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  Methods for quantitative proteome analysis of glycoproteinsUSPTO Application #: 20070202539Title: Methods for quantitative proteome analysis of glycoproteins Abstract: The invention provides a method for identifying and quantifying polyglycopeptides in a sample. The method can include the steps of immobilizing glycopolypeptides to a solid support; cleaving the immobilized glycopolypeptides, thereby releasing non-glycosylated peptides and retaining immobilized glycopeptides; releasing the glycopeptides from the solid support; and analyzing the released glycopeptides. The method can further include the step of identifying one or more glycopeptides, for example, using mass spectrometry. (end of abstract) Agent: Mcdermott, Will & Emery - San Diego, CA, US Inventors: Rudolf H. Aebersold, Hui Zhang USPTO Applicaton #: 20070202539 - Class: 435007100 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Antigen-antibody Binding, Specific Binding Protein Assay Or Specific Ligand-receptor Binding Assay The Patent Description & Claims data below is from USPTO Patent Application 20070202539. Brief Patent Description - Full Patent Description - Patent Application Claims
BACKGROUND OF THE INVENTION [0001] This application claims the benefit of priority of U.S. Provisional application Ser. No. 60/385,707, filed Jun. 3, 2002, and U.S. Provisional application Ser. No. 60/469,361, filed May 9, 2003, each of which the entire contents is incorporated herein by reference. [0002] The present invention relates generally to the field of proteomics and more specifically to quantitative analysis of glycoproteins. [0003] Complete genomic sequences and large partial (EST) sequence databases potentially identify every gene in a species. However, the sequences alone do not explain the mechanism of biological and clinical processes because they do not explain how the genes and their products cooperate to carry out a specific process or function. Furthermore, the gene sequence does not predict the amount or the activity of the protein products nor does it answer the questions of whether, how, and at what position(s) a protein may be modified. [0004] Quantitative protein profiling has been recognized as an important approach for profiling the physiological state or pathological state of cells or organisms. Specific expectations of quantitative protein profiles include the possibility to detect diagnostic and prognostic disease markers, to discover proteins as therapeutic targets or to learn about basic biological mechanisms. [0005] Not only do the amounts and type of proteins expressed vary in different pathological states, post-translational modifications of proteins also vary depending on the physiological or pathological state of cells or organisms. Thus, it is important to be able to profile the amount and types of expressed proteins as well as protein modifications. [0006] Glycosylation has long been recognized as the most common post-translational modification affecting the functions of proteins, such as protein stability, enzymatic activity and protein-protein interactions. Differential glycosylation is a major source of protein microheterogeneity. Glycoproteins play key roles in cell communications, signaling and cell adhesion. Changes in carbohydrates in cell surface and body fluid are demonstrated in cancer and other disease states and highlights their importance. However, studies on protein glycosylation have been complicated by the diverse structure of protein glycans and the lack of effective tools to identify the glycosylation site(s) on proteins and of glycan structures. Oligosaccharides can be linked to serine or threonine residues (O-glycosylation) or to asparagine residues (N-glycosylation), and glycoproteins can have different oligosaccharides attached to any given possible site(s). [0007] Among the many post-translation modifications of proteins, glycosylation is a modification that is common to proteins that are exposed to an extracellular environment. For example, proteins expressed on the surface of a cell are exposed to the external environment such as blood or surrounding tissue. Similarly, proteins that are secreted from a cell, for example, into the bloodstream, are commonly glycosylated. [0008] Among the diverse types of proteins expressed by cells, proteins that are integral to or associated with lipid membranes perform a wide range of essential cellular functions. Pores, channels, pumps and transporters facilitate the exchange of membrane impermeable molecules between cellular compartments and between the cell and its extracellular environment. Transmembrane receptors sense changes in the cellular environment and, typically via associated proteins, initiate specific intracellular responses. Cell adhesion proteins mediate cell-specific interactions with other cells and the extracellular matrix. Lipid membranes also provide a hydrophobic environment for biochemical reactions that is dramatically different from that of the cytoplasm and other hydrophilic cellular compartments. [0009] Membrane proteins, in particular those spanning the plasma membrane, are also of considerable diagnostic and therapeutic importance, which is further reinforced due to their easy accessibility. Antisera to proteins that are selectively expressed on the surface of a specific cell type have been used extensively for the classification of cells and for their preparative isolation by fluorescent activated cell sorting or related methods. Membrane proteins, as exemplified by Her2/neu, the abundance of which is modulated in the course of certain diseases such as breast cancer, are commonly used as diagnostic indicators and, less frequently, as therapeutic targets. A humanized monoclonal antibody (Herceptin, Genentech, Palo Alto, Calif.) that specifically recognizes Her2/neu receptors is the basis for a successful therapy of breast cancer, and antibodies to other cell surface proteins are also undergoing clinical trials as anticancer agents. Moreover, the majority of current effective therapeutic agents for diseases such as hypertension and heart disease are receptor antagonists that target and selectively modify the activity of specific membrane proteins. It is therefore apparent that a general technique capable of systematically identifying membrane proteins and of accurately detecting quantitative changes in the membrane protein profiles of different cell populations or tissues would be of considerable importance for biology and for applied biomedical research. [0010] In addition to membrane bound proteins, proteins secreted by cells or shed from the cell surface, including hormones, lymphokines, interferons, transferrin, antibodies, proteases, protease inhibitors, and other factors, perform critical functions with respect to the physiological activity of an organism. Examples of physiologically important secreted proteins include the interferons, lymphokines, protein and peptide hormones. Aberrant availability of such proteins can have grave clinical consequences. It is therefore apparent that the ability to precisely quantitatively profile secreted proteins would be of great importance for the discovery of the mechanisms regulating a wide variety of physiological processes in health and disease and for diagnostic or prognostic purposes. Such secreted proteins are present in body fluids such as blood serum and plasma, cerebrospinal fluid, urine, lung lavage, breast milk, pancreatic juice, and saliva. For example, the presence of increased levels of prostate-specific antigen has been used as a diagnostic marker for prostate cancer. Furthermore, the use of agonists or antagonists or the replacement of soluble secreted proteins is an important mode of therapy for a wide range of diseases. [0011] Quantitative proteomics requires the analysis of complex protein samples. In the case of clinical diagnosis, the ability to obtain appropriate specimens for clinical analysis is important for ease and accuracy of diagnosis. As discussed above, a number of biologically important molecules are secreted and are therefore present in body fluids such as blood and serum, cerebrospinal fluid, saliva, and the like. In addition to the presence of important biological molecules, body fluids also provide an attractive specimen source because body fluids are generally readily accessible and available in reasonable quantities for clinical analysis. It is therefore apparent that a general method for the quantitative analysis of the proteins contained in body fluids in health and disease would be of great diagnostic and clinical importance. [0012] A key problem with the proteomic analysis of serum and many other body fluids is the peculiar protein composition of these specimens. The protein composition is dominated by a few proteins that are extraordinarily abundant, with albumin alone representing 50% of the total plasma proteins. Due to the abundance of these major proteins as well as the presence of multiple modified forms of these abundant proteins, the large number of protein species of lower abundance are obscured or inaccessible by traditional proteomics analysis methods such as two-dimensional electrophoresis (2DE). [0013] The classes of proteins described above, membrane proteins, secreted proteins, and proteins in body fluids have in common that they have a high propensity for being glycosylated, that is, modified post translationally with a carbohydrate structure of varying complexity at one or several amino acid residues. Thus, the analysis of glycoproteins allows characterization of important biological molecules. [0014] Thus, there exists a need for methods of high throughput and quantitative analysis of glycoproteins and glycoprotein profiling. The present invention satisfies this need and provides related advantages as well. SUMMARY OF THE INVENTION [0015] The invention provides a method for identifying and quantifying polyglycopeptides in a sample. The method can include the steps of immobilizing glycopolypeptides to a solid support; cleaving the immobilized glycopolypeptides, thereby releasing non-glycosylated peptides and retaining immobilized glycopeptides; releasing the glycopeptides from the solid support; and analyzing the released glycopeptides. The method can further include the step of identifying one or more glycopeptides, for example, using mass spectrometry. BRIEF DESCRIPTION OF THE DRAWINGS [0016] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [0017] FIG. 1 shows a schematic diagram of an exemplary method of identifying and quantifying glycopolypeptides/glycoproteins and for determining quantitative changes in the glycosylation state of proteins. [0018] FIG. 2 shows oxidation of a carbohydrate to an aldehyde followed by covalent coupling to hydrazide beads. [0019] FIG. 3 shows representative chemical reagents that have been tested and proved to be able to label amino groups of glycopeptides. The structures of labeled peptide are listed in the right column. [0020] FIG. 4 shows total protein staining or glycoprotein staining of crude serum before (-) and after immobilization (+) of glycoproteins to hydrazide resin. Proteins were separated by SDS-PAGE and stained with silver (left) or Gel Code Blue glycoprotein staining reagent (right). [0021] FIG. 5 shows an outline and comparison of the results of glycopeptide analysis of serum proteins observed with three methods: cysteine capture with extensive separation, glycopeptide capture and single liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), and cysteine capture and single LC-MS/MS. Continue reading... Full patent description for Methods for quantitative proteome analysis of glycoproteins Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Methods for quantitative proteome analysis of glycoproteins patent application. Patent Applications in related categories: 20080182270 - Combinatorial artificial receptors including peptide building blocks - R6 can be hydrogen or any suitable blocking or protecting group for an carboxyl-terminal carboxyl group of a peptide. Suitable blocking or protecting groups include those described in Green, T W; Wuts, PGM (1999), Protective Groups in Organic Synthesis Third Edition, Wiley-Interscience, New York, 779 pp. In an embodiment, R6 ... ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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