WO2002060377A2 - The separation, identification and quantitation of protein mixtures - Google Patents
The separation, identification and quantitation of protein mixtures Download PDFInfo
- Publication number
- WO2002060377A2 WO2002060377A2 PCT/US2001/047754 US0147754W WO02060377A2 WO 2002060377 A2 WO2002060377 A2 WO 2002060377A2 US 0147754 W US0147754 W US 0147754W WO 02060377 A2 WO02060377 A2 WO 02060377A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- proteins
- protein
- slinker
- slinkers
- magnetic
- Prior art date
Links
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J19/0093—Microreactors, e.g. miniaturised or microfabricated reactors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00781—Aspects relating to microreactors
- B01J2219/00851—Additional features
- B01J2219/00858—Aspects relating to the size of the reactor
- B01J2219/00864—Channel sizes in the nanometer range, e.g. nanoreactors
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00781—Aspects relating to microreactors
- B01J2219/0095—Control aspects
- B01J2219/00952—Sensing operations
Definitions
- the invention generally relates to the rapid separation, identification, classification, and quantitation of proteins in a mixture of proteins.
- the invention provides a method of repeatedly using "separation linkers" to effect the separation, identification, classification, and quantitation of proteins in order to characterize patterns of protein synthesis patterns.
- the invention further provides general-motif antibodies for use as separation linkers in the methods of the present invention.
- the invention further provides a means of determining protein concentrations in a sample by measuring their differential migration in a magnetic field of magnetic beads containing bound protein.
- Healthy tissues typically differ from diseased tissues in their levels of protein expression. Development of a disease state may be characterized by:
- interactions among proteins may be initiated or enhanced, e.g. a subset of proteins may become activated and, as a result, trigger or mediate signaling pathways which involve new protein interactions.
- Levels of calcium and/or the intracellular pH also may change upon viral infection, resulting in new interactions among proteins or the modulation of existing interactions.
- diagnostic procedures for detecting and identifying disease states usually rely on the detection of a single or only a few protein markers. This approach is limited in that it does not take into account the fact that most disease states are characterized by a variety of alterations in several proteins.
- One result of this oversimplified detection methodology is the frequent occurrence of undesirable "false positives" and "false negatives” due to the utilization of a single or only a few markers.
- the invention provides a method of separating proteins that are present in a mixture of proteins by utilizing a set of binding agents, hereafter denominated “separation linkers” or “slinkers”. Separation is accomplished by exposing the mixture of proteins to the separation linkers (either concomitantly in, for example, an array of slinkers, or sequentially, i.e. one separation linker at a time), and then separating the proteins that bind to each linker from those that do not. The bound proteins may be released from the linkers if desired.
- separation linkers either concomitantly in, for example, an array of slinkers, or sequentially, i.e. one separation linker at a time
- the slinkers may be immobilized directly onto a substrate (for example, magnetic beads, microslides, rods, fibers, chromatographic columns, and the like) and the immobilization may be via a spacer molecule or a secondary antibody.
- the slinker may be an antibody
- the proteins that are separated may interact with each other or may be non-interactive.
- the proteins and the slinker may be labeled with a detectable label, for example a fluorescent, colorimetric or chemiluminescent label.
- the method also may include separating proteins using a slinker in which the affinity of a protein for the slinker is adjusted by changing the environment of the binding reaction, e.g. by adjusting the temperature, pH, ionic strength or composition of buffer, and the like.
- the method further may be coupled with the use of other protein separation techniques, e.g. various forms of chromatography or electrophoresis.
- the present invention also provides a method for quantifying the amount of protein in a sample.
- the sample is contacted with a magnetic bead that is coated with a slinker which binds the protein.
- the speed or the distance of migration in a magnetic field of the magnetic bead with attached protein is measured and correlated to the quantity of protein that is attached.
- Further differentiation of migrating beads may be effected by subjecting the beads to a transverse field (e.g. electric, flow, or magnetic) prior to, simultaneously with, or after placing the beads in the main magnetic field.
- a transverse field e.g. electric, flow, or magnetic
- different slinkers may be attached to beads of different sizes, providing an additional differentiation mechanism.
- an apparatus for measuring protein content in a sample is also provided.
- the apparatus includes a magnetic bead, slinkers associated with the bead, a means for monitoring the rate of migration of the bead in a magnetic field, and a means to calculate the quantity of protein bound to the bead based on the measured speed of migration.
- the apparatus may further include a substrate with microchannels, the cross sections of which are larger than the diameter of the magnetic beads, and sensors to sense the movement of the magnetic beads.
- the slinkers employed may be antibodies (general-motif or specific). However, other types of slinkers may be used in the practice of this invention (e.g. actin, tubulin, integrins, substances that bind phosphorylated groups, etc.).
- the magnetic beads may preferably be nanobeads having a radius in the range of about 5 to 1000 nm; however, other materials and size ranges may be suitable for use in the practice of the present invention.
- the invention further provides a classification database for proteins based on their binding signatures.
- the binding signatures reflect the pattern of binding/not binding to the set of slinkers utilized in the methods of the invention, i.e. the Repeated Classification Procedure data.
- the invention also provides a method of generating such a protein classification database. The method involves exposing each protein to be classified to a set of slinkers, detecting which of said slinkers bind to each protein, and compiling a database from this information.
- a database can be utilized as a way of cataloging information for normal and disease states. Such a database then would aid in the diagnosis of diseases by allowing the comparison of binding signatures from unknown samples to those of normal and of known disease states.
- binding signature differed from the normal binding signature, that could suggest the presence of a disease state, especially if the signature matched that of a known disease state.
- Such information could be used in concert with other diagnostic techniques for confirmation, many of which are well-known to those of skill in the art.
- the present invention also provides a method for quantitating the proteins present in a mixture of proteins using optical fibers.
- the protein mixture is labeled with a non-specific fluorescent dye.
- a bundle of optical fibers is then immersed in the protein mixture.
- Each fiber in the bundle having previously been coated with a different slinker.
- the bundle is removed from the mixture and light is shined on each fiber in turn, inducing fluorescence in those fibers which have fluorescently labeled proteins attached.
- the intensity of fluorescence is correlated with an amount of protein.
- the invention also provides a method for generating general-motif antibodies (a subset of slinkers). The method involves identifying a protein motif that is common to all the proteins in a given set of proteins and which is antigenic, and producing an antibody to this motif. Examples of such motifs include regions of primary, secondary, or tertiary structural homology in the proteins.
- FIG. 1 A schematic representation of the use of RCP to separate and categorize eight non- interacting proteins (a, b, c, d, e,f g, and h) using three different slinkers ( ⁇ , ⁇ , and ⁇ ). 0 is used to denote the subsets of proteins which were not bound by a slinker.
- FIG. 2 A schematic representation of the use of RCP to separate four proteins, a, b, c, and d, two of which (a and c) bind strongly to each other at the solution conditions being used, using slinkers ⁇ and ⁇ . 0 is used to denote the subsets of proteins which were not bound by that slinker. The asterisk is used to denote the amount of protein c which was not bound to protein a in the original mixture.
- Figure 3 Example of a two-level separation in which ten proteins are separated using five different slinkers. Each of the ten proteins binds strongly to two of the slinkers.
- FIG. 4 Example of a two-level separation in which ten proteins are separated using five different slinkers. This shows that it is possible to separate the ten proteins even when each binds weakly to two of the slinkers. Thus, not all of that protein is removed in a single step. Remaining protein is indicated by a "bar" (-) over the protein name.
- Figure 5 Example of a three-level separation in which ten proteins are separated using five different slinkers. Each of the ten proteins binds weakly to three of the slinkers.
- Figure 6 Example of a three-level separation in which twenty proteins are separated using six different slinkers. . Each of the twenty proteins binds weakly to three of the slinkers.
- Figure 7. Schematic depiction of a microarray for use in RCP.
- Figure 8. Schematic depiction of a device for quantitating the amount of protein bound to magnetic beads.
- Figure 9 Schematic depiction of a portion of the device shown in Figure 4 which includes a photographic device used to evaluate bead movement.
- Figure 10 Illustration of conversion of a grey-scale image of a packet in a channel to a thresholded black and white image.
- the present invention provides a procedure, denominated the Repeated Classification
- RCP Procedure to separate, identify, and classify macromolecules obtained from a biological sample rapidly and quantitatively.
- RCP allows the detection of the presence of new proteins, new or enhanced protein interactions, and/or different concentrations of proteins in a single procedure.
- the macromolecules which are detected may be nucleic acid molecules such as mRNA or DNA, or any ohter type of large molecules (glycoproteins, starches, fats, etc.) which can be attached to an appropriate slinker.
- RCP therefore, offers a robust test of a change in state (for example, from a normal to a disease state) by detecting a combination of differences between states that may result from many causes.
- Utilization of RCP provides a means to determine, (as desired, part or all of) the pattern of molecular expression for a biological sample.
- biological samples include but are not limited to extracts from the cytoplasm of (both prokaryotic and eukaryotic) cells (e.g. mammalian, plant, fungus, bacterial etc.), or from various subcellular organelles such as the nucleus, mitochondria, the endoplasmic reticulum, chloroplasts, and the like.
- other types of samples may be assayed as well, including blood, plasma, amniotic fluid, tissue samples, biopsy samples, and the like.
- pathological signature we mean the pattern of molecular (e.g. protein) expression which is characteristic of a particular disease state. This signature is comprised of a combination of markers and thus renders the diagnosis more reliable and more informative than currently used procedures which rely on a single or only a few markers.
- Epstein-Barr virus An example of a disease state that is amenable to diagnosis by the methods of the present invention is Epstein-Barr virus (EBV) infection.
- Epstein-Barr virus EBV
- naive B lymphocytes become infected by Epstein-Barr virus, many proteins change their expression levels. Some of these proteins include the two cytoskeletal proteins fascin and MARCK, as well as the C3d receptor, the 45 kDa-lymphoblastoid associated protein and the C-cell restricted activation antigen.
- Fascin expression is routinely used as a marker for EBN infection. Fascin, however, also becomes overexpressed in certain cancer cells upon transformation. Hence, the diagnosis of EBN infection would be more accurate if the entire pattern of protein expression was detected rather than a single protein.
- RCP involves the step- wise, sequential exposure of a mixture of macromolecules to a set of slinkers.
- the macromolecules are proteins.
- the macromolecules are mR ⁇ A or D ⁇ A.
- combinations of, for example, proteins and mR ⁇ A, or proteins and D ⁇ A may be detected by the methods of the present invention.
- the slinkers may be immobilized on a solid support or may be used without a support.
- the slinkers are "general-motif antibodies", a full description of which is given below. However, any suitable slinker may be utilized in the practice of the present invention.
- Table 1 summarizes the potential interactions of proteins a, b, c, d, e,f g, and h with slinkers ⁇ , ⁇ , and ⁇ .
- "1" indicates strong binding between the protein and indicated slinker, and "0" indicates no binding.
- a schematic representation of the use of RCP to separate and categorize these eight proteins (a, b, c, d, e,f g, and h, which do not interact with each other) using a set of three different slinkers ( , ⁇ , and ⁇ ) is depicted in Figure 1.
- the immobilized slinker used at that level separates the proteins in the mixture into two different protein subsets or protein pools.
- each reference to a single protein e.g. "protein -/') actually means "all proteins which satisfy the specified binding conditions".
- protein - ' may represent a single protein or a group of proteins.
- the words "protein d” represent all proteins in the mixture that bind slinkers ⁇ and ⁇ but not slinker ⁇ .
- protein h represents all proteins in the mixture that do not bind slinkers ⁇ , ⁇ and ⁇ .
- Processl of Figure 1 shows the binding of slinker a to proteins a, d, e, and g.
- the proteins which are bound to it also are removed, forming a subset pool that contains proteins a, d, e, and g.
- a second subset pool contains the proteins b, c,f, and h (i.e. those which do not bind ⁇ ) which were left behind (symbolized by the "0" arrow).
- both protein subset pools then are exposed to a second slinker ⁇ (i.e., Process 2 in Figure 1), which divides each of them into two additional pools, yielding a total of four subset pools (d,g; a,e; b,f; and c,h).
- the proteins in one of the four pools (d,g) bind both the first and second slinkers; the proteins in a second pool (a,e) bind only the first but not the second slinker; the proteins in a third pool (b,f) bind only the second and not the first slinker; and the proteins in the fourth pool (c,h) do not bind either the first or the second slinkers.
- Each of the four subset pools is then exposed to a third slinker ⁇ (i.e., Process 3 of Figure 1), and so on, until the desired degree of separation is achieved, i.e. until each subset pool created by exposure to the various slinkers being applied contains only a single protein or only a desired subset of proteins.
- the separation may be complete (i.e. the final protein pools each contain a single protein), the separation may be partial (the final protein pools contain more than one protein), or some final pools may contain only one protein while others contain more than one protein, depending on the purpose of a particular investigation.
- slinker ⁇ two or more different slinkers (e.g. ⁇ l and ⁇ 2) can be applied instead of a single slinker ⁇ .
- two or more ⁇ slinkers may be applied.
- RCP may be applied to mixtures of proteins in which the proteins do not interact with each other (e.g. Figure 1), and also to mixtures of proteins in which some of the proteins do interact with each other. In the latter case, RCP can provide valuable information about the strength of the interaction and the relative concentrations of the interacting proteins.
- Table 2 summarizes all possible interactions between the four proteins a, b, c, and d and two slinkers and, ⁇ , where "1 " indicates strong binding and "0" indicates negligible or no binding. Table 2. Possible interactions between 4 proteins, a, b, c and d, and 2 slinkers ⁇ and ⁇ .
- FIG. 1 A strategy for separating four proteins, a, b, c, and d, two of which (a and c) bind strongly at the solution conditions being used, using slinkers and ⁇ is depicted is Figure 2.
- Process 1 of Figure 2 a mixture of proteins a, b, c, dand ⁇ -c complexes are exposed to a first slinker ⁇ . Slinker ⁇ , together with its bound proteins, is then removed thus dividing the sample into two protein pools.
- One pool contains the proteins which bind to (a, d, and a-c complexes); the other pool contains proteins b and the molecules of c which were not bound to a.
- a second slinker, ⁇ which binds proteins b and d, is used to separate protein d from the first pool and protein b from the second pool.
- proteins d, b, and c are separated, except for those molecules of protein c that are bound in a-c complexes and thus also are present in the pool containing a.
- Proteins a and c then are separated from each other by breaking the a-c bond (Step 3. e.g. denaturing by a change in pH or ionic strength) and re-exposing the resulting a, c protein pool to slinker (Step 4).
- Step 3. e.g. denaturing by a change in pH or ionic strength
- Step 4 re-exposing the resulting a, c protein pool to slinker
- the ratio of the amounts of c obtained via the two different separation paths provides information about the ratio of concentration of a versus c whenever the a-c bond is strong, and otherwise (when the ratio of initial concentrations is known) information about the strength of the a-c interaction.
- a modification of RCP can be used to identify the proteins in a cell or tissue extract which interact with each other.
- a mixture containing the protein a and three proteins b, c, and d which interact strongly with a may be analyzed in the following manner: the cell extract is mixed with immobilized , a slinker with affinity for a.
- the protein pairs a-b, a-c, and a-d then are separated from the cell extract by removing this slinker with its attached proteins; all proteins that do not interact with a remain in the cell extract.
- the immobilized -a complexes are then exposed to slinkers which are highly specific for b, c, and d, and which themselves are differentially labeled, for example, fluorescently labeled antibodies.
- slinkers which are highly specific for b, c, and d, and which themselves are differentially labeled, for example, fluorescently labeled antibodies.
- a modification of RCP can be used to quantify the affinity of a protein for other proteins.
- exposing the sample to an slinker i.e. specific for a
- a-b pairs are removed but some proteins of type b are left behind.
- a slinker ⁇ which is specific for b then is used to remove the remaining b protein.
- free a is removed as well as all of the b, as a-b pairs. Proteins a and b are subsequently separated by, for example, changing the pH and/or ionic strength and using slinker ⁇ .
- the antibodies may be specific antibodies, the production of which is well-known to those of skill in the art, or general-motif antibodies. The production of general-motif antibodies is described in detail below. However, those of skill in the art will recognize that other types of slinkers may be utilized, either alone, or in combination with antibodies to carry out RCP. For example, any molecule which contains a binding site for a suitable fraction of the molecule(s) of interest (e.g. proteins in a protein mixture) may be utilized as slinkers in the practice of the present invention.
- Examples of molecules which may function as slinkers are specific antibodies, enzyme substrates and substrate analogs, co- factors, coenzymes, inhibitors, general-motif antibodies, protein-binding DNA and RNA sequences, nucleic acids, metal ions, saccharides, spacer molecules with accessible functional groups (e.g. amino or sulfhydryl functions), integrins, selectins, cadherins, intermediate filaments, vimentin, neurofilaments, keratins, components of receptor-ligand pairs, and the like.
- Slinkers also may be full length proteins and peptides. Some examples are cytoskeleton filaments such as actin and microtubule (a polymer of tubulin) which can bind many cytoskeleton proteins.
- spacer molecule we mean molecules typically utilized by those of skill in the art to provide additional distance between the surface of a substrate and an active moiety which is tethered to the substrate, but which is itself relatively inactive, for example an alkyl chain. Any molecule which is capable of binding a suitable fraction of the molecules in a sample of interest may be used in the practice of the present invention. Any combination of slinkers may be used to form a set of slinkers for use in the practice of the present invention, so long as the use of the set of slinkers results in a satisfactory separation and identification of the proteins in the sample of interest.
- protein samples may include: original samples of interest (e.g. tissue samples); subsequent pools of protein which result from an exposure of a sample to a potential slinker; samples of purified protein; mixtures of purified proteins, and the like.
- original samples of interest e.g. tissue samples
- subsequent pools of protein which result from an exposure of a sample to a potential slinker samples of purified protein; mixtures of purified proteins, and the like.
- proteins mixtures would be appropriate for testing the activity of potential slinkers, and for optimizing the order of a sequence of slinkers.
- the data which is obtained in this fashion is utilized to generate sets of slinkers, which may be used in the practice of the present invention. Further, the order in which potential slinkers are exposed to samples also should be varied in order to test and optimize the efficacy of the sets of slinkers with respect to a sample of interest.
- the sets of slinkers so developed may be utilized sequentially (i.e. in a given order), simultaneously in arrays, or a few at a time for a given application.
- slinkers to be used may be coordinated with and based on genomic information.
- genomic information e.g. by the use of gene microarrays
- microarrays or custom sets of slinkers can be designed which contain only slinkers corresponding to the macromoleucles (e.g. proteins or mRNA) produced under a given set of circumstances, e.g. in a specific tissue type, or during a particular disease process.
- macromoleucles e.g. proteins or mRNA
- RCP Relative to the use of, for example, specific antibodies
- each slinker used in a separation scheme binds about half of the proteins in a protein sample.
- the minimum number of slinkers, n, required to identify N proteins using RCP is (logN/log2).
- n the minimum number of slinkers required to identify N proteins using RCP.
- slinker binds to proteins a, d, e, and g but it does not bind to proteins b, c, f and h; similarly, slinker ⁇ binds to b, d, f and g but not to a, c, e, and h. If any of these conditions is not satisfied the procedure still will work, but it will require the use of at least one additional slinker to make a complete separation. For example, if ⁇ does not bind then after Process 2 one is left with a protein group consisting of c, h, and/ instead of only c and h. Process 3 (i.e.
- step 3 separates c from/and h and an additional step (i.e., the use of ⁇ ) would be required to separate/ from h.
- additional steps may be required and inco ⁇ orated into the RCP protocol.
- RCP is not limited to separations involving a large number of successive steps or processes of separation.
- RCP also works by successively applying slinkers to a given pool of proteins using only a small number of processes of separation. Examples of two- and three- process procedures are described below. Those of skill in the art will recognize that many multi -process combinations of procedures can be designed for use according to the methods of the present invention, and all such combinations are intended to be included in the scope of the present invention.
- Two-Slinker Proteins Consider a mixture consisting often different proteins, a -j, each of which binds to two (and only two) of five slinkers , ⁇ , ⁇ , ⁇ , and e (see Table 3 for the list of potential interactions; 1 represents strong binding, 0 represents negligible binding). These ten proteins can be fully separated by applying general-motif antibodies , ⁇ , ⁇ , ⁇ , and e. At each step of the separation procedure of this particular illustration, the use of sufficient slinker is assumed to remove essentially all of the desired protein (e.g., more than 99% or 99.9% or whatever purity is desired).
- applying slinker ⁇ to the protein pool removes a, e, /and from the pool. Then applying ⁇ to the remaining proteins in the pool (i.e., at the same level of processes) removes b, g, and j. (Note that in this and related figures, the order of the successive applications of different slinkers in a process should coincide with the order from left to right as depicted in the figure.) Slinker ⁇ then removes c and h, and ⁇ removes d. Note that if one were certain that there were only these ten proteins in that mixture, the ⁇ removal is not necessary. However using ⁇ allows this also to be the procedure for removing ten particular proteins from a mixture of ten or more proteins.
- FIG. 4 shows a separation identical to that shown in Figure 3, except that some protein is "left behind" by each of the slinkers utilized in the first process.
- the protein that is left behind is entirely removed on exposure to the next slinker which binds to it.
- the protein left behind after the first application of a general antibody is indicated by a bar over the roman letter which identifies it, e.g., a.
- the number of slinkers required to separate a given number of proteins when each protein binds to more than one slinker can be calculated. Assuming, as in the above example, that each protein binds to exactly two slinkers, if m equals the number of proteins one desires to separate and n is the number of slinkers, then n is related to m by the equation:
- slinkers ⁇ , ⁇ , and ⁇ are successively applied.
- process 2 three, two, and one slinker(s), respectively, are successively applied to the three subsets of proteins that were generated in the first level of separation.
- all proteins are separated by following the pattern of application of slinkers given in Figure 5.
- Figure 5 assumes that binding to the slinkers is sufficiently weak that some protein is left behind (as in the example illustrated by Figure 4). When only strong affinity slinkers are used, the procedure is the same except that (since no protein is left behind) the letters with the overbars disappear.
- the slinkers of the present invention are immobilized on a substrate.
- Possible substrates for immobilization include but are not limited to magnetic beads and wires, other types of beads, chromatographic columns
- slinkers e.g. affinity or ion-exchange type
- generally planar surfaces such as microslides, multiwell plates, chips, rods, tubes, fibers, optical fibers, and the like.
- the slinkers may be immobilized, for example, as microarrays on a chip, in parallel arrays or lanes on a glass microslide, or as "spots" on a glass microslide.
- microarrays coated with slinker spots or "wells” are used to apply RCP.
- Such an array is illustrated in Figure 1, where the circles represent the spots or "wells" of a microarray and the characters , ⁇ , ⁇ , 6 and e represent five different slinkers that have been immobilized on the spots.
- an aliquot of the protein mixture to be analyzed is placed on the ⁇ -slinker coated spot and allowed to incubate.
- the supernatant (containing proteins that did not bind to the ⁇ -slinker) is then removed, leaving behind proteins which are bound to the ⁇ -slinker.
- the supernatant is placed on the ⁇ -coated spot on the left and labeled " ⁇ ".
- Proteins that became attached to the ⁇ -slinker spot are detached from the ⁇ -slinker spot by suitable means such as changing the ionic strength, pH, etc.. They are then mixed with buffer and placed on the second ⁇ -coated spot, located at the right and labeled ⁇ * .
- suitable means such as changing the ionic strength, pH, etc..
- slinker spots which receive protein that are bound to and subsequently detached from a previous slinker are labeled with (*) throughout Figure 7, while those receiving supernatant are labeled only with the Greek character corresponding to the slinker.
- the procedure is repeated using all slinkers in this array, in this case the five slinkers ⁇ , ⁇ , ⁇ , ⁇ and e. If all proteins in the mixture have previously been labeled (for example, with a generic fluorescent label) the presence of labeled proteins in the last row of the array can be detected and the identity of the proteins established by correlation with known previously binding patterns.
- the immobilized slinkers may be covalently or physically linked to the substrate by techniques such as covalent bonding via an amide or ester linkage or by abso ⁇ tion, depending on the nature of the slinker. Further, the slinkers may be directly immobilized on the substrate, or they may be immobilized via
- linker or “spacer” molecules which serve to increase the distance between the slinker and the substrate.
- spacer molecules may be advantageous in promoting slinker accessibility and in decreasing steric hindrance during si inker-protein interactions.
- suitable spacer molecules include but not limited to secondary antibodies, peptides, alkyl chains, nucleic acid molecules, and the like.
- any spacer molecule which serves to increase the distance between the slinker and the substrate without interfering with the interaction of the slinker and target molecule e.g. a protein
- target molecule e.g. a protein
- the RCP methods of the present invention may be used as "stand alone” techniques, i.e. they may be used alone to effect the separation and classification of the proteins in a sample.
- RCP may be utilized in conjunction with other traditional separation techniques as well.
- Other techniques may be inco ⁇ orated either prior to, during, or after the use of a set of slinkers.
- cell extracts may be subjected to separation methods such as chromatography or electrophoresis in order to obtain a particular pool of proteins for analysis by RCP.
- any subset pool of proteins generated during the RCP method may be further analyzed by other analytical techniques after completion of RCP or in the midst of utilizing a set of slinkers.
- HPLC High Performance Liquid Chromatography
- FPLC Fast Protein Liquid Chromatography
- ion exchange chromatography affinity chromatography
- sizing column chromatography denaturing and non-denaturing polyacrylamide gel electrophoresis
- capillary electrophoresis ammonium sulfate precipitation, and the like.
- the RCP method can be used at different solution conditions, including pH, salt content, and ionic strength. These solution conditions can modulate the affinity of the slinkers for their ligands.
- fluorescence labeling of the molecules (e.g. proteins) contained in a protein mixture can be used.
- the extract to be analyzed may be mixed with a generic fluorescent dye such as fluorescein, or with several more specific dyes for greater selectivity.
- proteins which are separated by the practice of the present invention may be readily detected by well-established methods which are well-known to those of skill in the art.
- the proteins In order to analyze a subset pool of molecules (e.g. proteins) which have bound to a slinker, the proteins typically are released from that slinker prior to exposure to the next slinker. This can be accomplished in a variety of ways, for example by changes in ionic strength or pH. However, in some cases, it may not be necessary to release, the bound proteins from the slinker prior to exposure of the sample to the next slinker. This is especially desirable when the repeated use of some of the agents that detach magnetic beads from proteins runs the risk of permanent denaturation or other undesirable interactions with certain proteins. There are two limitations to this multiple bead-slinker attachment procedure.
- RCP provides a ready means of classifying the proteins which are separated in this manner. Each separated protein will possess a distinct "binding signature" based on:
- binding signature we mean a summary of the binding affinities of a protein (or group of proteins) to a series of slinkers. For example, if an the series of slinkers consists of the four slinkers ⁇ , ⁇ , ⁇ , and ⁇ , and a protein binds to slinkers and ⁇ but does not bind to ⁇ and ⁇ , then its binding signature can be given as: ⁇ +, ⁇ -, ⁇ +, ⁇ - .
- binding signatures may be established for proteins (or other macromolecules e.g nucleic acids) for any of a variety of reasons, for example, to assess the pattern of differential protein expression during various biological states, such as disease states, pregnancy, changes related to aging, changes related to the administration of drugs (e.g. during drug screening and drug development), and the like.
- RCP may be utilized to assess the effect on patterns of protein expression of pollutants, diet or exercise, gene therapy, and the like.
- binding signatures of individual proteins can be used to establish novel databases for classifying proteins according to their ability (or lack thereof) to bind a given set of slinkers. Such a database would allow the identification of the proteins or subsets of proteins contained in an unknown sample that is subjected to RCP. Individual proteins can be classified according to their binding signatures as described above, i.e. ⁇ +, ⁇ -, ⁇ +, ⁇ -.
- pathological signatures which are established based on RCP can be compiled into novel databases which include the entire pattern of protein expression for any suitable condition.
- a typical pathological signature may be comprised of the binding signatures of those proteins whose expression is changed by a particular disease state, or by exposure to a chemical (e.g. a drug) or for any reason of interest.
- the database thus produced can be used for the comparative analysis of unknown vs known samples in research or clinical applications in order to ascertain how patterns of protein expression change in response to a variable of interest (e.g. a disease, or administration of a drug, etc.).
- Such databases may be utilized in the diagnosis of diseases, or for drug screening and development, and the like.
- the amount of protein which is bound to a slinker at a given step in the RCP process is modulated by controlling such binding reaction conditions as pH, temperature, ionic strength, counterion concentration, buffer type, additives (e.g. chelating agents or metal ions) and the like.
- the protein sample is exposed to the slinker under defined conditions of, for example, ionic strength. Any proteins which bind to the slinker under those conditions are removed from the mixture.
- the condition of interest e.g. ionic strength
- any proteins which bind to the slinker under the altered conditions are removed, and so on.
- condition of interest may be adjusted after the protein mixture is exposed to the slinker.
- This process of adjusting a reaction or solution condition, and then removing the proteins that bind after each adjustment is continued until a desired level of separation of proteins is achieved.
- this method of separation via adjustment of a reaction condition may be coupled with other means of protein separation, e.g. with the use of several different slinkers, or by combining the use of the adjustment of several different reaction conditions, e.g. adjustment of ionic strength and pH, either simultaneously or sequentially.
- Proteins separated by the RCP method can subsequently be quantitated. This can be carried out by any of a variety of well-established methods which are known to those of skill in the art.
- the proteins may be released from the immobilized slinker and quantitated spectroscopically. In some cases it may not be necessary to release the proteins from the immobilized substrate.
- fluorescence detection may be carried out directly while the protein is still attached to the slinker.
- the slinker is a general-motif antibody that is immobilized on a magnetic bead. Protein quantitation is carried out (without releasing the protein) by monitoring the migration of the magnetic bead in a magnetic field. This embodiment of the present invention is described in detail below.
- Quantification of protein expression in a sample can be obtained only as long as binding sites of the immobilized slinkers remain unsaturated. Proteins expressed in large quantities may saturate the binding sites, while those which are expressed at low concentrations will not saturate the binding sites.
- the protein sample is simply diluted with an appropriate buffer. The sample is diluted and retested until an increase in protein binding is detected, which is the point at which less than complete saturation occurs. An additional dilution step is advisable to insure that a sufficient number of unsaturated sites remain available. Alternatively, the concentration of immobilized slinkers may be increased until the point of saturation is exceeded.
- Slinkers are substances (most often proteins or protein-polysaccharide combinations) which contain a functional domain that binds a selected fraction or subset of the different proteins contained in a protein mixture.
- a functional domain of a slinker binds a common motif in the subset of proteins.
- the proteins in the subset may or may not be otherwise related.
- the use of slinkers is therefore fundamentally different from the use of monoclonal and polyclonal antibodies, the latter of which routinely are used in biological applications to detect (e.g. in immunocytochemistry) or to separate (e.g. in affinity chromatography) specific proteins contained in cell extracts or mixtures.
- slinkers can be small molecules or existing proteins (e.g. actin tubulin, etc.).
- the slinkers which are utilized are "general-motif antibodies" which bind to two or more proteins.
- One way of creating general-motif antibodies is by first identifying amino acid sequences to use as antigens from which general-motif antibodies may be produced. This may be accomplished by comparing the amino acid sequences of those proteins which have been identified as belonging to, for example, a superfamily of proteins and identifying regions of homology among the proteins. Those of skill in the art will recognize that the amino acid sequences of such superfamilies are readily available from such sources as Gene Bank and Swiss Prot.
- Appropriate regions may contain identical amino acid sequences, or the amino acid sequences may be highly homologous, containing conservative substitutions.
- the homologous region comprises all or a portion of the protein which is generally accepted to be that which defines membership of the protein in the superfamily.
- the sequence YLLSSGINGSFL (SEQ ID NO: 1) is shared by all 78 members of the Src homology 2 (SH2) superfamily of proteins. This sequence is located within the SH2 domain (i.e. within the defining domain) of this superfamily of proteins and is identical in all protein superfamily members identified to date. In contrast, other parts of the SH2 domain display only 48% identity from member to member.
- Amino acid sequences identified in this manner may be produced synthetically or obtained by partial proteolysis of a purified protein according to methods which are well- known to those of skill in the art.
- This peptide sequence then serves as an antigen for generation of a purified, general-motif antibody, according to well-established methods which are well-known to those of skill in the art.
- the antibody may be conjugated to a dye molecule, thus making detection simple and reliable. Numerous suitable dye molecules and their method of use are well-known to those of skill in the art. This procedure allows the identification of many potential general-motif antibodies which then can be screened for efficacy in the RCP procedure.
- general-motif antibodies also can be based on the secondary and tertiary structure of protein motifs and folds.
- proteins are classified not only by their shared primary sequence homology/identity, but also on the motifs and folds shared among parts (e.g. functional domains) of proteins.
- General-motif antibodies may be generated against any protein motif which is antigenic, so long as the general-motif antibodies are useful in the practice of the present invention.
- Such motifs include primary structural elements (the primary sequence of amino acids), secondary structural elements (areas of local protein folding e.g.
- Such motifs may also include binding regions for molecules characteristically associated with a protein, or the bound molecules themselves (e.g. a coenzyme or cofactor).
- Such motifs include but are not limited to: homologous amino acid sequences such as those described above, DNA or RNA binding regions, co-factor binding regions or the cofactors themselves, sulfhydryl functions, saccharide binding regions or the saccharides themselves, sites of phosphorylation, and the like.
- General-motif antibodies may be generated against any antigenic motif which is characteristic of or common to a subset of proteins in a mixture.
- a slinker may bind a large fraction (e.g. about half) of the proteins in the sample to which it is being exposed.
- slinkers which bind smaller or larger fractions of the proteins in a sample may also be suitable in the practice of the present invention. Any substance which binds a fraction of the proteins in the sample of interest may be used as a slinker in the practice of the present invention, so long as the set of slinkers into which that slinker has been assigned is capable of effecting an acceptable separation of proteins.
- each protein bound to various numbers of slinkers.
- the proteins each bound to only two or to only three of the slinkers.
- the use of illustrative examples in which the number is constant (e.g. 2 or 3) is not a necessary condition.
- RCP schemes can be produced with the various proteins binding to different numbers of slinkers. In fact, in a particular mixture, some proteins may bind to only one slinker.
- the use of slinkers may be coupled with the use of other binding agents such as specific antibodies.
- specific antibodies may be utilized to identify specific proteins within the subset pool, or to remove specific proteins from the subset pool. Subsequently, the remaining proteins in the subset pool may again be subjected to RCP with additional slinkers.
- Other binding agents such as specific antibodies may be utilized at any step of an RCP procedure if so desired.
- a set of slinkers is two or more slinkers that can separate different constituents of interest in a sample containing a mixed population of constituents (e.g. proteins).
- the set will include two or more slinkers, and may include 5 or 10 or more slinkers.
- the set also may include at least one, and possibly two or more specific antibodies.
- slinkers may be used to separate non- interacting proteins as follows: Magnetic beads are coated with a slinker and are incubated with the sample of interest (e.g. a cell extract) in a suitable container, e.g. a single well of a multi-well plate. A set of proteins (optimally roughly half of those present in the sample) bind to the slinker on the beads and can be removed from the sample by removal of the beads via a magnetic field gradient. The slinker-bound proteins are released from the beads into, for example, a second well of the multi-well plate.
- sample of interest e.g. a cell extract
- suitable container e.g. a single well of a multi-well plate.
- a set of proteins bind to the slinker on the beads and can be removed from the sample by removal of the beads via a magnetic field gradient.
- the slinker-bound proteins are released from the beads into, for example, a second well of the multi-well plate.
- This first separation.step thus results in two pools of protein: one which binds to the first slinker and is removed with the magnetic beads, and one which does not, the latter pool being "left behind" when the magnetic beads are removed. Both protein pools are then incubated with a second slinker which is also bound to magnetic beads. Again, roughly half the proteins of each pool bind to the second slinker and are removed from the pool upon removal of the magnetic beads via a magnetic gradient. The proteins again are released from the magnetic beads.
- Each of the two protein pools thus is divided into two additional pools, resulting in a total of four protein pools, one of which binds both the first and second slinker, one of which binds the first but not the second slinker, one of which binds the second but not the first slinker, and one of which binds neither the first nor second slinker.
- This procedure may be repeated (e.g. with a third slinker in a set) as many times as is desired.
- Each subsequent exposure to a different slinker splits each protein pool which is assayed into two pools (one of which contains proteins which bind the slinker and one of which does not) until the desired level of separation is attained, e.g.
- binding signature indicates which slinkers it binds to, and which slinkers it does not bind to.
- the binding signature may yield enough information to identify the protein when the nature of the binding epitopes is known. For example, a protein which is bound by a general-motif antibody which was raised to the sequence YLLSSGINGSFL (SEQ ID NO: 1) most likely contains an SH2 domain. Confirmation of this identification may be made using specific monoclonal antibodies or by other means, such as an activity based assay.
- slinkers such as general-motif antibodies can be used in combination with specific antibodies to identify the proteins in a cell or tissue extract which interact with each other.
- a mixture containing the protein a and three proteins b, c, and d which interact strongly with a may be analyzed in the following manner: the cell extract is mixed with magnetic beads coated with ⁇ , an anti- ⁇ antibody.
- the protein pairs a- b, a-c, and a-d are separated from the cell extract by magnetically removing the beads; all proteins that do not interact with a remain in the cell extract.
- the beads are then sequentially exposed to antibodies which are highly specific for b, c, and d, and which are themselves differentially labeled.
- the beads When the beads are examined, it is possible to identify and quantify the concentrations of the a-b, a-c, and a-d pairs by detecting the differentially labeled specific antibodies.
- the ⁇ antibody may be immobilized on a substrate such as a glass microslide or rod and non-bound proteins removed by rinsing the substrate prior to exposure to antibodies that are highly specific for b, c, and d.
- the present invention provides a method and apparatus to quantitate the amount of protein in a sample once the protein is attached to a magnetic nanobead via a slinker.
- the method involves exposing a protein sample to a magnetic bead of relatively small diameter to which a slinker has been affixed.
- the number of molecules of slinker which are affixed to the bead is variable. For example, in some instances it may be advantageous to have only a single molecule of slinker attached. For other pu ⁇ oses, it may be advantageous to have a plurality of slinker molecules attached. Further, the molecules of slinker may all be the same (only one type of slinker) or different (more than one type of slinker e.g.
- the beads are removed from the sample and the quantity of protein that is bound to the bead via the slinker is determined by measuring the speed of migration of the bead in a magnetic field in comparison to the speed of migration of a control bead or to a known speed stored in a lookup table or a known speed otherwise defined.
- speed is distance traveled in a known amount of time, one can use either speed, or distance as the measurable.
- the beads are exposed to a sample which is obtained, for example, by lysis of a cell.
- the beads are exposed to a sample by introducing the beads into a cell prior to lysis.
- particles of a size similar to the beads of the present invention may be introduced into cells by a variety of established techniques.
- the macromolecules of interest e.g. proteins, mRNA, or DNA
- bind to the slinkers on the beads within the cell, and the cells may then be lysed by any of a variety of techniques which are well-known to those of skill in the art.
- FIG. 8 An apparatus for carrying out the measurement is illustrated in Figure 8 and comprises a substrate containing a plurality of microchannels with adjacent loading areas at the proximal end of the channels.
- the cross-section of the microchannels is larger than the diameter of the beads.
- Figure 8 depicts only two loading areas (1 A and IB) at the proximal ends of two channels, 2A and 2B.
- the channels are symmetrical about a longitudinal axis of the substrate.
- the exact dimensions of the channels and loading areas is not a crucial feature of the apparatus and may vary according to several factors, including the size of the beads being analyzed.
- the apparatus further comprises a plurality of electromagnets 4, 5A and 5B.
- substrates would be appropriate for containing the microchannels of the device of the present invention.
- substrates include but are not limited to: SiO 2 , Si, Al 2 O 3 , GaAs, TiO 2 , etc.
- the exact form of the substrate is not crucial to the practice of the invention.
- the channels may be fabricated on the horizontal surface of a glass microslide, or within a cylindrical-shaped substrate, and the like. Any substrate/channel design in which the beads may be deposited in a loading area and are free to migrate down a channel in response to a magnetic field gradient at a detectable rate, may be used in the practice of the present invention.
- the slinker used in the method may be, for example, an antibody (either a general- motif antibody or a specific antibody).
- the slinker-coated beads are incubated with a specimen of interest, typically extracts from tissues and/or cell cultures. During the incubation, a protein or set of proteins attaches to the slinker on the beads, forming beads coated with slinker-protein complex (complexed beads, 20B). The complexed beads are removed from the sample and loaded onto a loading area IB of a channel 2B.
- Identical control beads 20A i.e.
- Electromagnets e.g. coaxial magnetic coils
- electromagnet 4 is turned on, creating a magnetic field gradient, and electromagnet 4 is turned off.
- the magnetic field-gradient coils produce a force which causes movement of the beads 20A and 20B towards the distal ends (3 A and 3B) of the channels.
- a control bead 20A is depicted as migrating in a channel 2A ahead of complexed bead 20B, which is migrating in parallel channel 2B.
- the migration of both beads is tracked by a camera 10 with a lens 11 , appropriately mounted over channels 2A and 2B in order to record images of the migration of beads 20A and 20B.
- the camera 10 is further connected to a computer 12 for analysis of the acquired images. Images of beads traveling in the channels may be acquired using, for example, either a digital camera or a silicon-intensifier target (SIT) camera.
- Figure 9 illustrates a control bead 20A run simultaneously with a complexed bead 20B, it is possible to compare results for the complexed bead 20B against stored empirical results, against predicted results, or against some other measure.
- the movement of magnetic nanobeads within microchannels further may be differentiated by exposing the beads to a transverse field illustrated by arrow 22 during or prior to subjecting the beads to the final magnetic field.
- the transverse field may be, for example, a flow field,, an electric field, or a preliminary magnetic field.
- the transverse field can be used to enhance the separation of controls from complexed beads to affect the length of movement in the channel, and for other reasons.
- the movement of a single bead within a channel is monitored.
- the position of a bead can be defined by the centroid of the light intensity distribution or by the thresholded image of the bead using methods which are well- known to those of skill in the art. Both approaches offer a spatial resolution which is better than one pixel.
- the grey-scaled intensity image of the bead is "binarized", i.e. the grey-scale image is analyzed pixel by pixel; pixel intensities smaller than a threshold value, typically chosen by the user, are assigned white; intensities larger than the same threshold are assigned black.
- the grey-scale image is thus transformed into a black and white image.
- the spatial resolution that already has been obtained using video-enhanced light microscopy is about 3 nm (Apgar J., Tseng, Y., Federov, E., Herwig, M.B., Almo, S.C. and Wirtz, D. (2000) Biophysical Journal, 79, 1095-1106).
- the movement of several beads is tracked.
- the number of beads loaded into a channel must be small enough so that all of the beads in each channel can be tracked individually. From this data one can compute the position of, for example, the center of mass of the group of beads as a function of time. When displacement of the center of mass of the control beads is sufficiently different than that of the complexed beads, protein attachment can be detected. This approach is more precise than single bead tracking since it computes an average speed of migration from an ensemble of particles and thus eliminates much of the uncertainty due to differences in bead size.
- particle-packet centroid tracking instead of tracking individual beads, the entire group of beads in a channel is analyzed as if it were a single object, or "packet". The process is illustrated in Figure 10.
- the grey-scaled intensity image 20 of a bead packet migrating in a channel 2 is "binarized".
- the grey-scale image 20 is analyzed pixel by pixel; pixel intensities smaller than a threshold value, typically chosen by the user, are assigned white; intensities larger than the same threshold are assigned black.
- the grey-scale image 20 is thus transformed into a sha ⁇ ly delineated black and white image 30.
- the displacement of the packet's centroid 40 i.e. center of mass position
- Determination of the position of the centroid can be based either on light intensity measurements or on the threshold shape of the packet. Comparison of the migration of the centroids of control vs. complexed bead packets allow detection of protein binding to complexed beads because the complexed beads migrate more slowly due to their increased hydrodynamic drag, i.e. if the centroid of a complexed bead packet moves faster than its control, then protein is attached to the complexed beads.
- particle-packet centroid tracking is that it automatically integrates size polydispersity by tracking many beads at the same time. Moreover, subpixel spatial resolution can be obtained.
- images of beads traveling in adjacent channels are collected using either a digital camera or a SIT camera. These images are obtained with either bright field microscopy or fluorescence microscopy (for beads which have fluorescent tags attached, either directly to the bead or to the antibody). Distances traveled by the beads are computed by monitoring the center-of-mass displacements of the controls and, in the adjacent channel, of the beads that were incubated with the cell extract.
- the displacement of control beads and complexed beads are compared.
- the speed of movement of the beads will depend on the size of the bead, and when the beads are of an appropriate size, on the type and quantity of protein molecules that are attached to the beads. Further, measurements of complexed beads with known quantities of bound proteins whose size is known may also be used to generate standard calibration curves for use in quantitating the amount of an unknown protein which is bound to a magnetic bead.
- spatial resolution we mean the minimum measurable distance between individual beads or bead packets traveling in two different channels. This spatial resolution depends on:
- Calculation of the spatial resolution between control and complexed beads is carried out as follows for a magnetic bead of magnetic susceptibility ⁇ and volume V, subject to a constant magnetic field gradient dH/dx and moving in a buffer of viscosity ⁇ .
- the magnetic susceptibility can be rendered constant in the supe ⁇ aramagnetic beads employed in the present invention by applying a constant magnetic field of ⁇ 200 Gauss, which is readily obtained by magnets which are well known to those of skill in the art.
- the magnetic force is proportional to ⁇ VdH/dx, and the friction force to 6 ⁇ ⁇ v, where v is the velocity of the bead and a is its hydrodynamic radius. Therefore, v is proportional to ( ⁇ VdH/dx / 6 ⁇ a ⁇ , i.e. it is proportional to the inverse of the hydrodynamic radius of the particle.
- the volume V of the magnetic material that constitutes the bead is constant regardless of protein attachment to the bead. However, the hydrodynamic radius increases upon protein attachment. If a protein becomes attached to the bead, the radius increases by ⁇ to a + ⁇ .
- v is then proportional to ( ⁇ VdH/dx / 6 ⁇ (a + ⁇ ) ⁇ Therefore, the velocity of the complexed bead is less than that of the equivalent control bead. Note that, for a given magnetic field gradient and suspending buffer, the distance traveled by the centroid of the bead depends only on its hydrodynamic radius.
- the attached protein increases the hydrodynamic radius by 5nm) then the distance traveled by the control bead during 10 seconds of image collecting time is given by 10s x 0.1 pN / 6 ⁇ ⁇ or -265 ⁇ m, while for a bead which has protein attached, the distance traveled by that bead is reduced to 10s x 0.01 pN / 6 ⁇ ( + ⁇ ) ⁇ , or -212 ⁇ m
- the difference between the two distances measured in this manner is 53 ⁇ m, which is readily detectable by light microscopy.
- the force applied to the beads may be smaller by, for example, a factor of 100. Because this results in a smaller magnetic susceptibility, then one would simply apply the smaller force for a longer period of time (e.g.
- the relative distance is much larger for a small bead than for a large bead.
- the magnetic nanobeads to be utilized will have a radius in the range of about 5 to about 1000 nm.
- Magnetic beads which are currently commercially available fall outside this size range but may still be used in the practice of this invention.
- Dynal manufactures micron-size (2.8, 4.5, and 5.0 ⁇ m) spherical beads for protein-purification and cell-sorting applications. These beads are supe ⁇ aramagnetic, uniform in size, and contain a dispersion of magnetic material (Fe 2 C an d Fe 3 O 4 ).
- Beckman-Coulter also manufactures 1 ⁇ m-diameter supe ⁇ aramagnetic beads for cell-sorting applications. These beads also contain a dispersion of magnetic material.
- the present invention provides magnetic nanobeads which fall within the preferred radius range of about 5 to about 50 nm.
- the beads are hexagonal shaped crystals of ⁇ Fe 2 O 3 beads, which are made using the counterflow diffusion flame reactor in which iron carbonyl is utilized as the precursor.
- the counterflow diffusion flame reactor is described in United States Patents 5,268,337 to Katz et al. and 5,650,130 to Katz et al. and the complete contents of both patents is herein inco ⁇ orated by reference. Beads manufactured by this process are nearly all of exactly the same shape and have a very narrow size distribution. Further, the size can be extensively controlled.
- the small size of the magnetic beads of the present invention provides a distinct advantage over previously known magnetic beads for a number of reasons, for example: 1) Using small beads helps reduce the amount of undesirable contact between the bead surface and the attached protein, as compared to larger beads. Ideally, a protein bound to a bead does not contact the surface of the bead at all but makes contact only with appropriate atoms of the slinker. (The slinker itself is, of course, linked to the bead surface, either directly or indirectly via a spacer molecule.) Portions of the protein not directly involved with attachment to the slinker would remain free, tethered to the bead by the slinker but held away from the bead surface.
- a bead has a large radius (i.e. a small curvature) then a protein "perceives" the bead as being relatively flat, and the probability of contact between the protein and the bead surface is higher than if the bead's radius is small (i.e. if the bead's curvature is large). Using small beads therefore reduces the probability of contact between the bead surface and the attached protein, thus decreasing the possibility of protein denaturation.
- the relative levels of expression of proteins in different specimens may be obtained from the distances traveled by a bead in a fixed length of time. For example, two specimens (e.g. normal vs cancer cells) can be compared side-by-side. If proteins are expressed at different levels, the "pattern" of progression of the magnetic beads will be different; beads with more bound protein (i.e. from a sample which contains a higher amount of the protein) will travel a lesser distance than beads containing less bound protein. Further analysis of the bound proteins (e.g. amino acid analysis or sequencing) may also be carried out by sampling the beads at the ends of the channels.
- This method applies to the detection of proteins which not only are over-expressed in a sample (or which exist in higher concentrations in a mixture), but also to proteins which are down-regulated (or exist in lower concentrations) compared to a control.
- proteins which are down-regulated or exist in lower concentrations compared to a control.
- the corresponding protein a may be present or expressed at lower levels in a diseased specimen than in a normal, control specimen.
- an excess of particles in the case where protein a is down-regulated, a majority of ⁇ beads will travel a large distance when incubated with the disease sample than those that were incubated in the normal specimen due to the lower amount of attached protein.
- magnetic beads of different sizes may be utilized in order to enhance protein separation. This may be especially useful for proteins which are interactive.
- a protein mixture contains proteins a and b (plus ab complex) which bind antibodies ⁇ and ⁇ , respectively.
- Antibody a is immobilized on beads of a relatively large size.
- Antibody ⁇ is immobilized on beads of a relatively small size.
- the protein mixture is first exposed to antibody ⁇ , which will bind all of protein a.
- the protein mixture then is exposed to antibody ⁇ thus binding the protein b.
- the large beads the ⁇ beads
- the ⁇ beads will move much more rapidly than the ⁇ beads (regardless of the quantities of protein bound), because of the large difference in bead size.
- the attachment of a single (as opposed to more than one) slinker to a particle is greatly facilitated by using small (e.g., 5-50 nm-radius) magnetic particles.
- small e.g., 5-50 nm-radius
- a very large excess of small magnetic particles is incubated with a relatively small concentration of a given slinker. This greatly increases the probability that each particle becomes attached to either zero or one slinker, as opposed to more than one slinker.
- all particles are subject to a force; those particles which became attached to one slinker during the precedent incubation will move more slowly than the particles attached to no slinker.
- additional slinkers are positioned at or near the distal ends of the channels through which the beads migrate.
- One pu ⁇ ose for these additional slinkers is to "capture” the beads or a subset of the beads as they migrate through the channel.
- such "capturing slinkers” may be positioned (e.g. via immobilization) at or near the distal end of a channel. They may be, for example, specific antibodies or general -motif antibodies directed to a motif on a protein of interest. The protein of interest is already attached to a migrating bead or beads via a slinker as described above.
- the bead-protein complex will migrate through the channel and be stopped upon contacting the capturing slinker because the capturing slinker will bind to the protein of interest, sequestering the protein and the attached bead.
- beads with no bound protein, or beads bound to a protein which is not recognized by the capturing slinker can be separated from beads bound to a protein which is recognized by the capturing slinker. Beads with no bound protein, or beads bound to a protein which is not recognized by the capturing slinker, would freely migrate past the capturing antibody.
- binding events between immobilized slinkers and proteins may be detected using optical fibers.
- an array of optical fibers, each coated with a different slinker is utilized. This array of fibers is dipped into the cell extract to be examined, to which a generic fluorescent dye has been added. The optical-fiber array is removed from the cell extract and a laser beam is shined into each optical fiber. If the fiber end is fluorescent, then a protein that interacts with the slinker on the fiber is present in the extract.
- Kit The present invention also provides a kit for the determination of the binding signatures of molecules (e.g. proteins) via RCP.
- a kit comprises a set of slinkers for use in determining the binding signature of one or more molecules correlated with a given condition of interest.
- the condition of interest may be a disease state.
- the slinkers may be antibodies (e.g. specific or general-motif antibodies) or other slinkers as described above.
- the slinkers may be immobilized, for example on a substrate such as a magnetic bead.
- the kit may further comprise such items as buffers, instructions for use, and database materials (e.g. software) for carrying out comparative analyses of the results obtained with the slinkers.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2002246621A AU2002246621A1 (en) | 2000-12-15 | 2001-12-14 | The separation, identification and quantitation of protein mixtures |
US10/450,194 US20040067599A1 (en) | 2001-12-14 | 2001-12-14 | Separation identification and quantitation of protein mixtures |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US25539300P | 2000-12-15 | 2000-12-15 | |
US60/255,393 | 2000-12-15 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2002060377A2 true WO2002060377A2 (en) | 2002-08-08 |
WO2002060377A3 WO2002060377A3 (en) | 2003-01-03 |
Family
ID=22968122
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2001/047754 WO2002060377A2 (en) | 2000-12-15 | 2001-12-14 | The separation, identification and quantitation of protein mixtures |
Country Status (2)
Country | Link |
---|---|
AU (1) | AU2002246621A1 (en) |
WO (1) | WO2002060377A2 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004081575A1 (en) * | 2003-03-12 | 2004-09-23 | Bioinvent International Ab | Screening assay |
WO2008076139A1 (en) * | 2006-03-10 | 2008-06-26 | Tethys Bioscience, Inc. | Multiplex protein fractionation |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5922858A (en) * | 1994-05-11 | 1999-07-13 | Trustees Of Boston University | Method for the detection and isolation of protein |
US6096870A (en) * | 1994-01-05 | 2000-08-01 | Sepragen Corporation | Sequential separation of whey |
US6121428A (en) * | 1997-06-13 | 2000-09-19 | Genentech, Inc. | Protein recovery |
US6123821A (en) * | 1997-06-24 | 2000-09-26 | Large Scale Biology Corporation | Automated system for two-dimensional electrophoresis |
-
2001
- 2001-12-14 WO PCT/US2001/047754 patent/WO2002060377A2/en not_active Application Discontinuation
- 2001-12-14 AU AU2002246621A patent/AU2002246621A1/en not_active Abandoned
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6096870A (en) * | 1994-01-05 | 2000-08-01 | Sepragen Corporation | Sequential separation of whey |
US5922858A (en) * | 1994-05-11 | 1999-07-13 | Trustees Of Boston University | Method for the detection and isolation of protein |
US6121428A (en) * | 1997-06-13 | 2000-09-19 | Genentech, Inc. | Protein recovery |
US6123821A (en) * | 1997-06-24 | 2000-09-26 | Large Scale Biology Corporation | Automated system for two-dimensional electrophoresis |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004081575A1 (en) * | 2003-03-12 | 2004-09-23 | Bioinvent International Ab | Screening assay |
AU2004219906B2 (en) * | 2003-03-12 | 2010-11-11 | Immunovia Ab | Screening assay |
AU2004219906B9 (en) * | 2003-03-12 | 2011-06-16 | Immunovia Ab | Screening assay |
WO2008076139A1 (en) * | 2006-03-10 | 2008-06-26 | Tethys Bioscience, Inc. | Multiplex protein fractionation |
US8097425B2 (en) | 2006-03-10 | 2012-01-17 | Tethys Bioscience, Inc. | Multiplex protein fractionation |
Also Published As
Publication number | Publication date |
---|---|
WO2002060377A3 (en) | 2003-01-03 |
AU2002246621A1 (en) | 2002-08-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US5137609A (en) | Differential separation assay | |
JP4007459B2 (en) | High throughput test | |
AU773291B2 (en) | UPA, a universal protein array system | |
EP1687633B1 (en) | Use of particulate labels in bionalyte detection methods | |
US7232691B2 (en) | Bioassay and biomolecular identification, sorting, and collection methods using magnetic microspheres | |
EP2765424B1 (en) | Method for analyzing biomolecules | |
US5221454A (en) | Differential separation assay | |
AU5308000A (en) | Array cytometry | |
JP2004514114A (en) | Method for analyzing multiple analyte molecules using specific random particle arrays | |
EP2203749A2 (en) | Highly multiplexed particle-based assays | |
JP4533536B2 (en) | Multi-colored sign | |
US20030186465A1 (en) | Apparatus used in identification, sorting and collection methods using magnetic microspheres and magnetic microsphere kits | |
US20160320629A1 (en) | Fluidic Super Resolution Optical Imaging Systems With Microlens Array | |
JP4425640B2 (en) | DNA-binding protein detection method | |
US20050239076A1 (en) | Analysis system | |
US20040067599A1 (en) | Separation identification and quantitation of protein mixtures | |
WO2002060377A2 (en) | The separation, identification and quantitation of protein mixtures | |
US8084257B2 (en) | Methods for sorting dimorphic daughter cells | |
US7670833B2 (en) | High throughput analysis for molecular fractions | |
JP2009250652A (en) | Screening method for functional material by measuring cell migration speed | |
CN113687059A (en) | Protein target molecule digital quantitative detection method based on virtual segmentation method |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PH PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
AK | Designated states |
Kind code of ref document: A3 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PH PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A3 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 10450194 Country of ref document: US |
|
122 | Ep: pct application non-entry in european phase | ||
NENP | Non-entry into the national phase in: |
Ref country code: JP |
|
WWW | Wipo information: withdrawn in national office |
Country of ref document: JP |