated that the development of phosphoproteins and/or glycoproteins in plasma EV as disease biomarkers is highly feasible and may transform cancer screening and monitoring.

Phosphoproteins in extracellular vesicles as candidate markers for breast cancer

A novel imaging reagent and gel-based method for analyzing and comparing phosphoproteomes on the same gel are disclosed herein.

Difference gel electrophoresis of phosphoproteome

The invention generally relates to methods for identifying protein-protein interactions. In certain aspects, methods of the invention involve conducting a protein-fragment complementation assay on a sample to form a protein-protein complex between two proteins in the sample that only transiently interact under physiological conditions, separating the complexes from the sample, and analyzing a protein of the complex using a mass spectrometry technique.

Methods for identifying protein-protein interactions

The invention generally relates to methods for isolating proteins. In certain aspects, methods of the invention involve preparing a plurality of sample preparations, each preparation including one or more intact cells. A capture unit is introduced to a plurality of the preparations. The capture unit includes a member that transiently interacts with one or more proteins in the cells and a reactive functional group. The sample preparations are incubated, and a reaction is initiated at a different time in a plurality of the preparation. In this manner, a protein within the cell that specifically interacts with the member of the capture unit becomes bound to the capture unit via the reactive functional group to form protein/capture unit complexes. The cells are lysed and the protein/capture unit complexes are isolated.

Methods for isolating proteins

The present invention relates to a purified molecules, including antibodies, that bind specifically to murine &#x3b2;-catenin phosphorylated at amino acid position Serineand to orthologs thereof, such as mammalian orthologs, including human orthologs. Methods of making and using such purified molecules are also provided. Kits containing such purified molecules are further provided.

Method of detecting phosphorylation state of beta-catenin

Protein phosphorylation is a major post-translational modification and it plays a pivotal role in numerous cellular functions. We present a composition that includes a soluble nanopolymer core functionalized with groups having an affinity for either metal ion or metal oxides which can be used for phosphopeptide enrichment. Exemplary compounds including PolyMAC-Zr, PolyMAC-Fe and PolyMAC-Ti demonstrate outstanding reproducibility, exceptional sensitivity, fast chelation time, and high phosphopeptide recovery from standard mixtures that include phosphorylated peptides. The composition can be used for phosphoproteome isolation from samples of medicinal, diagnostic or biological interest such as malignant breast cancer cells. Such compositions were used for the quantitative analysis of the changes in the tyrosine phosphoproteome in highly invasive breast cancer cells after induction of Syk kinase, a potent suppressor of tumor growth and metastasis. The composition and method disclosed herein offers an efficient and widely applicable tool for phosphoproteomics.

Materials and methods for isolating phosphopeptides

Disclosed herein are reagents that include a moiety that includes a metal such as titanium and that readily binds to phosphorylated molecules the reagents also include at least one moiety that produces a signal or that binds to a molecule that produces a signal. The reagent may also include a moiety that binds to a larger molecule or to a surface. Some forms of the reagent include a dendrimer that can simultaneously bind to multiple metal moieties that include a metal such as titanium and multiple moieties that can be used to detected bound molecules. These reagents can be used in detection and/or measurement and/or at least partial purification of phosphorylated molecules. These reagents and methods using them are used to analyze proteins, polypeptides, nucleic acids, phospholipids and the like. They are readily adapted for use in gels, blots, plate based high through put assays and for mass spectrometry.

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West Lafayette, IN, United States of America

Weiguo Andy Tao