TWI240796B - Novel proteome analysis method and devices therefor - Google Patents

Abstract

The present invention provides a proteome analysis method including grouping a proteome into membrane proteins and compounds capable of interacting with the membrane proteins, while retaining their native structure and function, and analyzing both the membrane proteins and the compounds based on biological affinity, and devices therefor.

Description

1240796 A7 V. Description of the invention (2 (Please read the note on the back? Matters before filling out this page; > As a specific membrane transporter of physiological substances from inside the cell to the outside of the cell, on the other hand, as a support for the structure of the dynamic membrane Membrane-lined proteins. Difficulties in purification, isolation, and functional analysis have delayed the study of membrane-related proteins. Since the function (physiological effect) of a protein cannot be predicted by a nucleic acid sequence determined by genomics, the latter gene Science requires the establishment of a method for linking genetic information to new drugs. One of the notable ones is the goal of proteomics (proteomics). The goal of proteomics is the existence of all proteins with more than 10,000, isolated, Identification and functional clarification. According to the current situation, how to connect genomics information to understand protein function is a strategic goal of drug discovery in the 21st century. Generally speaking, the molecular structure and function of protein molecules covered in biological activity cannot be compared with DNA molecules. In this sense, the strategy of genomics (which is by applying only DNA sequencing methods Structurally similar to 24 human chromosomes and achieved success), it is impractical to directly apply to the diverse proteomics of the rich object. Using only DNA sequences, it is impossible to predict the activity of the source. Therefore, waiting to be clarified Proteomics of protein function. The relationship between molecular structure and molecular activity printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economics is the basis for studying biology. For understanding any biological response, the structure-activity relationship is strict, such as enzyme function 'Methods of intercellular communication, and cellular regulation and feedback systems. For vital phenomena, proteins are necessary to sustain life and are presented in interactions with other molecules containing protein molecules, DNA molecules, synthetic compounds or photons, etc. Its function. To understand a certain protein is more than just to detail the physical or chemical properties of this molecule. It includes influencing each other by identifying ^ paper size applicable Chinese National Standard (CNS) A4 specification (21〇) < 297 mm) '' — 2 313287 A7 1240796 V. Explanation of the Invention (Molecules of 3 and Clarification of Interactions ( What kind of interactions do you find with this molecule? (Please read the note on the back? Matters before filling out this page) Some species of macromolecules that are known to interact with and bind to other molecules have fame Specific three-dimensional and electron distribution. Any macromolecule with this specificity is considered as a receptor, whether it is an enzyme that catalyzes the hydrolysis of metabolic intermediates, or a cell surface protein that mediates the transport of ionic membranes, which can be used for self-proximity Cells identify glycoproteins of special cells, antibodies circulating in plasma, nucleotide sequences of nuclear DNA or others. Various molecules that receptors selectively bind to are called ligands. About half of the existing medicines are known The product acts on receptors on cells through receptors. Therefore, it is elucidated that novel proteins and their physiological ligands provide a revolutionary screening system for the development of novel therapeutic agents for various diseases. In addition, the construction of a database of new and known membrane proteins and ligands in diseases can elucidate the molecular dynamics in diseases that cannot be reached by pharmacogenomics, and it is expected to lead to new diagnostic methods and novel treatments. Agent development. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. Many methods can be used to discover unknown receptors and ligands, but the number of receptors or ligands obtained from self-learning concepts, methods, and experiments is sometimes affected by their characteristics. limit. It was found that a complex receptor composed of a plurality of peptides was related to a more difficult problem. Novel receptors and coordination systems are discovered through novel techniques such as X-ray crystallography or genetic recombination. However, these new methods, depending on accidental coincidence, require long-term biochemical research, or can only be applied to very limited molecular species. In consideration of the above, the paper size of studying egg proteins such as membrane receptors and membrane transporters, which are part of the physiological function (= identification of physiological ligands), is applicable to the paper standard of China National Standard (CNS) A4 (Issued by 210x297) --- ---- 3 313287 1240796 V. Description of the invention (4) The goal is of great significance. Conventional research in proteomics is limited to the use of two-dimensional electrophoresis to separate proteins. However, when analyzing a certain type of cells, the current method has the following five problems. First, when the entire biological sample was electrophoresed on a single colloid, the protein with a high molecular weight and insoluble protein in substance 2 still moved close to the original and caused the analysis itself to fail. Therefore, the conventional two-dimensional electrophoresis cannot provide analysis of the total proteins expressed in the cells, and is used to separate specific proteins (most of them are soluble, low molecular weight proteins). Due to the development of molecular biotechnology, biology in the 20th century has progressed violently, but most of the proteins are water-soluble proteins. Proteomics in the early stages The total number of so-called proteins measured in Yanjiu Xianxue Blood Paddle is about 200, but the number of proteins contained in the cell plasma is to the side. The total number of proteins expressed by approximately 3,000 human genes is expected to exceed 100'OOG. The idea is that even with the most sensitive proteomics techniques, only a few percent of the total protein can be detected. Printed by the Consumer Affairs Agency of the Intellectual Property Office of the Ministry of Economic Affairs, any life phenomenon can be interpreted as the function of protein, and life is generated by the cost of the membrane knife and not itself. Membrane proteins perform the task of discerning the non-self in the outside world and making itself respond to it. Nonetheless, most of the proteins that cannot be detected by current proteomics technologies are membrane proteins that play an important role in life activities, which show functions when they are associated with membranes or embedded in tadpoles. "A protein complex consisting of a plurality of proteins and performing unique functions in cells cannot analyze the structure (quaternary structure of the protein) and the actual paper size conforms to the national standard (CNS) ^^ ⑽NO --- 1 4 313287! 240796 A7 B7 V. Description of the invention (5) The function existing in the living body, because when the electrophoresis is performed in a buffer solution containing a detergent, the binding between proteins based on the hydrophobic interaction will separate The reason is that the second 'effective concept of grouping the total protein contained in a biological sample' has not been provided. The method or technique has reached a total number of proteins exceeding 100,000 expressed by a total of approximately 30,000 human genes. Since the same Genes are spliced after transcription to produce proteins with shorter peptide chains than others, and after translation, various modifications are applied to them by sugar, lipid, phosphate groups, etc. As a result, proteins (proteins The goal of somatology) consists of a group of molecules that are far more complex than DNA polymer molecules (the goal of genomics). Based on these facts, a A single method (determining the sequence of nucleotides) for one purpose (determining the sequence of nucleotides) cannot clarify the various structures and functions of the protein body. Therefore, it is very important that the protein body be included in a biological sample based on a certain concept before analysis of the protein body There have been many attempts on pretreatment in protein grouping. For example, Mouy et al. [Em j

Biochem. 267, 2871-2881 (2000)] and Santoni et al. [Electrophoresis 21, pretreatment of the sample with a strong dissolving agent, but not dissolving all proteins. Herbert et al. [Electrophoresis 21, 3639_3648 (2000)] and Zuo et al. [Anal · Biochem · 284, 266-278 (2000)] Separated pretreatment samples by their isoelectric points, but it was difficult to set the target protein. Proper isoelectric point range and prevent isoelectric focusing. It should be noted that these attempts are directed to a partial improvement of the electrophoresis method, and not to the analysis of total protein bodies by grouping the total protein bodies achieved by the present invention. However, as of now, there is no proposed grouping method *. The paper size from the sample to the subsequent ^ handle Hou Tian ^ paper size applies to China National Standard (CNS) A4 specifications (210 X 297 public meals Y ---:- --- 313287 (Please read the precautions on the back before filling this page) _ I n ϋ II n ^ OJ > ϋ ϋ I ϋ ϋ I i Printed by the Intellectual Property Bureau of the Ministry of Economic Affairs and Consumer Cooperatives 5 1240796

In the same way, the samples are not grouped. This has forced the research of protein f somatology to encounter the two problems mentioned above. (Please read the precautions on the back before filling this page) Fourth, in the study of proteomics, time is consumed, and multiple steps make the operation of segmenting the condensate into small fragments complicated and require use before milk analysis A special solution extracts proteins from each fragment. The complicated operation of this method cannot achieve miniaturization of the device, reduction of measurement time, processing of multiple samples, or automation of the entire device. The fifth problem is that there are many kinds of so-called "low-abundant proteins". Only one gene in yeast encodes 50% by weight of all proteins f, and this means that 50% of the proteins are products of thousands of genes. Many of the most important proteins, such as regulatory proteins or receptor-containing signals. Related proteins are included in the "low_sufficient protein" #, so current proteomic analysis methods based on electrophoresis cannot analyze them. In order to address the limited capabilities of this type of electrophoresis and to elucidate protein-protein parent interactions, many attempts have been made, such as the ICAT (Isotopically-Coded Affinity I * Health Heart Signature) method [Gygi et al., Nat. Biotech. 1 0,994- 999 Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs (1999)], the two hybrid system in yeast, bia_ms_ms, protein array method (solution or chip) [zh〇u et al. TRENDS in Biotechn〇1〇gy 19, S34-S39] or a peptide mixture of LC-MS-MS. However, these novel methods and technologies have not yet achieved the ultimate purpose of proteomics: total protein body performance analysis, interaction analysis, and network analysis. Even the most promising protein array methods among the above methods include solid-phase protein array methods (wafer method) [Fung et al. Curr. Opin · Biotechnoi 12, 65-69 (2001)] and liquid-phase protein array methods (such as , Fluorescently coded ball beads [Fult〇ji et al. Ciin This paper size applies to Chinese National Standard (CNS) A4 specifications (210 X 297 mm) 6 313287 1240796

Membrane proteins; and (4) analysis of membrane proteins and, by affinity, water-soluble proteins, or (please read the Note: Please fill in this page again.) ^^ It is preferred that at least one physical or chemical property of the protein in the t-hall can be analyzed by mass spectrometry. Therefore, this suitable ligand carrier is the mass spectrometer. Pull flat. The present invention specifically addresses the following problems. Can maintain the membrane protein's first-, third-, and fourth-order structures and physiological functions. This is because the membrane protein system is transferred to an artificial microlipid membrane that maintains its hydrophobic region and hydrophilic region / biological conditions, without using protein denaturation. Agents, proteolytic agents or any other processing conditions that deviate from the physiological conditions in the presence of membrane proteins. Regarding the receptor, it can preserve any structure and function of any biofilm-type receptor (including 3 GPI-type, GPCR-type, and oligo-type receptors). The preparation of each membrane protein at the same time maintains the structure and function, thereby eliminating the difficulties in membrane protein research in all biological fields including medicine and agriculture. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. As only water-soluble proteins containing ligands are separated by electrophoresis, any water-soluble protein can be caused by no contaminants in the water-soluble protein. All kinds of interferences are analyzed. Water-soluble proteins separated by one- or two-dimensional electrophoresis are directly retarded on the plates of the mass spectrometer, thus greatly reducing the operation time. By increasing the amount of proteins to be transferred to the mass spectrometer plates, it is possible to measure low-abundance proteins. The protein M device is reduced in size and mechanized, and the number of samples to be processed is increased. The paper size applies the Chinese National Standard (CNS) A4 (210 x 297 mm) 9 313287 Printed by the Intellectual Property Bureau of the Ministry of Economic Affairs and Consumer Cooperatives 10 1240796 A7-V. Description of the invention (10 meshes) By contacting the microlipids to which the membrane proteins have been transferred with various ligands fixed on the carrier to utilize its biological affinity purification, highly purified Target membrane protein-ligand complexes. According to the present invention, the pure interaction between the molecules of the peptone protein and the ligand (including the competitor) can be measured, regardless of whether the two molecules are highly purified or coexist with many other substances. Effects from extracellular signal transmitters or transcriptional regulators. In other words, interaction detection based on cellular responses leaves behind The physiological point of action of a predetermined ligand, regardless of its mesangial protein, extracellular signal transmitter, transcription regulator, or different points of action. Conversely, the present invention provides only detection of the relationship between the protein and the ligand. Interaction. Purification of membrane protein-ligand complexes on a mass spectrometry plate can be collectively detected by mass spectrometers. Formation of membrane protein-ligand complexes and detection of functions that clarify membrane proteins (and Interactions of ligands) The present invention also provides a method for identifying membrane proteins and / or their ligands that show a specific change in the quantity or properties of a disease, which method includes the use of organisms (including humans) (Plants, animals, and all other organisms) samples by the above method to analyze the protein body, and then compare the analysis data obtained with healthy organisms of the same kind. When this method is applied to multiple diseases and the obtained data can be collected, Construct a diagnostic database. In this way, by discovering the disease-specific membrane proteins and their ligands, the quantitative values are rounded to the data, and then compared This health data and the same species of ugly and Syria, based on the film, the paper scale applicable Chinese National Standard (CNS:)? A4 size coffee χ 29) -, 313287 (please read the back of the phonetic matters then fill out this page}

Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 11 1240796 A7 __ B7 V. Description of the invention (11) The diagnosis of diseases with white drug and ligand double drug proteomics data becomes applicable. Pharmacogenomics has boundaries set by patient genetic analysis or static etiology. Most of the results of pharmacogenomic analysis are not directly related to the patient's disease, and therefore cannot provide information on proteins that dynamically change and cause the disease. In contrast, pharmaceutical proteomics directly teaches the dynamics of proteins that control biological responses and dynamically shows the patient's current disease state through changes in the protein. The collection of pharmacogenomic information is inconsistent with personal information about the patient's genetic qualities, which can sometimes be an insolvable moral issue for individuals, races and countries. However, because pharmacoproteomics information is used to clarify the patient's disease, it is collected experimentally in the same way and diagnosis is usually performed in a hospital 'efforts between the biological science perspective and the 21st century ethical perspective do not cover the stakes. Since the dual data of membrane proteins and ligands can be collectively analyzed, when the disease is caused by signal transduction through the defect of membrane proteins (if it is caused by changing the amount and properties of membrane proteins, the amount of ligands is changed And nature, or both), to instantly diagnose the cause of the disease. Once the aforementioned constitution of the present invention is realized by a fully automatic device, it is possible to explore the revolutionary industrial field of utilization. One example is a fully automated membrane protein-ligand protein body analysis system, the elements of which are shown in the figure. It goes without saying that various other devices can be assembled based on the present invention based on the research purpose of membrane proteins. _The present invention can be applied to discovery, identification and analysis not only by covering the same paper size as the applicable Chinese National Standards (CNs) A4 specification (210 x 297 mm)------ 313287 -------- -------------- Order --------- (Please read the notes on the back before filling this page) 1240796

V. Description of the invention (l2) The search for the orphan ligand of the G protein-coupled receptor (GpCR) predicted from the genomic sequence by phylogenetic search is almost impossible to find, by using a computer and a prediction technology based on the homologous genomic sequence It is also almost impossible to find all other types of receptors (GPI type, oligomeric type) and their ligands. In addition, the present invention is considered to greatly contribute to the discovery, identification, and analysis of all kinds of membrane proteins (membrane proteins other than receptors) (currently still extremely difficult), because @ 能 发展 & The discovery-type "medicine" is the discovery type of protein-related drugs, and medicine, whose goals are to analyze diseases, develop diagnostic reagents and diagnostic methods, and develop therapeutic agents that cover all membrane proteins and ligands. The invention introduces a novelty The methodology of the times to the research of tritium protein (which is the greatest difficulty in the history of biological research) will not only help medical care but also become a driving force for discovering and utilizing new biological functions and new industries for its application. The goal is to resolve key issues regarding food and the environment. 'This is a global issue in the early 21st century. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. These and other objects and advantages of the present invention will be made clearer by the following description of the present invention. , And other features of the present invention. [Simplified description of the drawings] FIG. Representative example of the Ming protein f-body analysis system and its documentation. One example. White matter carrier (ligand) The second diagram is used in the mass spectrometry plate of the present invention The third diagram is used in the water-soluble egg of the present invention Various examples. _Li g W The ligand used in the present invention is a paper size suitable standard (CNS) A4 specification ⑵ G χ 297 public ^ 12 313287 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economy 1240796 A7 _ B7 Explanation (13) An example of one of the tables. Figure 5 shows the urokinase receptor (ligand: FiTC-UK) on the U93 7 cell membrane before and after Bt2cAMP stimulation (Figure 5B) and after (Figure 5A). Body (ligand: FITC-C5a) and interferon-r receptor (ligand: FITC_INF 7). Figure 6 is a photograph showing the separation membrane segment from U937 cells. The left side shows 40% sucrose density. Before gradient centrifugation, the right side shows 40% sucrose density after gradient centrifugation. Figure 7 is a photo showing the microlipids embedded with protein isolated from U937 membrane, where A: microlipids (200 to 1) + membrane fraction; B : Liposome (50 // 1) + 臈 Division; C: Membrane Division; D: Liposome (2 00 # 1). Section 8 The results of the FACS analysis of FITC-labeled membrane sections (Figure 8A), FITC-labeled, membrane protein-embedded microlipids (Figure 8B) and simple microlipids (Figure 8C) are shown in Figure 9. Results of a protein lag analysis showing the expression of urokinase receptors on U937 cells. Figure 10 shows urokinase receptors in the presence (Figure 10A) and absence (Figure 10B) of anti-urokinase receptor antibodies. Isofocus laser photomicrographs of the embedded microlipids. Figure 11 shows the results of the protein slip analysis of the dissolved membrane protein-embedded microlipids. Figure 12 illustrates the binding capacity of the urokinase receptor ligand (FITC-labeled urokinase) in the membrane segment (Figure 12A), with FITC-labeled HSA as the control group (Figure 12B). -IHI ϋ II n ϋ I (210 X 297 mm) 13 313287 1240796 Α7 Β7 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. 5. Description of the invention (U) Figure 13 The ligand of the urokinase receptor not embedded in the microlipids ( FITC's urokinase binding ability (Figure 13-8), using the identified HSA as a control group (Figure 13b). Figure 14 Molar ratio dependence of the unbound labeled ligand with U937 cells (Figure i4A) and the microlipids embedded in urokinase receptors (Figure 14β). Figure 15 does not show the binding capacity of ligands (FITC-labeled urokinase) for microlipids embedded in membrane proteins prepared from U937 cells stimulated by PMA (Figure 1SB) and non-stimulated (Figure i5A). Figure 16 shows the appearance of microlipids embedded in urokinase receptors as the size of microlipids decreases. Figure 17 is a photo of a coomasie bright blue stained polypropylene amidamine gel with a urokinase-stable mass spectrometer plate and a pre- and post-coomasie plate. Figure 18 shows the results of mass spectrometry analysis of urokinase that is retarded on the mass spectrometer plate. Figure 19 shows the quantitative results of urokinase retarded on the mass spectrometer plate. Figure 20 shows the results of mass spectroscopic analysis of rhodopsin, 20A shows the results of rhodopsin at various concentrations, and 20B shows the rhodopsin alone and rhodopsin and simple microlipid. The coexistence of granules and the result of rhodopsin in microlipids. Figure 21 shows the results of mass spectrometry analysis of urokinase receptors on a mass spectrometer plate. Figure 21A is a photograph of electrophoretic purified urokinase receptors, and Figure 21b is an explanatory diagram of a complex formed on a mass spectrometer plate. And Figure 21C The paper size applies to the Chinese National Standard (CNS) A4 specification (21 × 297 mm) 14 313287

1240796 A7 _________ B7 5. Description of the Invention (I5) Shows the results of mass spectrometry analysis when anti-urokinase receptor IgG (top) and control IgG (bottom) are used as ligands. Figure 22 shows the results of mass spectrometry analysis of bovine rhodopsin with IAA or DHB added. Conditions: Bovine rhodopsin: 2.56 pmol membrane suspension (in 20 mM tri-HC 10m MyS-mercaptoethanol, 100 β M EDTA, ρΗ7.5 ·) matrix: 0 · 17 mg / eLDHB (in ethanol 0.5 // L ) Or IAA saturated solution (in ethanol 1.0 // L). Figure 23 shows a selective measurement of the mass spectrum of urokinase receptors as a denatured protein model (Figure 23a) and urokinase as a physiological protein model (Figure 23B). Figure 24 shows the binding of microlipids to various embedded receptors and their ligands immobilized on Sepharose (Agarose) 4B gel. Figure 25 shows a mass spectrometric analysis of urokinase as a ligand and urokinase receptor as a receptor. [Detailed description of the invention] 1. Terms and general examples of the present invention When used in this specification, the following terms generally have the following meanings. General examples of the invention are as follows. (i) The term "complementary" means the anatomic harmony or consistency of the surface of the receptor with which its ligand interacts. In other words, the receptor is complementary to its ligand, and therefore the properties of its contact surfaces are complementary to each other. (ι〇 "Ligand" is a molecule recognized by a specific receptor. In the present invention, f Jing first read the phonetic on the back? Matters before filling out this page} · 111111.

Printed by the Consumers' Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs, the scale is applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 meals) 15 313287 1240796 a7 B7 V. Description of the invention (16) The ligand is not limited to Physicians, including full agonists, partial agonists, antibodies and agonists for cell receptor conversion, toxins, viral epitopes, hormones (eg, sedatives, anesthetics, steroids, etc.), peptides, enzymes Substances of enzymes, cofactors, drugs, exogenous lectins, carbohydrates, oligonucleotides, nucleic acids, oligosaccharides, proteins and monoclonal antibodies. The ligand can be a natural molecule or an artificial molecule. The location of the natural ligand is not limited, and it can be any substance existing on the surface of the earth, including the atmosphere, substances secreted by living things, intracellular substances, organelle substances, nuclear substances, and others. (iii) A receptor is a molecule that has an affinity for a particular ligand. The receptor can be a natural molecule or an artificial molecule. This can be done alone or in a complex with other molecular species. The receptor system forms complex receptors (oligomeric receptors) directly through covalent or non-covalent bonds or through specific binding substances. Artemisiae used in the present invention include, but are not limited to, antibodies, cell membrane receptors, monoclonal antibodies, and specific epitopes (for example, on viruses, cells or other materials), drugs, polynucleotides, nucleic acids, cells Antisera for peptides, cofactors, exogenous lectins, sugars, polysaccharides, cells, cell membranes and organelles. In the appropriate field, receptors are sometimes referred to as "anti-ligands". When this note uses the term "receptor", it is not intended to mean a different meaning. μ ★ In the invention, regardless of its structure and function, any membrane receptor, membrane pipeline, membrane pump, membrane transporter, membrane proteins and proteins attached to these eggs are called receptors or membrane proteins. This is because those skilled in the art can easily recognize that the method of the present invention can separate and identify

set. I Standard for timely payment of paper size (ci ^ 4 size ⑵Q Χ 29) 313287 (Please read the precautions on the back before filling this page)

-------- Order --------- Line | Printed by the Consumer Cooperatives of the Ministry of Economic Affairs Intellectual Property Bureau 16 Printed by the Employee Cooperatives of the Ministry of Economic Affairs Intellectual Property Bureau 1240796 A7 B7 V. Description of Invention (17 ) When two macromolecules form a complex through molecular recognition binding, a "ligand-receptor complex" is formed. The location of the receptor is not limited to the narrow common sense cell membrane (so-called plasma membrane that forms the outer layer of cells). Receptor means a molecule that binds to any membrane having a lipid bilayer in general. For example, a DNA polymerase complex bound to the nuclear membrane is important for DNA replication and repair ’RNA polymerase is important for transcription, and ribosomes bound to the endoplasmic reticulum membrane are important for protein translation. A group of oxidoreductases that bind to the mitochondrial membrane play an important role in the production of ATP. A group of metabolisms related to the enzymes of the peroxidase membrane participate in the metabolism and production of heat in the lysosomes. A group of degrading enzymes is involved in the degradation of proteins, nucleic acids, hearings and lipids', and Golgi ’s membrane proteins have important functions for protein synthesis and glycosylation after the transport of synthetic proteins or lipid membranes. Furthermore, the possibility of participating in the surface of various cell membranes as a foothold in which a group of phosphorylated and dephosphorylated enzymes are deeply involved in intracellular signal transduction is suggested. The foregoing example does not limit the important function of the receptor (membrane protein) to the scope of the above example. Membrane proteins and their functions determined by the present invention are all included in the present invention. The positional relationship between the receptor and the lipid bilayer varies. The most common is a penetrating type (() protein-coupled receptor) that is folded several times and stabilized by the hydrophobic region of the protein and the hydrophobic bilayer of the lipid bilayer: the hydrophobic interaction of the region. An immobilized receptor (glycosylphosphatidylinositol-immobilized receptor) in a lipid layer outside the bilayer. Examples include glycoproteins, lipids, and oligosaccharides (which are immobilized on the outer surface layer of cells, The paper size of this article is applicable to the Chinese National Standard (CNS) A4 specification ⑵G χ 297 male f) ----- 313287

17 1240796 A7 B7 V. Description of the invention (I8) A polymer-receptor containing a GTP / GDP coupled protein group immobilized inside the cell constitutes a molecular group, and a membrane-lined protein group that plays an important role in maintaining and changing the shape of the membrane ' The functional protein base with which it binds. The foregoing is an example of the positional relationship between the receptor (membrane protein) and the lipid bilayer. The examples are not restrictive, and the present invention encompasses any positional relationship as the present invention clarifies a wide variety of such relationships. There are many receptors that can be used as the research object of the present invention (including the unknown, the following examples only list a part of them. A) Cancer-specific membrane proteins are identified by analyzing membrane proteins that behave and function in cancer cells, and analyzing their functions. The development of agents with the mechanism of action of cancer cell growth inhibition, apoptosis induction, metastasis inhibition is expected. Antibodies specific for the membrane protein itself can be used as effective medicaments and can also be applied to targeted therapies that deliver toxins and the like to target cancer cells. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs b) By identifying membrane proteins expressed in the cell 臈 of tissues attacked by autoantibodies (ligands) in autoimmune diseases or organ transplanted cytotoxic lymphocytes, It can develop related therapeutic agents or medicines that can be used to block the binding of autoantibodies or autoantigen-specific tissue toxic lymphocytes. In particular, a wide variety of diseases in autoimmune diseases are considered to be based on tissue specificity and if they can be isolated and identified to attack Addison's anemia, filamentous nephritis (primary, IgA), exophthalmic thyroid Hyperfunction, insulin-dependent diabetes, multiple sclerosis, malignant anemia, rheumatoid arthritis, dry keratitis syndrome, psoriasis, systemic lupus erythematosus, thyroiditis, white spot disease, local ileitis, spontaneous platelets Reduction of purple spot disease and other standards are applicable to the Chinese National Standard (5NS) A4 specification (⑵297 x 297 meals) ------ ~-18 313287 1240796 A7-B7 V. Description of the invention (19) Autoantibodies or self of the disease Membrane proteins of histotoxic lymphocytes can develop medicines that are specific to various autoimmune diseases and have slowing side effects. C) By specificizing benzodiazepine receptors, cannabinol receptors, sigama receptor 1 and Sigama receptor 2 as endogenous ligands (there are artificial ligands (competitors) but Endogenous ligands are unknown), and endogenous ligands that bind to the Phen cyclidine binding site of the NMDA receptor through specificization can develop innovative therapeutic agents for central nervous disease. d) Receptors expressed in microorganisms Determining ligands that bind to membrane transporters that are essential for microbial survival can be used to develop antibiotics with new mechanisms of action. Of particular value are antibiotics against opportunistic fungi, protozoa, and bacteria that are resistant to antibiotics currently in use. e) Receptors of nucleic acids as ligands. When synthesizing nucleic acid sequences and isolating, identifying, and functionally analyzing membrane proteins that hybridize to DNA or RNA sequences, elucidate completely new interactions between endogenous nucleic acids and cell membrane functions. Effect, which in turn leads to the development of useful related diagnostic reagents or medicine. For example, anti-meaning technology-based medicine can be developed to effectively transport systems or DNA, RNA virus new infection defense mechanisms in the cells of the consumer cooperatives of employees of the Intellectual Property Bureau of the Ministry of Economic Affairs. f) Receptors for lipids or lipid metabolites of ligands When these low molecular weight compounds are synthesized and isolated, identified, and functionally analyzed for cross-reactive membrane proteins, useful diagnostic reagents or agents can be developed. For example, by discovering completely new receptors involved in smooth muscle contraction and the relaxing effects of many metabolites in the arachidonic acid cascade and reference paper sizes, the Chinese National Standard (CNS) A4 (210 x 297 mm) f) 19 313287 1240796

V. Description of the invention (20) / ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, a new type of edg (endothelialization gene) ^: developed in vivo in the central nervous system, circulatory disease, and cancer "Medical medicine with a completely new mechanism of action in the field of diseases of the Shaw system or immune system. g) Membrane protein protein In order to clarify the conversion mechanism from normal protein to pathogenic protein, a reconstruction system of GPI-type protein membrane protein embedded in microlipids with its original structure was used. The release mechanism, analysis of the conversion rate of the release-promoting molecule's single-protein protein and free protein to pathogenic protein. As a result, a method for measuring a pathogenic protein can be developed, a method for removing a pathogenic protein, a method for defending against a pathogenic protein infection, a cjd start delay method, a CJD patient treatment method, and the like. (⑺ "Biological 臈" means any membrane with a lipid bilayer as a component, including cell membranes and membranes that make up organelles, such as the endoplasmic reticulum membrane, Golgi apparatus, nuclear radon of any organism, etc .. Consumption by employees of the Bureau of Intellectual Property, Ministry of Economic Affairs Printed by a cooperative (v) "Lipolipids" refer to particles separated from the outside world by a lipid bilayer. The lipid bilayers of microlipids are physically, chemically and biologically similar to biological maggots. Microlipids The granules are preferably made of membrane components such as those extracted from plants. 膜 "Membrane-related substance" refers to any substance that penetrates and binds to the inside or outside of a biological membrane. Membrane-related substances include the transduction of signals from outside the cell to the cell Internal membrane receptors' are used to transport physiological substances between the outside and inside of the cell. The membrane proteins that make up the membrane channels, the membrane proteins that maintain the dynamic membrane structure, white matter translation enzymes, ribosomes, and via covalent bonds or non-covalent The paper size of the paper is applicable to the Chinese National Standard (CNS) A4 size (Q X 297 meals) ...... One-_ 20 313287 A7 1240796 _______B7_ V. Description of the invention (22) In the example It is in the form of magnetic particles, non-magnetic particles, bundles, sediments, gels, plate-shaped 'tubular' spheres, containers, capillaries, pads, sheets, membranes, flat plates and other various surface structures. Basically, optional and convenient forms can be used in the present invention. The surface to be subjected to can be biological, abiotic, organic or inorganic, or a combination of these 'and is formed on the surface of a rigid support and The reaction described in the present invention occurs thereon. The Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs prints selected surfaces to provide suitable protein adsorption properties. Examples include functionalized glass, Si, Ge, GaAs, GaP, Si02 , SiN4, reconstituted polyoxylate's wide range of gels and polymers, such as polytetrafluoroethylene, polyvinylidene fluoride, polystyrene, polycarbonate, or a combination of these. Has multiple monomers or Examples of substrates of polymer sequences include linear or cyclic polymers of nucleic acids, polysaccharides, lipids, peptides with alpha-, non-, or omega amino acids; gels for chromatography Surface carrier Ionic / cationic compound, a hydrophobic compound consisting of 1 to 18 carbon chains, and a carrier for cross-linking hydrophilic compounds, such as dioxide crushing, nitrocellulose, cellulose acetate, agarose, etc.) ; Synthetic homopolymers such as polyurethanes, polyesters, polycarbonates, polyureas, polyimides, polyethyleneimines, polysulfide 'polysiloxanes, polyimides, polyacetates, etc., · A heteropolymer of any of the above compounds with a conjugate of a known drug or natural compound bound thereto (covalently or non-covalently); and other polymers that will be appropriately discovered after a general review of this disclosure (VII) Mass spectrometry plate "refers to a substrate (ligand carrier) used to bind soluble molecules commonly known as ligands, which can be applied in accordance with Chinese national standards by high-precision analysis methods such as this paper scale (CNS) A4 specification (0x297)) " 22 313287 1240796 A7 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 5. Description of the invention (23) One-dimensional and two-dimensional electrophoresis, high-efficiency liquid chromatography and other separation ligands Automatically and immediately after adoption and Transfer directly to the surface of the substrate, and subsequently to the substrate of the highly sensitive mass spectrometer. The substrate is composed of basic results to maintain its shape and properties, and is composed of a ligand adsorption material (substrate surface) to optimize the binding mode and quantity of the ligand. The ligand adsorption material may be a covalently bonded or non-covalently bonded adsorption material 'depending on the binding mode of the ligand. The latter adsorption mode usually includes normal phase, reversed phase, hydrophobic, anionic, cationic and other non-covalently bonded adsorption materials. ^ The adsorbent material may contain spacer molecules to form a distance between the ligand and the surface of the basic structure of the substrate (10A to 10000A, preferably about 100 people). ° The material for this spacer molecule can be a reticulated or porous biopolymer or a synthetic polymer. In any case, it is formed to provide a membrane protein with ligand 1 antibody affinity rather than affinity, so that the membrane is embedded in microlipids having a diameter of 10111 to 5,000 nm The protein binds to a ligand with a large binding capacity. The surface of the substrate may be biological, abiotic, organic or inorganic, or a combination thereof, and is formed on the surface of a rigid support on which the reaction according to the present invention occurs. The substrate is selected to provide suitable protein adsorption properties. Examples include Lunenghua's glass, Si, Ge, GaAs, Gap, SiO2, and SiN4, reconstituted polysilicon oxide, a wide range of gels and polymers, such as polytetrafluoroethylene, polyvinylidene fluoride, poly Styrene, polycarbonate, or a combination of these. Examples of substrates with a plurality of monomer or polymer sequences include nucleic acids' paper size applicable to China National Standard (CNS) A4 specifications (2〗 〇297 catering) " --------- -23 313287 Please read the notes of the back first

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1240796 A7 B7 V. Description of the invention (24) Linear or cyclic polymers, polysaccharides, lipids, peptides with, or 0-amino acids; gel surface carriers used in chromatography (anionic /% Ionic compound, a hydrophobic compound composed of 1 to 8 carbon chains, a carrier cross-linked with a hydrophilic compound, such as silica, nitrocellulose, cellulose acetate, agarose, etc.) ; Synthetic homopolymers such as polyurethane, poly S, polycarbonate, polyurea, polyimide, polyethyleneimine, polysulfide, polysiloxane, polyimide, polyacetate, etc .; A heteropolymer of any of the above compounds and a known drug or natural compound bound thereto (covalently or non-covalently); and other polymers that will be appropriately discovered after a general review of this disclosure . (V111) Mass spectrometer means that by ionizing a sample into a gaseous state, casting the ionized molecules and their molecular fragments into an electromagnetic field, separating them based on the mass / charge number based on the moving state, and measuring the spectrum of the substance. Device for measuring and detecting the molecular weight of a substance. Several are better used, but others can also be used. Μ In a typical example (Figure 1), the protein body analysis method of the present invention is printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs, including the separation of only water-soluble proteins by colloid electrophoresis, and the separated water-soluble proteins from colloid The step of directly retarding on the mass spectrometer plate (step A), and the step of attaching or penetrating the protein to the artificial liposome membrane (step B ”to make the membrane egg self-embedded from the liposome of the liposome Xiaoqi #, The steps of contacting a water-soluble protein mass spectrometer plate with a bio-affinity (antibody affinity) to form a ligand-receptor complex (step 0, and collectively measuring the complex by mass spectrometry. The body S is collected and made Steps in the database of the obtained data (step D) 〇G-scale scale stab Zhongguan standard (CNS) A4 specifications ⑵Qx 297 -------------- 313287 24 A7 B7 1240796 5 2. Description of the invention (A specific example of 25 steps of AU and other aspects of the invention of each self-constituting element implementing these steps are described in detail below. / D 2. Fast protein lag device (device a) This device is made by electrophoresis Device, protein lagging device and quality It is composed of main components. The electrophoresis device can be a marketer or a special designer. Depending on the purpose, both-dimensional colloid electrophoresis and two-dimensional colloid electrophoresis are used. In the two-dimensional colloid electrophoresis towel, the first movement is based on the basis The isoelectric point of the protein is separated, and the second movement is based on the separation of the molecular weight of the protein. There is no particular restriction on the size of coagulation A used for electrophoresis. Although it is usually 10 cm x 10 cm, it is 20 cm x 2 when necessary. cm or other sizes can be used. Although the basic material of the gel is polyacrylamide, different substrates such as agarose gel, cellulose acetate film, etc. can also be used, depending on the purpose. The gel concentration can be Constant or may have a gradient. Water-soluble proteins printed by employees' cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs to be subjected to electrophoresis can be prepared from biological samples by any known method. Examples of samples include, but are not limited to, selective Cells, tissues or extracellular fluids of organisms such as plants, animals, microorganisms, etc. (eg, blood, plasma, urine, bone marrow fluid, ascites, etc.) For example, in obtaining the target After the cells, the soluble fractions can be homogenized in an appropriate buffer solution in the presence of various protease inhibitors, or suspended using a cell homogenizer such as pOlytrón, or the cells can be disrupted by low-tension shaking Or, the cell membrane is ruptured by ultrasonic disruption, and then obtained by centrifugation to obtain a supernatant. The second step includes transferring proteins from a colloid to a mass spectrometer plate after electrophoresis. In a preferred embodiment of the present invention, the mass spectrometer plate The shape is designed to fully match the sample inlet of the mass spectrometer to be used afterwards. For example, it refers to the "quality paper size applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 meals) 25 313287 1240796 A7 B7 V. Invention Explanation (26) :: Cluster plate ", which has a pre-made split line that can easily separate groups into split-type plates. Its size is suitable for the sample inlet of the mass spectrometer. After electrophoresis, the protein is transferred to the complex. Gel-sized mass spectrometers on clustered plates. Figure 2 shows an example of a preferred mass spectrometer plate of the present invention. In conventional mass spectrometers, one sample is measured one at a time, or only a limited number of samples are measured at a time. This is due to the one-dimensional transfer of a mass spectrometer plate (such as the aforementioned split-type mass spectrometer plate). In contrast, the fully automatic protein body knife analysis according to the present invention enables continuous overall scanning by moving the laser nozzle or the platform with a mass spectrometer in two dimensions (intermittent scanning leaves proteins without laser beam irradiation, also (Ie, unanalyzed proteins) and all proteins that are two-dimensionally coated by electrophoresis are analyzed. The combination of this method and intermittent scanning makes it possible to place the 96 samples allocated to the% -well plate on the mass spectrometer as a whole (96 samples are arranged on a rectangular wafer with the same pitch) and then apply this wafer directly to the mass spectrometer. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. In a better example, the material of the mass spectrometer plate is composed of a nameplate as a base and sulphur dioxide as an adsorption material. The slab is used for stagnation of electric power. It is also possible to use different conductors, such as stainless steel. In order to retard the diffusion, insulators (ceramic, plastic, etc.) can be used. As for the adsorbent material, dioxin on which any protein can be transferred is used, but other materials mentioned above can also be used depending on the purpose. Various methods (diffusion, electricity, etc.) are used to transfer the protein coated on the colloid to the mass spectrometer plate. This step is often referred to as a lag. For measurement limits in subsequent mass spectrometry, slew efficiency is an important factor. In addition, try to avoid denaturing proteins during migration and lag. This is because the current paper size 1¾ uses the Chinese National Standard (CNS) A4 specification ⑵G X 297 male f) '26 313287 Ϊ 240796

V. Description of the invention (27 Member of Intellectual Property Bureau of the Ministry of Economic Affairs X Consumer Cooperatives printed water-soluble proteins bound to membrane proteins' If one of them is denatured or unable to retain the natural three-dimensional structure, the binding will be incomplete. For this device, which is widely used in the study of proteomics, the composition of the molecules covering the surface of the mass spectrometer plate is of great significance. In order to improve the sluggish efficiency, a larger protein adsorption capacity is needed to adsorb any protein (not the specific protein) Adsorption capacity) and the constant adsorption rate of any protein, and in order to achieve binding to plutonium protein, it is basically necessary to maintain the three-dimensional structure of the protein after being delayed and adsorbed on the mass spectrometer plate. MASS analysis can be used to detect and identify = Quantitative collective membrane proteins and ligands, or two membrane proteins alone or one ligand alone, depending on the surface material. When membrane proteins are analyzed by MASS mass spectrometry in the present invention, they are polymerized by proteins or other organisms The spacer made of a polymer or a synthetic polymer is preferably interposed between the ligand and the carrier. To achieve an effective binding of the membrane protein and the ligand based on its biological affinity (antibody affinity). This is because the membrane protein is immobilized on the liposome and the ligand is also immobilized on the carrier. For example, " The terms and general examples of the present invention, as mentioned, need to improve the avidity of antibodies. The biopolymers used in the examples are protein G, antibodies, and biotin. The ligand and the surface of the basic structure of the substrate The distance is between 100 and 100 Å, preferably about ⑽A. The shape of the spacer may be mesh or porous. The mode of binding between the carrier and the spacer, and between the spacer and the ligand is described in detail. Later, but covalent or non-covalent bonds can be used, depending on the purpose of protein analysis ^ ° Based on this, mass spectrometry can be used to collectively or individually measure membrane proteins and water-soluble proteins with high sensitivity. Applicable to China National Standards (CNS) specifications (21〇X 297 meals) 5 Read Sj Note first

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27 313287 1240796

V. Description of the invention (28) According to the method of the present invention, the protein moving in the gel can be retarded on the mass spectrometer plate by a single-step process. Therefore, after electrophoresis in the conventional protein-knife analysis <lt; And all complicated steps before the start of mass spectrometry analysis can be omitted, which significantly shortens the operation time. Therefore, it is possible to achieve the processing of a huge amount of samples required for protein body analysis. In addition, in the present invention, it is not necessary to gel the gel after electrophoresis. The step of cutting into small pieces. As a result, the protein on the cut point (line) and the part lost without MASS measurement can be eliminated. This makes it possible to add all the proteins added to the gel to the mass spectrometer plate. Also, the use of electricity Or the direct retardation of the diffusion force replaces the step of extracting the protein from the gel (extraction efficiency is less than 10%), which can achieve 100% retardation efficiency, and the amount of protein retarded on the mass spectrometer plate is the amount of conventional protein extraction. Several times to dozens of times. In a typical example of the present invention, after forming a complex of a membrane protein-embedded microlipid and a ligand as follows, the membrane is collectively detected and analyzed White matter and ligands, and in various examples, the present invention provides a ligand analysis method by performing mass spectrometry by mass spectrometry plates on which only the ligand (water soluble protein) is stagnated. The proteins on the mass spectrometer plate can be analyzed by performing mass spectrometry on the mass spectrometer plate. The knife analysis of the protein immobilized on the mass spectrometer plate according to the present invention can be performed using any kind of commercially available mass spectrometer, but it is more preferable to use the following The mass spectrometer of the method is performed by the matrix_assisted laser desorption / ionization (MALDI) (where the sample is mixed with the matrix that absorbs the laser beam and then dried to crystallize, and then transferred by the energy from the matrix and The high-intensity laser pulse is used to ionize and introduce the crystallized sample into the vacuum), and this paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 public hair) '------ 313287 dm (Please read the notes on the back before filling this page) Order --------- Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 28 1240796 V. Description of the invention (29) Fei time spectrum (TOFMS) (its MALDI-TOFMS method consisting of analyzing mass numbers based on the difference in time of flight of sample molecular ions caused by initial stage acceleration; where a protein is placed in a droplet and ionized directly and electrically from the liquid Method; wherein the sample solution is electrically sprayed into the air, and each unfolded protein polyvalent ion causes a nano-ESMs method in the gas phase, etc. Various examples are used as the ligand carrier of the present invention in combination with the carrier most suitable for the purpose and purpose of proteomic analysis and the ultra-highly sensitive detection system. The details are as follows (for example, Table j and Figure 3), except _ Non-magnetic particles and magnetic particles are also exemplified outside the plate. Μ Various examples of combinations with ligand carriers most suitable for the target and purpose of protein body analysis can also be adopted as the detection system of the present invention. The details are as follows. In addition to mass spectrometry, fluorescence measurement, radioactivity measurement, etc. are also exemplified. When the ligand carrier is a particle, the membrane protein-ligand complex can be made in advance; the titanium lipid particles are highly sensitively detected, isolated and quantified. This step (step A) can be applied not only to water-soluble protein f, but also to extracellular fluids (for example, various body fluids such as blood, plasma, urine, fluid, ascites, etc.), intracellular fluid, or cells Peptides, carbohydrates, RNA, etc. in the internal fluids of the body, do not adhere to or penetrate the cell's cell membrane, nuclear membrane, endoplasmic reticulum membrane, etc., any other derivatizable compounds derived from living organisms, any synthetic compounds' Substances (for example, oxygen molecular nitrogen dioxide, etc.) and the like. In other words, the "use of property" is used to highly purify the target substance of each analysis, and to analyze the interactions with the microlipids that encapsulate the membrane protein and the like using the most suitable body.乂 + a @ (210 χ 29 line 313287 A7 B7 1240796 V. Description of the invention (M) 3. Device for manufacturing microlipids embedded in membrane protein (device B) This device contains a membrane separation section from cells to prepare microfat Granules, fusion membrane segment and microlipids, adjust the particle size of the fusion microlipids (membrane protein-embedded microlipids), and if necessary, a device for storing the fusion microlipids. To extract membrane segments from cells' Conventional methods can be used. For example, target cells can be obtained, suitable buffered in the presence of various protease inhibitors, homogenized in six fluids, or suspended in a cell rupture device such as Polytron, etc. Rupture, or destroy the cell membrane by ultrasonic disruption. After that, the cell membrane segment and the organelle membrane segment are prepared by density gradient centrifugation using various media. As for the method of preparing the microlipids, various known methods can be used. Typical But it is not limited to making the mixture of selected lipids uniformly dissolved in an organic solvent, and completely evaporating the solvent in argon and then hydrating it into a buffer solution to produce liposomes. The composition of is important. Generally, the cell membrane contains cholesterol, but the lipid bilayers that make up organelles such as the endoplasmic reticulum contain little or no cholesterol. Therefore, the composition of the microlipids that receive membrane proteins is determined Which membrane protein of the cell is to be transferred to the liposomes becomes a critical factor. Those skilled in the art can decide the appropriate lipids to constitute the liposomes, depending on the derivation of the membrane proteins. As for the membrane division and the liposomes For the fusion method, various known methods can be used. For example, a method including mixing the two in an appropriate ratio and then repeatedly freezing and thawing the method 'including placing an aqueous solution containing a membrane segment in a liquid cadaver made of selected lipids The method of transferring the membrane egg _ty to the lipid bilayer by hydration, or it can be made into different papers for this purpose ---- 313287 -------- ^ --------- ^ (Please read the notes on the back before filling out this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 30

5. Description of the invention (1240796 method. The freeze-thaw method is preferred, and using this method can achieve a 100% embedding ratio (transfer ratio) of membrane protein to microlipids. Or, when the microlipids are formed, In the fusion of membrane proteins, the fusion of microlipids and membrane protein fractions can be improved by adjusting the particle size to 10 nm to 5000 nm, preferably 1011111 to 500 nm. The prepared membrane protein-package Buried artificial microlipids can be sized by ultrasonic disruption, homogenization or other methods. In the present invention, it is preferred to use filtration (extruder method) to minimize the denaturation of membrane proteins and adjust the particle size to 10nm to 5000nm, preferably 100nm to 500nm. By carefully adjusting the mixing ratio of the membrane segment and the liposomes, the desired type and number of membrane proteins to be embedded in the liposomes can be controlled. This technique is absolutely important for facilitating the analysis by reducing the interference proteins (interference peaks) in the measurement and measuring the molecular weights of both the membrane protein and the ligand forming the complex of the present invention with the device C described below. hair The method for stably storing the microlipids (the microlipids embedded in the membrane protein) obtained by attaching or penetrating the target membrane protein to or through the lipid bilayer is effective to prevent the disappearance of a mass spectrometer anywhere, anytime, and without a mass spectrometer. It is extremely important for any institution to be able to conduct proteomics research. The development of several additives for the preservation of micromoisture particles in samples can be used for this purpose. The method of the present invention can be applied to any membrane protein. Therefore, its Can be used for discovery 'identification and analysis of all other types of receptors (GPI-type, oligo-type) and their ligands that are almost impossible to find by using computer-based genome sequence homology-based predictions, and search for borrowing The orphan of the G protein coupled receptor (GPCR) predicted by the homology search from the genome sequence. The paper size applies the CNS A4 specification (G x 297 public love) --- 313287 -------- Order --------- (Please read the notes on the back before filling out this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 31 1240796 A7 B7 V. Description of the invention (32 ligands By target Medical development of receptor-based drug discovery for all kinds of receptors is possible. In addition, the present invention greatly contributes to the discovery, identification, and analysis of membrane proteins other than receptors. Therefore, the goal is to analyze all membrane proteins and compounds. The development of medicine through membrane protein ^ -based drug discovery, the development of diagnostic reagents and diagnostic methods 'and the development of therapeutic agents have become achievable. The method of the present invention can be applied to discovery' identification and analysis in general Intracellular membrane proteins that do not appear on the surface layer of the cell membrane, such as those that form part of the lipid that is fixed (penetrated) on the inner layer of the lipid bilayer, and is commonly referred to as the membrane lining protein. The upper layer of the double layer. Furthermore, the present invention greatly contributes to research aimed at antibody-based drug discovery (including antibody drugs) using antibodies to analyze, diagnose, and treat diseases. Antibodies that are effective for analyzing, diagnosing, and treating diseases are membrane proteins. For the preparation of corresponding antibodies, membrane antigens (membrane proteins) that maintain structure and function are indispensable. Only the method of the present invention (in which the membrane antigen is embedded in the liposomes while maintaining the structure and function) can meet the above-mentioned conditions in which the discovery of antagonist-based objects is essential. In the goal of developing analytical, diagnostic reagents and diagnostic methods for diseases covering all membrane proteins and ligands, and general methods of developing therapeutic agents (the present invention also covers this), antibody-related drug discovery methods are to replace antibodies with antibodies One of the special methods of this body, which is obvious to all. Therefore, in addition to the development of receptor drug discovery medicine, the present invention also provides the development of antibody drug discovery medicine. For proteomic analysis of membrane proteins and their ligands, the present invention can collectively discover and quantify collectively, as well as functional (interaction) analysis of membrane paper. The scale of paper 1 is applicable to the Chinese National Standard (CNS) A4 specification (210 X 297). Li) 'a' · 313287 ------------- $ {Please read the phonetic on the back? (Please fill in this page for matters) Order --------- Line · Printed clothing for employees' cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 32

V. Description of the invention (33 Proteins and their ligands, or the interaction between the discovery and quantification of any one of them and the analysis of both. In this case, are membrane proteins and their ligands known or not known? It can be explored. This ligand contains natural endogenous ligands, competitors (full agonists, partial agonists, antagonists, transformation agonists), printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 33 1240796 Medical Agents' reagents, antibodies, and any other substances that have been artificially modified to bind to membrane proteins. By administering normal membrane proteins as new dosage forms, membrane protein-embedded artificial microlipid medicine, in this step (step B) The obtained membrane protein and embedded microlipids can be directly administered to patients with diseases caused by defects, mutations and other abnormalities of membrane proteins. When the lipid bilayer is composed of biological species other than animals When obtained, humans can be prevented from being infected by the disease source. The 臈 protein obtained in this step is a complex of embedded artificial liposomes and endogenous ligands, or membrane eggs The complexes of artificial microlipids and competitors (whether full agonists, partial agonists, antagonists or conversion agonists) can have new dosage forms with new mechanisms of action. Proteins_Embedded artificial micro Lipid drugs are administered directly to patients with diseases caused by abnormalities in both or both of the membrane protein and the ligand. The present invention uses single or combined use of covalent or non-covalent bonds to make fluorescent The method of cross-linking light substances and other labeling substances to the lipid bilayer of artificial microlipids through the method of embedding hydrophobic parent interactions and the method of encapsulating soluble labeling substances in the water phase in artificial microlipids, also Can provide the theory of detection of novel and ultra-sensitive membrane protein-ligand (including competitor) interactions. The method and device may also be used by using avidin-bioxin 1 paper size Applicable to liTi National Standard (CNS) A7iiHFx 297 Meal) --- ~ 313287 (Please read the precautions on the back before filling out this page) ------- Order ------- A7 B7 1240796 V. Invention Description (34) system, nickel-histidine or other cross-linked system To cross-link, embed or encapsulate substances that produce a second signal (for example, enzymes such as alkaline phosphatase) (instead of fluorescent substances (please read the precautions on the back before filling this page) and other identifying substances) and The artificial microlipids were then amplified and tested for sensitivity. As shown in the following examples, when artificial microlipids (including fluorescent molecules and other markers that can be identified by FACS)

Molecules are introduced at the DNA stage, RNA stage or protein stage by genetic engineering or other imaginable methods), and the size is set to 10nm to 5000nm by extruder method or other methods, preferably 500nm or less, FACS The analysis clearly shows the population of identified membrane protein-embedded microlipids in the corresponding size regions. Therefore, for example, when preparing a cDNA encoding a prion protein from a gene sequence, using an expressive vector containing the cDNA to transform a host cell and make a membrane protein (in which other molecules used for the identification of fluorescent molecules that can be recognized by FACS have been introduced) At the time of expression, adjust the labeled membrane protein-embedded non-labeled microlipids prepared from the cell membrane section of the expressive host to this size and analyze by FACS to detect the presence or performance of the membrane protein, and Performance. Unlike other currently used methods, the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs prints DNA sequences that do not have membrane proteins or reagents (antibodies, ligands, competitors, cellular responses to membrane proteins or other detection agents). Debt testing under the conditions of information becomes possible. The use of the presence of a detection agent of a membrane protein, such as an antibody, makes it possible to identify at the protein stage, and similarly, it is possible to analyze performance by Facs. It goes without saying that similar theories can also be applied to study ligands. This step can be applied in addition to membrane proteins to make sugar paper containing lipid bilayers attached to or penetrating cells, such as cell membranes, nuclear loops, endoplasmic membranes, etc. The paper size applies to Zhongguanjia Standard (CNS) A4 ⑵0x 297 public love) — 1 ~ 34 313287 1240796

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A7 B7 36! 24〇796 V. Description of the invention (increased concentration. Microlipids that are not specifically bound to the mass spectrometer plate can be removed by intermediate washing with a washing buffer with an appropriate salt concentration and composition. Simultaneous washing The temperature conditions of the prior period are also important, and those skilled in the art can determine suitable conditions when needed. The method of removing liposomes by cytolysis can include adjusting its agricultural degree as a buffer, and then An example of a suitable organic solvent (glycerin, ethyl acetate, alcohol, dioxan, DMSO, DMF, etc.) in contact with a mass spectrometer plate is exemplified. The use of a mild detergent (eg, octyl glucose, etc.) is also effective. Mass spectrometry is a manual and explanation of a fully automatic 臈 protein-ligand proteome analysis device shown in FIG. 1 of the detection system. Examples of carriers of water-soluble proteins that are consistent with the various purposes of the present invention are considered. It should be diversified in nature, as shown in Figure 3 and Table 1, which exceeds the range shown in Figure 3 and Table 1. Table 1 Types and uses of water-soluble protein (ligand) carriers (please first Read the note on the back 咅 ^ Matters and fill out this page) Order --------- Line 1 Printed by the Intellectual Property Bureau of the Ministry of Economic Affairs Employee Consumer Cooperatives Types of ligands Target carriers Binding methods Ligand receptors Application scope tube Column magnetic particles covalent 〇LC-MS-MS [Overall automatic] Flat magnetic particles 〇HTS (competitive agent screening) Flat (direct) covalent 〇HTS (competitive agent screening) Wafer magnetic particles covalent / non-covalent 〇 〇 Ligand (non-covalent) detection / identification chip (direct) covalent / non-covalent 〇 〇 Non-magnetic particles are covalently detected by SELDI, plasmid genome, fluorescence, RI, etc. Paper sizes from fluorescent, RI, etc. are applicable to China National Standard (CNS) A4 (210 X 297 mm) 36 313287 1240796 A7

V. Description of the invention (37) (Please read the notes on the back before filling out this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. In the example shown in Figure 3, a conductive magnetic metal is used as the electrical X The basic structure of the carrier to be retarded, and the use of magnetic particles bound to a spacer developed for the purpose of improving the antigenicity of antibodies as the surface material of the carrier is used. The magnetic particles are combined with water-soluble proteins separated by two-dimensional electrophoresis with covalent or non-covalent bonds. In the case of using a column, after being divided into 625 regions, the magnetic particles are held in individual 625 microfiber cavities. Membrane protein-embedded microlipids were passed, rinsed, and the eluted fractions were sequentially and separately measured by LC-MS-MS. In different applications, 'using a plate' uses magnetic force to keep magnetic particles in individual 625 compartments after being divided into 625 areas. Add membrane protein-embedded microlipids and competitor to achieve reaction in HTS mode, rinse and detect. The wafer application is an example of a mass spectrometer plate that can be applied to the previous example. In Table 1, one example of the applicable range of the present invention depends on the kind of carrier. The last example shows that the water-soluble protein is covalently bound to the non-magnetic particles (polysaccharides) through the spacer molecule of the present invention. Gels, synthetic polymer gels, etc.), the combination of water-soluble proteins and microlipids embedded in membrane proteins. As for the detection system, it is possible to flexibly use fluorescence, radioactivity, and second signal amplification systems other than mass spectrometry systems according to the purpose of proteomics analysis, thereby providing search for unknown ligands, especially orphan ligands. Optimization system. 5. Device for mass spectrometry The detection of physiologically active substances used in the present invention is not limited to mass spectrometers. Nonetheless, mass spectrometers are considered important detection devices in the present invention because the molecular weight (which is one of the physical quantities of a substance) can be directly measured, the detection limit is close to picograms, and MS- MS method analysis of amine paper size is applicable to Chinese National Standard (CNS) A4 specifications (210 X 297 mm) 37 313287 A7 1240796 ------- -B7__ 5. Description of the invention (~ ^ 一 '&quot;'- The reason for the amino acid sequence. The analysis of the ligand- 臈 protein complex on the mass spectrometer plate of the present invention by the above device C can be performed with any kind of commercially available mass spectrometer. For example, similarly using the above device A is preferably used for analyzing a mass spectrometer in which only a ligand is fixed on a mass spectrometer. In the present invention, when a membrane protein and a water-soluble protein coexist on a mass spectrometer, both or only one of them can be used simultaneously. Highly sensitive measurement by mass analysis, depending on the choice of solvents to be added to the sample. 6. Device for database construction and analysis (device D) The device A, B &amp; C obtained by the above-mentioned device of the present invention will be used. Membrane protein The measurement results of the complexes are entered in the previously set "ligand-receptor (membrane protein) matrix table" (Figure 4) and new data is added at any time to build a database of diagnostic assays. Specify the number of rows ( 1 to 25) and column symbols (A to γ) to cover the entire area of the mass spectrometry plate introduced in the above example, so that the number of one-to-one correspondences (A1 to Y25) is allocated to a total of 625 (25χ 25) areas Each region (4mm x 4 mm) (Figure 2). As a result, the entire ligand transferred to the mass spectrometer plate after electrophoresis can be selected by the number of ligand_receptor (membrane protein) matrix regions. Needless to say, Corresponding receptors obtained by reacting with the receptor-embedded liposomes can be selected with the same number of regions, and it is speculated that the substance groups gathered under a certain number of regions are physically and physiologically combined with each other. In a special example, two-dimensional gel electrophoresis is first applied to the target body fluids of healthy subjects (considered to contain soluble ligands) such as serum and the same target body fluids of patients with specific diseases. After applying to the mass spectrometer plate Measured by mass spectrometry, etc. At this point, due to disease ligands, the Chinese paper standard (CNS) A4 (210 X 297 mm) applies to this paper size ~-313287 (Please read the precautions on the back before filling this page ) II ----- Order ---- I ----. Printed on the clothing of the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. A4 specification ⑵〇χ 297 公 爱 39 1240796 Α7 ------ B7 V. The increase and decrease of invention description (39) become clear and can be added to the database. Then 'target body fluids of healthy subjects, such as Serum and the like are subjected to electrophoresis and then transferred to two gels to prepare two mass spectrometry plates (on which ligands of healthy subjects have been transferred). Separately, the same kinds of cells were taken from the healthy subject and the patient, and then the above-mentioned device B (membrane protein separation and transfer to microliposome) was used to transfer individual membrane proteins to microliposome to prepare a membrane protein package. Buried microfat particles. Use device c (water-soluble protein and peptone protein binding device) to separate microlipid particles embedded in peptone protein derived from healthy subjects and patients with individual mass spectrometer plates on which ligands have been transferred from healthy subjects Ground contact, followed by a ligand-receptor binding reaction, followed by washing, etc., to obtain a ligand-receptor complex highly purified on a mass spectrometer plate. Mass spectrometry can be added to the database as the increase and decrease in disease receptors becomes clear. The items in the database are the name of the disease, the name of the body fluid from which the ligand was obtained, the name of the cell from which the protein was obtained, and the number of molecular, quantitative, or quantitative regions of the ligand-receptor (membrane protein) matrix from the mass spectrometry results. (Or the intensity of the signal output from the analysis device, such as peak height, peak area, etc.). The results of the mass spectrometry analysis are entered into the database according to an automatic program, and the results are represented by a differential display, as shown in Fig. 4, in which the "ligand / receptor (membrane protein) matrix table" In the half, the increase in ligand is shown in = color, the decrease is shown in blue, and the change is not shown in yellow. Similarly, on the left half of the triangle, the increase in receptor is shown in red, and the decrease is shown in blue and not in yellow. * No change. In Figure 4, the pattern is used. The operating time described is about: 4 pieces of electricity at the same time, forever | iffl Chinese National Standard (CNWA4 楣 抆, '二 二 ... -------— 色 夜 ( About 1 2 small 313287 -------- 1 --------- (Please read the precautions on the back before filling this page)

V. Description of the invention (at 40 1240796 hours), the simultaneous production of 2 kinds of membrane sections is about 6 (please read the precautions on the back before filling this page) to the microfat granules about 2 hours, 2 kinds of simultaneous heart 1 sample at the same time After shifting the hours (the total time of the above steps is about 1 to 8 hours [b], hours), analyze the 625 area with a commercially available TOF_mass spectrometer for about 52 hours. ♦ ^ φ. 〇1 When Tian uses a mass spectrometer, the total number of steps required is 21 + 52χ 4 = 229 hours, and each of you + „When Tian uses 4 mass spectrometers, 21 + 52χ 1 = 73 hours. Rate The boat steps are determined to be electrophoresis, membrane segment preparation, and mass spectrometry analysis. When these improvements and automatization are automated, the operation time can be significantly shortened. For example, the 'electrophoresis device can process the sub-samples and membranes once in 5 hours. Preparation of the sections' transfer to liposomes and ligand / receptor binding reactions can be fully automated and integrated into the 奘, 1 、, 1 、, I, I, 芏, 千, 千 device, which can simultaneously process 20 tests in 3 hours In addition, the analysis time of the mass spectrometer can be reduced by five times. By rough calculations, it is estimated that the approximate time required to construct a disease determination database after such a shortening (with 10 sets of the device of the present invention and 100 sets) The mass spectrometer analyzed 100 samples of each of the 100 disease groups by the method described above, using serum and a target cell of the disease as samples. The electrophoresis was 1000x 100 × 3 3 5/99/10 (15 2 ) Hours, the mass spectrometry plate was prepared as 100 ΙΟχχ 2/19/10 (105) hours, and the mass spectrometric analysis was ι〇χχ 100 &gt; &lt; Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 10.4χ 3/100 (3 120) hours. The rate is defined as 31,200 hours (130 days). Therefore, a database for measuring 100 scheduled diseases can be constructed within half a year, and then one day can be provided to diagnose 100 diseases. From the foregoing, this device and this database The impact in the medical field is unlimited. The ligand samples to be used for this purpose can be body fluids such as blood, serum, urine, cerebral bone marrow fluid, semen, saliva, sweat, tears, etc., all fine paper sizes Applicable to China National Standard (CNS) A4 specification (210 X 297 mm) 40 313287 1240796 A7 B7 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 41 5. Description of the invention (Intracellular fluid 'All intracellular organelle fluids etc. The intended membrane protein sample is any cell constituting a living body and all organelles contained in the cell. According to the disease classification table of ICD-10, about 10,000 diseases are shown. When all organisms are When targeted, the number of records in the database constructed using this device is expected to be astronomical images. However, rapid progress in the field of information engineering including computer hardware and software will allow the construction of such a thorough database. The invention also Provides useful tools for the development of therapeutics for ligand- / protein-related membrane-related diseases. Functional analysis of ligand / fluorene-related proteins has occupied important areas of biology (including medicine and agriculture) in the 21st century. Molecular biology will shed light on a larger part of biological functions that will clarify the relevance of certain membrane-associated proteins. Although the above diagnostic databases (the data up to this point are used in diagnostic assays) to clarify the increase and decrease of ligands and the increase and decrease of receptors associated with specific diseases, a further advantage of the present invention is that the The two corresponding molecules of the aptamer and the peptone protein are physiologically meaningful specific ligands and specific membrane proteins. One of them was discovered by accident or for some other ㈣ (eg #, ligand), and then the corresponding 胄 was found using the identified ligand, or 胄 was found by other means-for the ligand and Receptors have discovered new substances in this field. To this point, accidental coincidence and long-term patience are required. There are many ligands and membrane proteins with unknown functions, and receptors that bind to artificial f (medicine) but whose physiological ligands have not been identified. When a number of molecular species (including one coordination) are recognized somewhere in the number of ligand-receptor (membrane protein) matrix regions obtained by entering the measurement results of this method and increasing or decreasing the presence of disease specificity Body and a relative 庑 xi as the threat 々, at least two types of paper scale thorn cap θ family body 313287 (Please read the precautions on the back before filling this page)

1240796

V. Description of the invention (42) Sub-species), all molecules located in this area can be provided as disease-related ligands / peptone proteins, and as highly promising candidates for successful research in drug discovery. For this purpose, a tandem f spectrometer is used. This can be speculated that the amino acid sequence that can be attributed to &amp; Xiao "All of the number of regions". Since this method can analyze any membrane protein of a certain kind of cell, it can analyze the relevant diseases and target cell membrane proteins systematically in a cyclic manner, instead of relying on accidental coincidence to find individual membrane proteins involved in the disease one by one. In addition, as shown in Table 2, when the focus is on membrane proteins of organelle membranes, such as mitochondrial membranes, changes in ATP-based manufacturing capacity can provide drug discovery strategies that have not been imagined until now, such as the classification of diseases, Clarification of relationships between diseases, or development of population-specific therapeutic agents. Table 2 Attractive targets for drug discovery of various membranes (please read the note on the back? Matters before filling out this page) Printed by the Intellectual Property Bureau of the Ministry of Economic Affairs, Consumer Cooperatives, m intracellular signal transduction of extracellular effector molecules of target cell membranes Nuclear membrane DNA replication, RNA transcription / conjugated mitochondrial membrane oxidation-reduction, ATP production of peroxisome membranes, peroxisome metabolism, lysosomal membrane proteins, degradation of nucleic acids, carbohydrates and lipids Basement and membrane transport Endoplasmic reticulum protein / lipid synthesis and membrane transport, MHC X membrane signal transducer molecule response site? 7. Other preferred examples The present invention also provides the ability to observe and analyze insoluble solutions in water-soluble paper. Applicable to China National Standard (CNS) A4 (210 X 297 mm) 42 313287 1240796 Λ7 ------ -—_____ B7_ V. Description of the Invention (43) (Please read the notes on the back before filling this page) The theory and method of measuring the interaction between molecules in the liquid and other soluble or insoluble molecules. Therefore, the scope of the present invention is not limited to the discovery, quantification, and analysis of membrane protein-ligand complexes derived from living organisms, but rather the discovery, quantification, and analysis of artificially produced substances that interact with membrane proteins, and can interact with each other. Discovery, quantification and interaction analysis of two or more membrane proteins, analysis of insoluble substances other than membrane proteins derived from living organisms, analysis of interactions with insoluble or soluble substances, and non-derivation of living organisms Discovery, quantification and analysis of substances (subjects). It is in sharp contrast to conventional methods that require the use of protease, phospholipase, esterase and other previously processed MS analysis of proteins including sugar bonds and lipids as constituents, by changing only the solvent of the sample to be added to the mass spectrometer plate In the present invention, a spectrum of a simple protein completely (or partially, as appropriate) without sugar chains or lipids can be obtained. Example 8 detailed below. The present invention further provides an ultra-highly sensitive method for detecting denatured non-water-soluble proteins such as pathogenic protein infectin protein and the like. The details are described in Example 9 below. The significance and purpose of the invention printed by the Employees' Cooperatives of the Intellectual Property Bureau of the Ministry of Economics is not limited to the completion of a fully automatic proteomic analysis device for proteins and ligands, but rather to help make the critical part of life phenomena negative. Responsible but technical contributions to the prosperous development of membrane protein science that have left research far behind due to methodological and technical obstacles. As described above, the present invention provides a theory, a method, and a result of dividing a protein body into a membrane protein and a water-soluble protein, and then embedding the membrane protein in the microlipids, which can be performed in a similar manner to the treatment of water-soluble proteins. Process membrane proteins. This paper size applies to China National Standard (CNS) A4 specifications (210 X 297 public love) -------- 43 313287 1240796 A7 B7 44 V. Description of the invention ((Please read the note on the back? Matters before filling in (This page) — This Rina and method combined with the new trend of drug discovery explored by the methodology of genomic drug discovery 'will be recognized in the near future as being closely related to receptor-related drug discovery, antibody-related drug discovery, hts-related Pleasure Discovery 'analysis of membrane protein drugs based on techniques of protein three-dimensional structure analysis, gene expression analysis, other proteosome analysis, etc. The present invention is described in detail with reference to the examples below, but the examples are only examples and are not limited to the present invention. [Examples] The following shows specific examples and results to establish the method and device of the present invention that can effectively screen the interactions of both linked proteins and their ligands and analysis, and is novel for proteomic analysis Methods and devices: The Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs printed structure-based classification of receptors into three types: GPI fixed type, gpcr type Oligomeric type. In order to demonstrate that the present invention can be used for proteomic analysis of membrane proteins, the interactions of specific ligands of plaques should be analyzed with all these types of receptors as targets. Still 37 cells are derived from human monocytes Cell line, and many receptors are displayed on its surface. Among them, the urokinase receptor was selected as the GPIgI type, the ▲ clear complementary MC5a receptor was selected as the GPCR $ ', and the interferon receptor was selected as the recruit type. Example 1: Confirmation of the performance of the three receptors Before and after Bt2CAMP stimulation, U937 cells were reacted with three ligands pre-labeled with FITC and analyzed by FacSt to observe the presence of receptor performance. As a result, all The performance of the three receptors is shown in Figure 5. Example 1: Preparation of urokinase receptors-embedded microlipids —— ^ --- | / n

It ί 旦 p 田 由 阂 关 这 作 隹 λ / 1 la 44 / om ^ οπ ^ / \ ------- 44 313287 1240796 A7 B7 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs (45 (1) Preparation of membrane segment U937 is a cell line derived from human monocytes and is expressed at a high concentration by expression of urokinase receptors by phorbolester (PMA) stimulation. It was used as a sample of the separation membrane section. After rinsing, the cells were ruptured with pOlytrón at 1 minute intervals under ice cooling. After 2 to 5 seconds X 3 times, the membrane section was 40% sucrose density. Gradient centrifugation (95,000 gx for 60 minutes) accumulates on the interface (Figure 6). (2) Preparation of protein-embedded microlipids Purified egg yolk lecithin (1.25 g) and cholesterol (0 · ι25g) suspended in 25 mL physiological saline, and then treated with a probe-type ultrasonic crushing device for 15 minutes under cooling. The obtained lipid particles have an average particle diameter of 80 nm. The U93 7 film prepared in advance was divided into Add this portion to this microlipid solution, and then freeze and thaw three times at -80 ° C and room temperature. The mixture becomes cloudy When the solution containing the individual liposomes was treated in the same way, the liposome solution became cloudy. Conversely, the U937 membrane section remained translucent even after repeated freezing and thawing. Gasifiers were added to these samples The final concentration was adjusted to 40%, and a solution having a concentration of 30% and 15% of cesium vaporized and physiological saline was layered on this solution in this order and then subjected to density gradient centrifugation (95,000 g X lh). As a result, the U937 membrane segment formed a band at the interface between the 30% and 15% vaporized cesium concentration, and the turbid microlipids formed a band at the interface between the 15% cesium vaporized concentration and physiological saline. In the mixture with the U-937 membrane segment, a band was observed at a position similar to the turbid microlipids and no band was observed at the position of the U-937 membrane segment. From this result, it is assumed that the U-937 membrane segment has been Fused with microlipids to form membrane-embedded microlipids (Figure 7). This paper size applies the Chinese National Standard (CNS) A4 (210 X 297 mm) 45 313287 — — — — — — — — — — — ------- ^ · III I ---- (Please read the notes on the back before filling (This page) 1240796 A7 ___B7 V. Description of the invention (46) (3) Confirmation of the microlipids embedded in the membrane protein (please read the precautions on the back before filling this page) After separating and preparing the membrane segment, The protein is fluorescently labeled (FITC). By the method (2) above, this membrane segment is reacted with the microlipids to form the membrane protein-embedded microlipids. The membrane segments are analyzed by FACS, and the membrane proteins are embedded Samples of microfat particles and simple microfat particles. As a result, as shown in Figure 8, the highest fluorescence intensity (y_axis) of each particle is the membrane segment, and the lowest is the simple microlipids (no protein is present, weak scattered light is detected instead of FITC fluorescence) ) And the microlipids embedded in the membrane protein are located in between. Because the membrane protein-embedded liposome sample contains almost no weak scattered light from the particles detected by the simple liposome, it is assumed that the membrane segment and the simple liposome fraction are almost fused at 1000/0. That is, the membrane segment is fused with simple liposomes to form multilayered liposomes, and the membrane proteins are also transferred to the surface of the internal liposomes in the multilayered liposomes. Therefore, the number of membrane proteins on the outer membrane surface is reduced, and the fluorescence intensity of the microlipids embedded in the membrane proteins is thereby reduced. From the foregoing, it was shown that the membrane protein is bound to the cell and the cell membrane fraction thus prepared is transferred to the lipid bilayer of the microliposome by this preparation method. (4) Identification of urokinase receptors (U937 cells) Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. U937 cells cultured in the presence or absence of PMA were immunoprecipitated with goat anti-human urokinase receptor antibodies. After electrophoresis, protein hysteresis was performed to confirm the presence of urokinase receptors on U93 7 cell membrane. The first reaction of protein retardation is the response of biotinylated goat anti-human urokinase receptor antibody at 250 times dilution over 2h and the second reaction is streptococcus labeled with alkaline phosphatase at 100 times dilution Avidin response. After the reaction, rinse 3 times with PBS / 0.1% Tween20 and apply the Chinese National Standard (CNS) A4 specification (210 X 297 mm) at this paper scale. -------— B7 —_ 5. Description of the invention (P) B IP / NBT to detect. As a result, a broad band with a molecular weight of 50 kDa stained with an anti-human urokinase receptor antibody was observed, as shown in FIG. 9. Thus, the presence of urokinase receptors with different sugar chain structures was found on the surface of U937 cells. (5) Confirmation of microlipids embedded in urokinase receptor The microlipids embedded in the membrane protein obtained by the method (3) above and the goat anti-human urokinase receptor antibody as a primary antibody in 4 Reaction at ° C for lh 'and then at 4 ° C for 1h with FITC-labeled rabbit anti-goat IgG antibody. The primary and secondary antibody systems were reacted at a 10-fold dilution. After the reaction, four rinses with 0.01% BSA_pBS (containing a protease inhibitor) were performed. It was then observed with a 320-power confocal scanning laser microscope (LSM410, Carl Zeiss) (Figure 10). Fluorescence was observed in fusion lipolipids with anti-urokinase receptor-specific antibodies (Fig. 10A), but in fusion lipolipids without anti-urokinase receptor-specific antibodies (Fig. 10B), No fluorescence was observed. From the foregoing, transfer of the urokinase receptor to the lipid bilayer of microliposome is shown. In the same way, the obtained membrane protein-embedded microlipids were dissolved, immunoprecipitated with goat anti-human urokinase receptor antibody, and then protein retardation was performed. The first reaction of protein retardation is the reaction with biotinylated goat anti-human urokinase receptor antibody at 250-fold dilution over 2h, and the second reaction with 100-fold dilution at 1h with alkaline phosphatase labeling Streptococcus Avidin Response After the reaction, the samples were washed 3 times with PBS / 0.1% Tween20 and detected by BCIP / NBT. As a result, a broad band with a molecular weight of 50 kDa stained with an anti-human urokinase receptor antibody was observed, as shown in FIG. 11. The results show that the original paper size of U937 cells is in accordance with Chinese National Standard (CNS) A4 specifications (210 X 297 public meals Γ 313287 -------------------- Order --- ------ Line (Please read the precautions on the back before filling this page) 1240796

V. Description of the invention (you) The molecular weight distribution is the same as confirmed before. This shows that the transfer to membrane proteins (please read the precautions on the back before filling this page) The urokinase receptor of the lipid bilayer embedded in the microlipids maintains the sugar chain structure existing in natural cells. (6) Ligand binding capacity of urokinase receptors Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. Since the tritium segment prepared by U937 cells showed that urokinase carcass were maintained in the lipid bilayer, the study of The body's urokinase (ligand) binding capacity. FITC applies a fluorescent label to urokinase and reacts with the membrane segment to examine the binding of the receptor to the ligand. Human gold albumin (HSA), fluorescently labeled with Fitc, was used as a control group. The results are shown in Figure u. From the figure, it can be seen that the membrane segment prepared by this method binds to the endogenous ligand urokinase (Figure 12A), while others bind to hsa (Figure 12B). This means that the urokinase receptor retained in the membrane fraction obtained by this membrane protein preparation method maintains a three-dimensional structure and a physiological function (ligand binding ability). Secondly, the urokinase binding capacity of the microlipids embedded in the membrane protein was examined. FITC was used to apply a fluorescent label to urokinase, and it reacted with the membrane protein-embedded microlipids (including urokinase receptor-embedded microlipids) obtained by the previous method to examine the receptors and ligands. Combined. Human serum protein (HSA), which was fluorescently labeled by FITC, was used as a control group. The results are shown in Figure 13. It can be clearly seen from the figure that the microlipids embedded in the urokinase receptor prepared by this method bind to the endogenous ligand urokinase (Figure 13A) and others bind to HSA (Figure 13B) . In order to confirm the binding specificity between the urokinase receptor-embedded microlipids and urokinase, a mixture of RI-labeled urokinase and non-labeled urokinase was prepared at various Mohr ratios of 1: i to 1: 10000, U937 cells and urokinase receptor embedded in this paper are applicable to Chinese National Standard (CNS) A4 specifications (210 X 297 mm) 48 313287 1240796 A7 ________ _________ B7 V. Microlipid response of the invention description (49), Radioactivity bound to cells and microlipids embedded in urokinase receptors was measured. As shown in Figure 14, as non-labeled ligands are increased, both show reduced radioactivity bound to the receptor. Therefore, it is assumed that the binding of urokinase receptors and urokinase embedded in the liposomes is specific. In addition, changes in the expression level of urokinase receptors stimulated by PMA were studied based on the binding amount of urokinase to the membrane-embedded microlipids. As shown in Figure 15, compared to those prepared from untreated cells, the lipid-embedded microlipids prepared from PMA-stimulated U937 cells clearly showed binding to fluorescently labeled urokinase The number of microlipid particles increased. The above results show that the structure and function of the membrane proteins embedded in the microlipids are the same as those of the membrane proteins expressed on the membrane of living cells. Therefore, it is shown that the method of the present invention can replace living cells to evaluate changes in the amount and properties of receptors and ligands during illness. Example 2: Appearance of Receptor-Embedded Microlipids After Reducing the Particle Size The extruder method was used to change the particle size of the membrane-protein-embedded microlipids and then examined by fluorescent (FITC) -labeled urokinase Appearance of microlipids embedded in urokinase receptors. As a result, the number of microlipid particles embedded with the target receptor was significantly increased in the microlipid solution passing through a filter having a filter hole size of not more than 0.6 µm, as shown in FIG. It is presumed that this is caused by the fact that most of the target receptors are entrapped in the large microlipids in the multilayer microlipids immediately after fusion, and the fluorescence cannot be detected. More importantly, the size of the microlipids becomes larger. "Small" means that the number of receptors embedded in a microlipid is smaller. Therefore, it is derived between the microlipids with the target receptor embedded and the microlipids without the target receptor. (CNS) A4 specification (210 x 297 mm) 313287 (Please read the precautions on the back before filling out this page) Order --------- Employee Consumption Cooperation of Intellectual Property Bureau of the Ministry of Economic Affairs

1240796 V. Description of the invention (50) (Please read the precautions on the back before filling this page) The strict difference, and the number of embedded microfat particles increased. When the size of the liposomes was set to 10 nm to 5,000 nm, preferably not more than 500 nm by an extruder method or other methods, the identity of the labeled lipid-embedded lipoproteins was found by FACS analysis. Groups clearly appear in the corresponding size areas. Thus, for example, when cdNA is prepared from the gene sequence of a membrane protein and a host cell is transformed with a expression vector containing the cDNA sequence for membrane protein expression (incorporating fluorescent molecules and other identification molecules recognizable by FACS), and When the non-labeled microlipids with embedded labeled membrane proteins prepared from the cell membrane fraction of the expressing host according to the method of the present invention were adjusted to the corresponding size and analyzed by FACS, it was clear that membrane proteins could be detected The existence or performance and the amount of performance. In this case, unlike the currently available methods, 'Information other than DNA sequences containing membraneless proteins, printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs and without reagents (antibodies, ligands, competitors, cellular responses) Reagents and other detection reagents for membrane proteins). In addition, if there are detection reagents for membrane proteins, such as antibodies, identification at the protein stage becomes possible, which can be expressed by FACS analysis. However, this method can be applied to search for ligands based on similar principles. Example 3: Sliding urokinase onto a mass spectrometer plate The paper size is in accordance with Chinese National Standard (CNS) A4 (210 X 297 public copy) Urokinase (10, 13, 16, 20, 33pg) ) Loaded in the same well of a polyacrylamide gel and electrophoresed. After the movement is completed, the gel is peeled off from the glass plate, and the gel buffer solution is replaced with a sludge buffer solution (5% acetonitrile / 125mM Nacl / PBS) for 5 minutes. Then a thermal sludge (diffusion sludge) is applied to the surface. 16 carbon hydrophobic material plate. 313287 50 1240796 ------- ~ --- 5. Description of the invention (51) A phosphate buffer containing 5% acetonitrile and 125 mM Nacl was used as a slugging buffer, and slugging was performed at 35 ° C for 4 h. Fig. 17 is a photograph showing a spectrum plate of the retardation and gels before and after the retardation stained with Coomassie brilliant blue. The urokinase band on the mass spectrometer plate cannot be seen with the naked eye, but because the urokinase band on the gel after the transition is blurred, it is speculated that a certain amount of urokinase has been transferred to the mass spectrometer plate. Example 4: Identification of urokinase on a mass spectrometer plate by mass spectrometry analysis After polyacrylamide gel electrophoresis, direct measurement of urokinase transferred to a mass spectrometer plate by thermal retardation (diffusion retardation) by a mass spectrometer, results Shown in Figure 18. As for the conditions of mass spectrometry, erucic acid (sinapinic-like sentence (a saturated solution dissolved in 50% acetonitrile / 0.5% trifluoroacetic acid) is used as an energy absorbing substance ', which is added at 0 · 5 // 1 / point and then applied. Air-dried, then added the same amount and air-dried. Measured with the SELDI Proteinchip® system from Ciphergen Biosystems. The quality is corrected using cold-lactoglobulin A (bovine), itchy peroxidase and convalescent protein (chicken ). The measurement parameters of the SELDI Proteinchip® system are: electrical waste of the detector 2,200V 'detector sensitivity 10, laser intensity 285. As a result, a peak was observed at a molecular weight of 49461.2. Because it is used for inspection Urokinase is a precursor and the theoretical molecular weight predicted from the amino acid sequence is 48,525, so it is recommended to add a sugar chain molecule. Example 5: Transfer rate of urokinase to a mass spectrometer plate Quantitatively transferred to the mass spectrometer plate by mass spectrometry analysis Kinase. Using standard urokinase 25, 12.5, 6.25ng, draw a standard curve of peak height versus concentration obtained by mass spectrometry. Then, urokinase ( 2,500ng) Loading on this paper scale Applies to Chinese National Standard (CNS) A4 specification (210 x 297 public copy) 313287 —--------- 1— c Please read the note on the back first? (This page) Xin — — — — — — — .sm Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 51 1240796 A7 η -------- Β7 S, Description of Invention (52) on Polyacrylamide Gel Electrophoresis was performed. Urokinase transferred to the mass spectrometer plate with thermal hysteresis (diffusion hysteresis) was directly measured by a mass spectrometer. As a result, a peak height corresponding to 25 ng was detected, as shown in FIG. 19. From this result, the transfer rate of the gel from the gel to the mass spectrometer plate by thermal hysteresis was about 1%.

Example 6: Mass spectrometric analysis of bacteriorhodopsin_embedded microlipids on a mass spectrometer plate H The results obtained by measuring bacteriorhodopsin (Sigma, B3636) using a mass spectrometer are shown in FIG. 20. The sample was placed on the wafer and air-dried. After adding 2,5 · dihydroxybenzoic acid (i70mg / mL in ethanol) at 0.5 / z L / point, the sample was air-dried and then SELDI Proteinchip® system ( Ciphergen). The quality correction was performed using a protein other than lactoglobulin A (bovine), itchy peroxidase, and albumin (chicken). The measurement parameters of the SELDI Proteinchip (R) system are a detector voltage of 2100v and a detector sensitivity of 10 'and a laser intensity of 285. As a result, two peaks at 27027.3 and 28120.2 were observed for each example. From the theoretical molecular weight of bacteriorhodopsin 27068 0, the former is bacteriorhodopsin and the latter is presumed to be a bacteriorhodopsin-like protein, because it shows the elimination of retinal when treated with an organic solvent . Figure 20A shows the relationship between the protein concentration and the peak intensity when measuring rhodopsin alone, and from the proportional relationship, it can be seen that the debt measurement limit is 20 fmol. Figure 20B shows the measurement results of bacteriorhodopsin alone and bacteriorhodopsin under the condition that simple microlipids and bacteriorhodopsin embedded in microlipids coexist. Under the same solvent conditions, at the same concentration, these three Thai paper sizes are applicable to China National Standard (CNS) A4 specifications (210 X 297 mm) 313287 (Please read the precautions on the back before filling this page) _---- --- Order --------- line. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economy 52 1240796

This mode shows different MS signal intensities of bacteriorhodopsin, which indicates that mass spectroscopic analysis of bacteriorhodopsin can be performed even in the presence of simple microlipids and bacteriorhodopsin embedded in microlipids. V. Description of the invention (53) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 53 Example 7: Mass spectrometry analysis of urokinase receptors on mass spectrometer plates Protein G is linked with covalent bonds on the mass spectrometer plates, and Urokinase receptor antibodies bind non-valently to prepare mass spectrometry plates. The purified product of the urokinase receptor shown in Figure 21 (Figure 21A) was applied to this plate and mass spectrometry was performed by conventional methods (Figure 21B). As a result, a peak of the urokinase receptor was found at the molecular weight position of 49978 ″ (above FIG. 21C), and an anti-urokinase receptor IgG antibody regarded as a ligand of the urokinase receptor was also found in the region of the theoretical molecular weight. . When a non-specific IgG was used as a control group, no mass peak of urokinase receptor was observed (below Figure 21C). Mass spectrometry was then performed on the U937 cell membrane fraction. As a result, a peak of the urokinase receptor was found at a similar position. It is assumed that the urokinase receptor from the U937 cell membrane segment can also specifically bind to the concept of anti-urokinase receptor IgG. In addition, protein G was covalently bound to a mass spectrometry plate to bind an anti-urokinase receptor IgG (goat) with the protein G / antibody complex as a spacer on the plate. After the purified product of urokinase was applied to a plate, a protein liposome solution prepared from U937 cells by a conventional method was subjected to overnight reaction at 4 ° C on the plate. Mass spectrometry was performed after removing liposomes using a buffer containing octyl glucoside. As a result, a peak composed of both the urokinase receptor and urokinase was found around the area belonging to 50,000 Da (top of Fig. 25). Although the wave peaks were found in the area where simple lipoproteins without protein were used instead of protein liposomes (Figure 25, this paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 public love) 313287 ---- ---------------- Order --------- Line (Please read the precautions on the back before filling this page) 1240796

V. Description of the invention (between M), but the peak intensity is obviously reduced. That is, the difference between the two peaks is due to the presence or absence of urokinase · ㈣, which is buried in the protein microlipids (please read the precautions on the back before filling this page). The inflammatory kinase can be combined with Combine the fact of urokinase on a plate. In the case of the inability to combine urinary excitement: At At ^, dumb cow 1gG instead of anti-urokinase IgG, two peaks attributable to serotonin and urokinase were not observed (bottom of Figure 25) . Special attention should be paid here to the fact that the use of a ligand (urokinase) to detect and identify the urokinase receptor embedded in the lipid bilayer by mass spectrometry proves the basic principle of the present invention. Example 8: Remove the sugar component from the mass spectrometer plate Printed on the mass spectrometer plate to capture the membrane protein (urokinase receptor, bovine rhodopsin) and soluble protein (urine with sugar chains) Kinase, bryophyta peroxidase ", as well as glycoprotein-free rhodopsin (bacteriorhodopsin) and soluble protein (cold _ ritocin 'cytochrome c), air-dried, and added to the solution at 0/5 to dissolve in Saturated erucic acid (spA), saturated 2,5-dilight benzoic acid (DHB) or indole acrylic acid (IAA) in 50% acetonitrile_0.5% trifluoroacetic acid and air-dried. Use SELDIPrOteinchip ® system (Ciphergen) for measurement. Mass correction is performed using ubiquitin (bovine), myoglobin (female), and serum albumin (bovine). Results are shown in Table 3 and Figure 22. Thai paper scales are applicable China National Standard (CNS) A4 (210 x 297 mm) 54 313287

Glycan chain comparison MW about 3,000 Da no ODa about 3,000 Da exist about 1,000 Da exist about 1,400 Da no about 45 Da no about 45 Da 1240796 V. Description of the invention (55, Table 3 Sugar chain protein

臈 protein UkrT rhodopsin

Bovine rhodopsin soluble protein ProUK

HRP-lactoglobulin, cytochrome c Regardless of membrane protein or soluble protein, the molecular weight obtained using SPA or IAA is similar to the theoretical value expected from the amino acid sequence, and those obtained using DHB are regarded as protein. Depending on the presence of sugar bonds. For example, cytochrome c, a protein without sugar chains, shows almost the same molecular weight in both cases (similar to i2 25i and 12,297 in DHB), while protein urokinase receptors with sugar chains use DHB and spA (spA 47 466 and 50,465 of DHB) showed a difference of about 3,000 Da. The molecular weight of other proteins with sugar chains obtained using dhb is also higher than those of those obtained using SPA or IAA by up to 300 Da. For mass spectrometry analysis of proteins containing sugar chains or lipids as components, the method of the present invention can obtain the spectrum of simple proteins, and all can be removed only by changing the composition of the solvent of the sample to be added to the mass spectrometer plate ( Or part, depending on the conditions) sugar chains or lipids, in sharp contrast to conventional methods that require pretreatment with proteases, phosphatase, esterases, etc. The present invention can be applied to any protein, regardless of its mesangial protein or water-soluble protein, and the solvent may contain a catalyst containing protein in its composition. ^ Chinese National Standard (CNS) A4 Specification— (210-χ 297) ^ 7) 1 &quot; β-、 313287 -------------------- Order ---------- Line Qin (Please read the back Note? Matters need to be filled out on this page} Printed by the Intellectual Property Bureau of the Ministry of Economic Affairs, Consumer Affairs Group 55 1240796

Any other substance is also a substance of enzyme, among which the concentration, order of addition, etc. and (please read the precautions on the back before filling this page). There are no restrictions on the types, etc. "Detecting denatured proteins by mass spectrometry ^ Μ '# ㈣fenziric acid (SPA) as a solvent to measure water can == such as cytochrome ° 4 lactoglobulin, vegetable peroxidase, urine ^ When waiting, normal MASS signal strength appears. However, when red benzoic acid (DHB) was used as the rhenium solvent, the MASS peak became extremely weak (right side of Figure 23). For membrane proteins (bacteriorhodopsin, urokinase receptors), this solvent effect is reversed (many _ j, t _ (Figure 23 on the left). Therefore, only the membrane protein group or Sensitive detection of the water-soluble protein population only becomes sensitive: The important aspect of the present invention is to provide-"when the protein is soluble under natural physiological conditions for some reason (release of its own pupal fragment, gene mutation , Transfer to each other with a heterostructure molecule with the same sequence in a more thermodynamically stable structure, etc.) Provides a method for selectively detecting water-soluble and non-water-soluble proteins when denatured to become water-insoluble proteins. This It cannot be achieved by any existing technology. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. Therefore, the present invention provides more than sensitive detection of pathogenic protein proteins (whether human, sheep, cattle, enzymes and other biological species). And quantification. This measuring principle is not only applicable to other neurodegenerative diseases, such as Alzheimer's disease caused by denatured (insoluble) powdery protein-protein (Ay5) Familial amylolytic polyneuropathy caused by denatured (insoluble) thyroxine-binding preprotein (transthyretin) can also be used to detect caused by denatured (insoluble) protein, animal quarantine, meat inspection, etc. This disease can be widely used in the selection of denaturing conditions (insoluble) of soluble molecules and the size of this paper is applicable to China National Standard (CNS) A4 (210 X 297 mm) &quot; 56 313287 1240796 Λ7 B7 5 Explanation of the invention (57) Method for measuring physiological conditions (soluble), such as measuring the renaturation of protein in a test tube, etc. Example 10: Between a ligand bound to a carrier and a microlipid embedded in a membrane protein Combine the use of liposomes, biofilms (membrane segments) or biofilms (cells) as a carrier for membrane proteins by a combination as shown in Table 4, mass spectrometry plates, Sepharose 4B gel or magnetic iron particles as ligand carriers And protein G or biotin as a spacer to form a membrane protein-ligand complex. Table 4 Between the lipid particles embedded in the membrane protein and the ligand bound to the carrier Bonding ligand receptor (Read precautions to fill out the back of the page) Name Name spacer support vector detection method UK, IF, C5a flat plate S4B gel None

UK PoAb PoAb UK S4B Gel UK, IF, C5a S4B Gel

UKR, IFR, C5aR PoAb / Protein G UKR PoAb / Protein G UKR No UKR No UKR No Biotin Microlipid Fluorescence Microlipid Mass Spectrometry No Mass Spectrometry No Protein Sluggish Microlipid Fluorescence Microlipid Fluorescence Employees ’Intellectual Property Bureau Employees’ Consumption Printed by a cooperative

UK Magnetic Fe particles Biotin SSIFR ’Microlipids Glowing UKR Biofilm fluorescence As a result, it was found that it is better to have spacer molecules between the carrier and the ligand. Depending on the type of ligand, when the spacer is absent, the membrane protein-embedded microlipids are always unable to bind or the binding force is weak. This is because multi-point binding of the affinity effect of the antibody's avidity effect is required rather than one-point binding of the affinity effect. This is because both molecules are immobilized on different solid surfaces. In order to provide multi-point binding, the interference of suitable spacer molecules is required. This paper size applies the Chinese National Standard (CNS) A4 specification (210 x 297 mm) 57 313287 1240796 A7 ------______ B7 V. Description of the invention (58) It weakens steric obstacles and increases the frequency of collisions during the association of two molecules. It is composed of biopolymers (proteins, etc.), synthetic polymers, metals, etc. (Please read the precautions on the back before filling this page) The spacer molecule existing between the ligand and the carrier can be covalently bonded or non-covalently Valence bonds are combined (depending on the purpose of the analysis), and can be micro-mesh, porous, etc. with three-dimensional space. Example 11: Binding between ligands immobilized on agarose 4B gel and microcapsules embedded in receptors. The Ministry of Economic Affairs and Intellectual Property Bureau employee consumer cooperatives printed three kinds of ligands for biotinylation (urokinase , Interferon-7, complementary C5a) binds to avidinized agarose 4B gel. In this example, the ligand carrier was apolipo 4B gel and the spacer was avidin (protein). FITC-labeled fluorescent microlipids and U937 membrane fractions were used to prepare membrane protein-embedded microlipids according to the method described in Example 1 (2), and reacted with three ligands immobilized on agarose 4E gel. Rinse three times with pBS, and then observe with light and fluorescence. As a result, as shown in Fig. 24, all gels were observed to be white under visible light (B). Under fluorescence (A) in the dark domain, no color spreading was observed in the Sepharose 4B gel alone, but fluorescence was emitted using other gels that combined three ligands. This result shows that the principle of the present invention can be applied to any ligand carrier / any detection system in addition to the aforementioned mass spectrometry / mass spectrometry, such as particles / fluorescence, particles / radioactivity, particles / second signal generator, etc. Of combination. In the above embodiments, the urokinase receptor (GPI-immobilized type), activated complementary C5a receptor (GPCR type) and interferon-r receptor (oligomeric type) and their ligands (urokinase, C5a, IFNr) Detection results of interactions (functions) show that the present invention can be applied to any kind of receptors. From these results, it is well known that this paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 58 313287 1240796 A7 B7 V. Description of the invention (59) Those skilled in the art can easily understand the principles and Methods: Regardless of molecular structure and function, ligands can be used to isolate and identify any membrane-related stylistics, membrane-related channels, gadolinium-related pumps, membrane-related transporters, and substances bound to these proteins. . Although it shows the basic explanation and inspection of the individual components of a full-automatic protein body analysis device that uses mass spectrometry as a measurement method for membrane proteins and ligands ... but those skilled in the art can easily understand that the present invention can be applied to a wide range of configurations The uses are as shown in the examples using fluorescently labeled microlipids and ligand particles conjugated with fluorescent analysis. Although the present invention has been described with reference to the preferred embodiments, it will be apparent to those skilled in the art that variations from the preferred embodiments can be used and the invention is intended to be practiced other than as explicitly described herein. Accordingly, the present invention includes all modifications encompassing the spirit and scope of the present invention as defined by the scope of the following patent applications. Printed by the Intellectual Property Bureau Employee Consumer Cooperative of the Ministry of Economic Affairs, this application is based on the provisional patent applications No. 60 / 260,433 and 2000iF filed in the United States on September 2, 2001, No. 60 / 272,981 , The contents of which are incorporated herein by reference. All references cited herein, including patents, patent applications, and literature, are incorporated herein by reference in their entirety. Month ’This paper is a Chinese National Standard (CNS) A4 specification ⑵〇 χ 297 Meal 59 (Please read the notes on the back before filling this page} 313287

Claims (1)

No. 90 1 33 1 55 Patent Application Amendment Scope 2 (March 25, 1994) Preparation methods of protein preparations, including analysis of membrane proteins based on bioaffinity and compounds capable of interacting with their parents For both, this method includes: (1) applying gel electrophoresis to the analyte containing the compound, and then transferring the analyte containing the compound from the gel to the carrier and fixing the analyte thereon while maintaining its natural function; ) Prepare a lipid bilayer by isolating the membrane segment with membrane protein from the biological sample or fusing the membrane knife part with the microlipids; (3) Make the membrane embedded in the lipid bilayer according to (2) The protein is contacted with the analyte containing the compound on the carrier according to (1) to use the lipid shell to capture the membrane protein capable of interacting with the compound; and, (4) by being able to obtain the basis (3) Both the membrane protein captured by the lipid bilayer and the at least one physical or chemical information of either or both of the compounds immobilized on the carrier according to (1) are analyzed. 2. The analysis method according to item i in the scope of patent application, wherein the method is selected from the group consisting of mass spectrometry, fluorescence method, RI method and surface plasmid genomic resonance method. 3. The analysis method according to item 丨 of the patent application range, wherein the carrier is selected from the group consisting of a flat plate, non-magnetic particles and magnetic particles. 4. A carrier, which is used in the analysis method of patent application No. 丨, is used to fix proteins that can interact with membrane proteins after gel electrophoresis 313287 (revised) 124〇796 'This carrier is in the basin Acoustically covalently bind the compound while having a spacer to covalently or non-5 · -type protein body analysis device: two natural functions of the compound. This includes the following devices: (a) Transfer of compounds containing electrophoretic gels capable of interacting with the membrane protein enthusiasm from the electrophoretic gel to a device for holding the natural functions of the compounds; (b) A device for preparing a lipid bilayer for embedding membrane proteins temporarily and Shi Jiubei; (c) The essence can be trapped by contacting the membrane protein with the membrane protein, and can be captured and fixed on the carrier m. Device that interacts with a membrane protein embedded in a lipid bilayer; (d) obtains at least one physical or chemical information of the membrane protein and the dagger or both installation. 6. If the protein in the scope of the patent application No. 5 _ to obtain at least one kind of physical or chemical ^ of the membrane protein or the compound, the device obtained is selected from the quality green species Yu Bei Λ, Tian Bei. The group consisting of the method of art, fluorescence method, RI method and group resonance method. &Quot; Tu Ru declared that the protein body system of item 5 of the patent patent was selected from the one contained in the Hua Zhufei # ^ π clothing set. Group consisting of 1000 plates, non-magnetic particles, and magnetic particles 8. The analytical method of item 2 is in contact with the mass spectrometer plate, which is used in the scope of the patent application, and includes the use of coagulation after electrophoresis. Protein should be fixed on the gel tray. A method for determining the quantity or properties and interacting with the maxillary non-disease-specifically altered membrane protein compounds, the method includes (revised) 313287 2 9 · 1240796? The collected samples were subjected to the method of protein # 8, local specialty, and range-by-month analysis and knife analysis of application No. 1, and then compared the data of the healthy same species. According to a method for detecting a disease of a target organism, the method includes the following steps: Applying a patent claim to a sample collected from an organism suffering from a disease! The proteomic analysis method of the item ^ specifically analyzes the data and the data of healthy homologous organisms, and then determines the amount of membrane proteins that are not disease-specific changes and can be combined with: compounds; external use 2 apply at least Γ diseases Step 're-assemble the obtained data to construct a database; and 施加 apply the data collected in the step of the sample collected from the self-diagnostic target organism and collect the data collected in the step ^ ^ ^ ^ ^ 'The data from the skeletal library of the corpse for diagnosis of the dead body were used to determine the target disease. &quot; • -A system and system for measuring the disease of target organisms, including at least the following devices: ⑷ Transferring an analyte containing a compound capable of interacting with a membrane protein from an electrophoresis gel to a carrier for fixing the compound Device that maintains the natural function of the compound; (b) Device that prepares a lipid bilayer for embedding membrane proteins; (C) Captures the embedded lipid by contacting the membrane protein with the compound immobilized on a carrier Device for a membrane protein in a double layer; (d) Device for obtaining at least one physical or chemical information of the membrane protein and at least one of either or both of the compounds; (e) Database of the scope of patent application . (Revised version) 313287 3! 24〇796 12. A method for measuring the disease of the target organism, including the method for proteomic analysis of the organism suffering from a certain disease: Item 丨 of the scope of interest, and then apply for special, according to Re-examination of the data of the same kind of organisms as health: Second :: Fractionates: Membrane proteins that are specifically altered or can interact with them (2) Apply step data to at least one disease to build a database; (3) Passed from the collection (3) Apply a step (!) to the sample collected from the self-diagnostic target organism, and then compare the obtained "data with the data collected in the diagnostic ^ tribunal constructed according to step ，, The target disease is thereby determined. (Amended) 313287