Method to detect proteins

Details Method to detect proteins Method to detect proteins

1998-07-30

2001-03-06

Housel, James C. (Department: 1641)

Chemistry: analytical and immunological testing

Involving an insoluble carrier for immobilizing immunochemicals

C424S009340, C424S134100, C435S007100, C435S007800, C435S007900, C435S007920, C435S970000, C435S971000, C435S973000, C435S975000, C436S807000, C524S900000, C530S387300

Reexamination Certificate

active

06197599

ABSTRACT:

BACKGROUND
This invention relates generally to the field of protein detection.
Proteins are the major components of cells. They determine the shape, structure, and function of the cell. Proteins are assembled by 20 different amino acids each with a distinct chemical property. This variety allows for enormous versatility in the chemical and biological properties of different proteins. Human cells have about 100,000 genes for encoding different proteins. Despite the fact that new proteins are being discovered at an unprecedented rate, protein structure and function studies are lagging behind, mainly due to a lack of high throughput methods.
Antibodies and recombinant proteins are powerful tools for protein studies. Antibodies are a large family of glycoproteins that specifically bind antigens. A protein can be identified by its specific antibodies in immunochemical methods such as Western blot, immunoprecipitation, and enzyme linked immunoassay. Monoclonal and polyclonal antibodies against most known proteins have been generated and widely used in both research and therapy. Genes can be readily expressed in organisms like bacteria and yeast and this has made recombinant proteins convenient and indispensable tools in protein structure and function studies. There is a growing demand for recombinant proteins, especially in large scale screening of drug targets and in clinical medicine. Today, numerous antibodies and recombinant proteins have been produced. One important issue is how to analyze proteins in large scale by using a large number of antibodies or recombinant proteins in a single experiment.
It is often necessary to immobilize proteins on a solid support during the process of studying proteins. In Western blot analysis, proteins of interest are first separated by electrophoresis and then transferred and immobilized onto a nitrocellulose or a polyvinylidene difluoride (PVDF) membrane. In the phage display screening of a protein expression library, several hundred thousand proteins expressed by phages are immobilized on membranes. In both Western blotting and phage display screening, proteins are immobilized non-covalently. The protein of interest is then selected by its unique property, i.e., interaction with an antibody. In some other applications such as immunoprecipitation and affinity purification, agents (e.g., antibodies, ligands) are covalently conjugated onto solid supports (e.g., agarose beads) through their primary amines, sulfhydryls or other reactive groups. In general, proteins retain their abilities of interacting with other proteins or ligands after immobilization.
Monitoring the expressions and properties of a large number of proteins is desired in many important applications. One such application is to reveal protein expression profiles. A cell can express a large number of different proteins. And the expression patterns (the number of proteins expressed and the expression levels) vary in different cell types. This difference is the primary reason that different cells have different functions. Since many diseases are caused by the change in protein expression pattern, comparing protein expression patterns between normal and disease conditions may reveal proteins whose changes are critical in causing the disease and thus identify appropriate therapeutic targets. Methods of detecting protein expression profiles will also have other important applications including tissue typing, forensic identification, and clinical diagnosis. Protein expression pattern can be examined with antibodies in an immunoassay, but usually in a small scale. Therefore, one major obstacle in profiling protein expression pattern is a lack of large scale protein screening methods.
Protein posttranslational modifications (e.g., phosphorylation, glycosylation, and ubiquitination) play critical roles in regulating protein activity. One of the modifications is phosphorylation at either serine, threonine or tyrosine residues. Protein phosphorylation is an important mechanism in signal transduction. Aberrant protein phosphorylation contributes to many human diseases. Among the methods of detecting protein phosphorylation, metabolic labeling of cells with radioisotopes and immuno-detection of phosphoproteins with antibodies are the most commonly used. However, these methods are only applicable to analyzing one or several proteins each time. Antibodies specific for phosphorylated amino acids, such as PY20, can reveal multiple phosphorylated proteins, but fail to identify them. A new method for simultaneously detecting and identifying multiple phosphorylated proteins is highly desirable for signal transduction studies and clinical diagnosis.
Protein-protein interaction is an important way by which a protein carries out its function(s). Currently, there are several methods to detect protein—protein interactions. Among them, co-immunoprecipitation (Harlow and Lane, 1988, Antibodies, a laboratory manual. Cold Spring Harbor Laboratory), yeast two-hybrid screening (Fields and Song, 1989, Nature, 340:245-246) and phage display library screening (Smith, 1985, Science 228:1315-1317) are the most commonly used. However, there are severe limitations in these methods. In co-immunoprecipitation, a protein of interest can be precipitated with its antibody which is immobilized on agarose beads. Any other protein(s) that co-immunoprecipitated with the protein of interest can be identified by either blotting with its antibody when it is known or purification and sequencing when it is a novel protein. However, this method can not be applied to large scale identification of protein—protein interactions. Yeast two-hybrid screening is a recently developed technique for detecting protein—protein interaction. Although a single yeast two-hybrid screening assay can detect many interacting proteins, it is time-consuming and prone to false positive results. Moreover, many protein—protein interactions only occur in the presence of additional cellular factors or after posttranslational modifications, which may not be present in yeast. Therefore, yeast two-hybrid screening fails to identify many important protein—protein interactions that only take place in mammalian cells. Phage display screening of protein—protein interaction suffers similar limitations.
Therefore, there is a need for improved techniques which allow rapid and detailed analysis of multiple proteins for both basic research and clinical medicine. Such techniques will be extremely valuable in monitoring the overall patterns of protein expression, protein posttranslational modification, and protein—protein interaction in different cell types or in the same cell type under different physiological or pathological conditions.
SUMMARY
We have designed a device (protein array) which has many agents immobilized on a solid support. Agents are immobilized in a predetermined order, i.e., each agent is immobilized at a specific position so that it can be identified by its unique position on the support. The device can capture and identify the specific proteins from a mixture (e.g., cell lysate). After capture and separation, the proteins can be further characterized. Therefore, protein array makes it possible to study a wide variety of proteins in a single experiment by a large number of antibodies and/or recombinant proteins immobilized on a support.
Protein array and the methods presented here have several significant advantages over the current methods. First, protein array allows rapid detection of many proteins and thus makes it possible to compare protein expression profiles from different sources or those from the same source but under different conditions. Information on protein expression profile is very useful in identifying diagnostic and therapeutic targets. Second, protein array makes it possible to detect posttranslational modifications of numerous proteins and provide a valuable tool to investigate protein and cellular regulations. Third, it can screen a large number of potential interactions directly; and it can detect interactions that take place only

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