Microfluidics Assays for Proteomics
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A critical need exists for the development of novel technologies that will enable high-throughput studies of the proteins encoded by the approximately 30,000 genes encoded by the human genome. The wide-ranging studies of protein expression, structure, and function, termed 'proteomics', has been limited by largely inadequate methods for the detection and characterization of large sets of protein-protein interactions. Such technologies include ELISA; Western blot analysis; two-dimensional gel electrophoresis and mass spectroscopy; the yeast two-hybrid interaction trap; and protein and peptide microarrays generated on solid surfaces either by photolithography or microcapillary spotting ide array. Protein microarray technology (generated by microcapillary spotting onto glass surfaces) has been recently developed by several labs, at Harvard and at Stanford University (see Project 3). The Steinman. Robinson, and Utz laboratories in the School of Medicine have utilized protein microarrays to characterize the autoantibody profile in a variety of disease states. These include autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), systemic sclerosis (SS), and multiple sclerosis (MS); as well as chronic infections with viruses such as simian immunodeficiency virus (SIV) and hepatitis C virus (HCV). During the course of these studies it has become clear that solid surface protein arrays have several important limitations for the detection and quantification of protein-protein interactions. This project is exploring a novel method of leveraging microfluidic systems technology to overcome these limitations and provide new functionalities for proteomics research.
Rationale:
Microfluidic systems leverage microfabrication technologies borrowed
from the microelectronics industry, including lithography and chemical
etching. These systems offer the ability to realize on-chip microchannel
networks that can be computer-controlled to transport, mix, separate,
and detect chemical species using both pressure forces and electrokinetics.
A key advantage offered by these systems is that an automated series
of reactions can be performed on 1-1,000 picoliter samples and have
all reactants and products of the assay remain suspended in a buffered
solution at all times. This is in contrast to solid-phase detection
systems of microarrays, a leading candidate in combinatorial proteomics
research, where the small spot sizes dictated by the needs for high
sample density result in evaporation of solvents within seconds.
A second advantage of microfluidics is that it provides a method
to combine assays with simple binding event detections such as post-reaction
electrophoretic assays or a post-binding measurement of catalytic
rates. In addition, microfluidic channel bifurcations and methods
of concentration (e.g., sample stacking or chromatographic separation)
provide methods of purifying candidate species of known reactivity
for subsequent testing off-chip. Finally, microfluidic systems offer
a method of cross-reacting each member of a set of proteins with
each other member in the set, such that n proteins processed by a
fluidic chip yield information on n(n-1)/2 reactant pairs. For competitive
immunoassays and other cross-reactive systems involving three or
more reactants, an automated on-chip mixing and post-mixing assay
scheme, even if performed in series, would quickly outperform array-based
schemes. Consider that array production is limited by the spot dispension
of array fabrication and by the fact that only one analyte solution
(e.g., a single combination of analyte proteins) can be tested per
array run.
We propose to develop novel biomolecular microfluidics systems that will enable rapid, high-throughput screening of biological samples and purified proteins for identification of protein-protein interactions. We are demonstrating that we can perform microfluidics-based detection of antigen-antibody interactions, as well as other protein-protein interactions that are currently detectable using more laborious techniques such as coimmunoprecipitation analysis and Far Western assays. We are utilizing eTag technology developed at ACLARA Biosciences (Mountain View, CA) to perform multiplex analysis of cytokine profiles, large scale autoantibody isotyping profiles, and analysis of signaling pathways. We will also develop a system for the rapid, multiplexed detection of three or more interacting molecules, a technique that is currently not possible using any of the traditional solid-phase assays described above. Although the initial studies will be specifically focused on protein targets of autoimmune diseases such as RA, SLE, and other connective tissue diseases (CTDs), the technology developed as part of this project will have broad applications to many areas of medicine, immunology, and biology.
These projects represent a well-established collaboration between several scientists from different disciplines. Drs Utz and Santiago have encouraged a true interdisciplinary experience. Students, technicians and fellows attend laboratory meetings of both laboratories involved in this project. Our initial involvement with Bio-X was driven by the realization that the clinical questions being pursued in labs in the School of Medicine cannot be easily answered using currently-available techniques. A number of emerging technologies are under development for proteomics applications, including arrays of proteins bound to planar surfaces such as microtiter plates or glass microscope slides; bead-based methods in which individual antigens are bound to distinct particles; and combinations of two dimensional electrophoretic separation followed by mass spectroscopic analysis of individual molecules. In collaboration with ACLARA Biosciences, we are developing a CE-based method for multiplex detection of two different groups of secreted proteins: cytokines/chemokines and autoantibodies. This technique will be broadly useful for many proteomics applications, will yield results that will help elucidate the pathogenic mechanisms contributing to blood diseases, and can be performed easily by any laboratory with access to a standard CE DNA sequencing apparatus. It should be obvious that microfluidics systems described above have the potential to rapidly accelerate scientific discoveries in many fields, particularly proteomics. Much more importantly, we believe that we can separate eTags on-chip in seconds, enabling high-throughput microfluidics assays to be developed. We envision that the ACLARA platform, particularly if performed on-chip, will emerge as a useful platform for the other 9 NHLBI proteomics centers that are each discovering novel biomarkers using 2D gels and/or mass spectrometry.
Our goals for Project 2 include:- To create and validate multiplexed cytokine assays for human and murine systems. As many as 20-40 molecules will be measured simultaneously, depending on the availability of reagents from commercial vendors.
- To develop isotyping assays to study multiple autoantibodies simultaneously.
- To analyze dozens of signaling molecules simultaneously to characterize cellular defects in SLE and RA
- To move from CE-based assays using large instruments to "on-chip" separations. Our goal is ultimately to measure single molecules, and to analyze the proteomes derived from single cells.
