Chemistry: analytical and immunological testing – Nuclear magnetic resonance – electron spin resonance or other...
Reexamination Certificate
2001-02-16
2004-11-16
Ceperley, Mary E. (Department: 1641)
Chemistry: analytical and immunological testing
Nuclear magnetic resonance, electron spin resonance or other...
C436S056000, C436S057000, C436S086000, C530S408000, C530S409000, C530S412000, C530S413000, C530S410000, C530S811000, C530S806000
Reexamination Certificate
active
06818454
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to reagents and methods for detecting and/or quantifying the phosphorylation state and/or level of proteins in samples.
BACKGROUND OF THE INVENTION
The availability of complete genome sequences is moving biological research to an era where cellular systems are analyzed as a whole rather than analyzing the individual components. While genome sequences and global gene expression measurements at the mRNA level opens the door to important biological advances, much of the understanding of cellular systems and the roles of the constituents will arise from proteomics (Wasinger et al., Electrophoresis 16:1090-1094, 1995). Proteomics, the analysis of the entire complement of proteins expressed by a cell, tissue type, or organ, provides the most informative characterization of the cell because proteins are the primary players that carry out nearly all processes within the cell. A key aspect to successful proteomic measurements is the ability to precisely measure protein abundance changes in a high throughput manner so as to allow the effects of many “perturbations” upon, or changes to, a cell type, tissue type or organ, to be determined in a rapid fashion (Pafterson,
Curr. Opin. Biotechnol.
11:413-418, 2000). An inherent goal of proteomic studies is to provide a greater understanding of the function of proteins in a global, cellular context, along with the more conventionally delineated molecular function. A greater understanding at the level of cellular systems will provide, for example, a stronger basis for understanding complex biological pathways and the nature of diseases. The global understanding of cellular systems provided by proteomic investigations will provide numerous opportunities unlikely to originate from the present paradigm of “single” protein characterization methodologies.
Though methods to allow global measurements of gene expression at the mRNA level have been developed (Schena et al.,
Science
270:467-470, 1995 and Khodursky et al.,
Proc. Natl. Acad. Sci. USA
97:12170-12175, 2000), such methods do not provide direct measurements of protein abundance (Gygi et al.,
Proc. Natl. Acad. Sci. USA
97:9390-9395, 2000). Clearly, only global analysis of gene expression at the protein level provides information about the roles of individual gene products and their involvement in cellular pathways. Information relating solely to the abundance of a particular protein within a cell fails to provide information relating to the “processed state” of the protein. The “processed state” refers to the level and/or type of post-translational modifications that are displayed by the functional protein. For example many proteins are initially translated in an inactive form and upon subsequent proteolysis, the addition of sugar moieties, phosphate groups, methyl groups, carboxyl groups, or other additional groups so they gain biological function. Information relating to the “processed state” of a given protein is necessary and, hence, methods of detecting the active state of proteins are important for furthering the understanding of intercellular signaling and for developing new and useful interventions and therapeutics.
The reversible phosphorylation of proteins plays a key role in transducing extracellular signals into the cell. Many proteins that participate in cell signaling pathways are phosphorylated via enzymes known as kinases and dephosphorylated via phosphatases. Phosphate groups are added to, for example, tyrosine, serine, threonine, histidine, and/or lysine amino acid residues depending on the specificity of the kinase acting upon the target protein. To date several disease states have been linked to the abnormal phosphorylation/dephosphorylation of specific proteins. For example, the polymerization of phosphorylated tau protein allows for the formation of paired helical filaments that are characteristic of Alzheimer's disease, and the hyperphosphorylation of retinoblastoma protein (pRB) has been reported to progress various tumors (Vanmechelen et al.
Neurosci. Lett.
285:49-52, 2000, and Nakayama et al.
Leuk. Res.
24:299-305, 2000, respectively).
The ability to quickly screen for irregularties in the phosphorylation state of proteins will further the understanding of intra and inter cellular signaling and lead to the development of improved diagnostics for the detection of various disease states.
SUMMARY OF THE INVENTION
The present disclosure provides reagents and methods for characterizing the phosphorylation state and/or level of a protein. Proteins can be post-transcriptionally modified such that they contain phosphate groups at either some, or all serine, threonine, tyrosine, histidine, and/or lysine amino acid residues. In many cases the extent to which a protein is phosphorylated determines it bioactivity, i.e., its ability to effect cell functions such as differentiation, division, and metabolism. Hence, the present disclosure provides powerful methods for diagnosing various diseases and for furthering the understanding of protein—protein interactions.
One aspect of the invention provides methods of comparing the phosphorylation state of one or more proteins in two or more samples. Moreover, the disclosed methods allow for the generation of phosphorylation profiles, which detect changes in the phosphorylation states of individual proteins within samples, rather than merely detecting overall increases in the phosphorylation level of all of the proteins within a sample. These methods involve providing a substantially chemically identical and differentially isotopically labeled protein reactive reagent for each sample wherein the protein reactive reagent satisfies the formula:
B-L-PhRG
wherein B is a binding agent that selectively binds to a capture reagent (CR), L is a linker group having one or more atoms that are differentially labeled with one or more isotopes, and PhRG is a phosphate reactive group that selectively reacts with amino acid residues that were formerly phosphorylated. Each sample is then reacted with one of the protein reactive reagents to provide proteins bound to the protein reactive agent, whereby the bound proteins are differentially labeled with the isotopes. The bound proteins of the samples are captured using the capture reagent that selectively binds the binding agent and the captured bound protein is then subsequently released from the capture reagent by disrupting the interaction between the binding agent and the capture reagent. The released, bound protein is then detected.
Another aspect of the invention provides methods for screening for therapeutics that alter the phosphorylation state of one or more proteins. These methods involve contacting at least one test sample containing a protein with the therapeutic and providing at least one control sample also containing an amount of the protein. One or more phosphate groups from one or more of the amino acid residues in the proteins in the test sample and control sample are removed and the proteins in both the test sample and the control are tagged with isotopically distinguishable B-L-PhRG that are substantially chemically identical. The level of phosphorylation can then be detected in the at least one test sample and the at least one control sample and the ability of the therapeutic to alter the phosphorylation of the protein can be determined.
Another aspect of the invention provides methods of comparing the level of tyrosine, serine, and threonine phosphorylation states of one or more proteins from two or more samples. Generally, these methods are practiced by sequentially removing phosphate groups from either the tyrosine residues, or the serine and threonine residues (the phosphate groups can be removed from either the serine and threonine first or the tyrosine phosphate groups can be removed first). Regardless of which phosphate group is removed first, a differentially isotopically labeled B-L-PhRG is subsequently used to “tag” the protein at the site formerly occupied by the phosphate group. The term “isotopically labeled” means
Conrads Thomas P.
Goshe Michael B.
Panisko Ellen A.
Veenstra Timothy D.
Battelle (Memorial Institute)
Ceperley Mary E.
Klarquist & Sparkman, LLP
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