WO2006085958A2 - Integration of direct binding sensors with mass spectrometry for functional and structural characterization of molecules - Google Patents
Integration of direct binding sensors with mass spectrometry for functional and structural characterization of molecules Download PDFInfo
- Publication number
- WO2006085958A2 WO2006085958A2 PCT/US2005/022719 US2005022719W WO2006085958A2 WO 2006085958 A2 WO2006085958 A2 WO 2006085958A2 US 2005022719 W US2005022719 W US 2005022719W WO 2006085958 A2 WO2006085958 A2 WO 2006085958A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- molecules
- optical sensor
- colorimetric resonant
- resonant reflectance
- reflectance optical
- Prior art date
Links
Classifications
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
-
- 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
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
-
- 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
-
- 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
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
Definitions
- Detection assays combined with structural analysis can provide complementary information on function and structure of molecules. Previous work has demonstrated the utility of this combined approach for applications such as ligand fishing, epitope mapping, and amino acid sequencing, but only in the low throughput sample-limited context of a microfluidic channel-surface plasmon resonance (SPR)-based systems. See, Nelson et al, BIA/MS of Epitope-Tagged Peptides Directly from E. coli Lysate: Multiplex Detection and Protein Identification at Low-Femtomole to Subfemtoniole Levels. Analytical Chemistry 1999, 71:2858-2865; Nelson et al., Biosensor chip mass spectrometry: A chip-based proteomics approach.
- SPR microfluidic channel-surface plasmon resonance
- Electrophoresis 2000, 21:1155-1163 Previously known methods of combining detection assays with structural analysis are extremely limited in utility by the sub-femtomole quantities of bound material that can be recovered from a flow cell, and by the many sample injection/detection/elution cycles required to generate sufficient quantities of detectable material. See e.g., Williams & Addona, The integration of SPR sensors with mass spectrometry: possible applications for proteome analysis. Tibtech 2000, 18:45-48.
- One embodiment of the invention provides a method of analyzing or identifying one or more molecules.
- the method comprises contacting a sample comprising the one or more molecules with a colorimetric resonant reflectance optical sensor such that one or more of the one or more molecules become immobilized to the colorimetric resonant reflectance optical sensor.
- the immobilized one or more molecules are eluted from the colorimetric resonant reflectance optical sensor and subjected to mass spectrometry analysis.
- the one or more molecules immobilized to the colorimetric resonant reflectance optical sensor can be detected.
- the one or more molecules immobilized to the colorimetric resonant reflectance optical sensor can be directly detected by a shift in peak wavelength value (PWV).
- PWV shift in peak wavelength value
- the one or more molecules immobilized to the colorimetric resonant reflectance optical sensor can be detected using a label.
- a peak wavelength value (PWV) signal can also be detected.
- the detecting can comprise use of an indicator molecule of equal, greater, or lesser molecular mass of the one or more molecules immobilized to the colorimetric resonant reflectance optical sensor.
- the one or more molecules can be quantified.
- the one or more molecules can be immobilized to the colorimetric resonant reflectance optical sensor by one or more moieties on the surface of the colorimetric resonant reflectance optical sensor.
- the one or more moieties can be TiO, RaM Fc, avidin, biotin, an antibody, an antibody fragment, a nucleic acid molecule, protein A, hybrids of protein A, protein G, hybrids of protein G, protein L, hybrids of protein L, high density PVA, CHO or a combination thereof.
- the colorimetric resonant reflectance optical sensor can be coupled to a flow system for detection.
- Another embodiment of the invention provides a method of analyzing or identifying one or more molecules.
- the method comprises contacting a sample comprising one or more molecules with a colorimetric resonant reflectance optical sensor such that one or more of the one or more molecules become immobilized to the colorimetric resonant reflectance optical sensor; obtaining any molecules that do not become immobilized to the colorimetric resonant reflectance optical sensor; and subjecting the any molecules that do not become immobilized to the colorimetric resonant reflectance optical sensor to mass spectrometry analysis.
- Yet another embodiment of the invention provides a method of analyzing or identifying one or more molecules.
- the method comprises contacting a sample comprising one or more molecules with a flow based surface plasmon resonance sensor such that one or more of the one or more molecules become immobilized to the sensor; eluting the immobilized one or more molecules from the sensor; and subjecting the one or more molecules to mass spectrometry analysis.
- the one or more molecules immobilized to the sensor can be detected and/or quantified.
- Still another embodiment of the invention provides a method of analyzing or identifying one or more molecules.
- the method comprises contacting a sample comprising one or more molecules with a surface plasmon resonant sensor such that one or more of the one or more molecules become immobilized to the sensor; obtaining any molecules that do not become immobilized to the sensor; subjecting the any molecules that do not become immobilized to the sensor to mass spectrometry analysis.
- the one or more molecules immobilized to the sensor can be detected and/or quantified.
- Figure 1 shows a sequence of steps for a colorimetric resonant reflectance optical sensor/MS analysis of a protein using an external MALDI-TOF plate.
- the colorimetric resonant reflectance optical sensor microplate is prepared with immobilized ligand that specifically detects a target protein from the test sample, registering a positive PWV shift. After binding, the bound target is eluted, mixed with MALDI matrix, and applied as a 1 ⁇ l spot to a standard MALDI plate for analysis.
- Figure 2 shows data from a colorimetric resonant reflectance optical sensor system showing the positive normalized PWV shift (ordinate values) for the attachment of antibody (hlgG & clgG) and binding of antigen (Fab) and reduction of PWV shift as antigen (Fab) is eluted as a function of time (abscissa values) for the experiment.
- the elution volume with 1OmM glycine buffer pH 2 was -20 ⁇ L.
- Figures 3A & Figure 3B show MALDI-TOF spectra for control of 2 pmol of Fab.
- Figure 3B shows MALDI-TOF spectra for l ⁇ L of material eluted from a single well of the colorimetric resonant reflectance optical sensor in Figure 2 spotted onto the MALDI surface with 1/xL sinapinic acid.
- the main peak for the expected material at 22,300 Da molecular weight is labeled.
- the two other significant peaks (11057 & 44967) are related to the major product peak.
- Figure 4 shows data from a colorimetric resonant reflectance optical sensor showing the positive normalized PWV shift (ordinate values) for the binding of antigen-A (Biogen pou proprietary molecule and Ab) and reduction of PWV shift as antigen-A is eluted as a function of time (abscissa values) for the experiment.
- the elution volume with 1OmM glycine buffer pH 2 was -12 ⁇ L.
- Figure 5 shows ESI-MS data for 6 ⁇ L of eluted material from the colorimetric resonant reflectance optical sensor shown in Figure 4. The only significant peaks are related to the primary 17,900 expected product.
- Figure 6A shows a cross-sectional view of a sensor wherein light is shown as illuminating the bottom of the sensor; however, light can illuminate the sensor from either the top or the bottom.
- n substrate represents substrate material
- ni represents the refractive index of an optional cover layer.
- n 2 represents the refractive index of a grating.
- Figure 6B shows another view of a sensor.
- Figure 7 shows an embodiment of a colorimetric resonant reflectance optical sensor comprising a one-dimensional grating.
- Figure 8 shows a resonant reflection structure consisting of a set of concentric rings.
- Figure 9 shows a resonant reflective structure comprising a hexagonal grid of holes (or a hexagonal grid of posts) that closely approximates the concentric circle structure of Figure .8 without requiring the illumination beam to be centered upon any particular location of the grid.
- Direct binding assays such as those supported by colorimetric resonant reflectance optical sensors, are able to monitor biomolecular or cellular interactions as they occur between an immobilized receptor and a ligand in solution.
- the technology can determine the functional characteristics of proteins, and can be used to determine affinity parameters.
- MS-based techniques such as matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), and electrospray ionization (ESI), are used in the structural characterization of organic molecules, such as proteins, and inorganic molecules with analysis ranging from protein sequence verification through an accurate mass determination to protein identification via peptide mass mapping combined with database search.
- MALDI-TOF matrix-assisted laser desorption/ionization time-of-flight
- ESI electrospray ionization
- Mass spectroscopy is not a universal detection method and often is not able to detect the presence of many materials if not properly tuned or focused on detecting them. With the present invention, the mass spectroscopist would know that material was indeed present and in what quantities and would thus be able to develop a better method to detect its presence, leading to the ability to identify the material by its mass and structure. hi one embodiment of the present invention, a method for the simplification of mass spectra can be achieved. Current trends in pushing mass spectroscopy technology to its greatest ends involve the mass analysis of very complex samples. The present invention allows for the deconvolution of the most complex samples by a sensor while preserving the mass analysis. A sensor allows for the specific selection and quantification of materials from complex media thus "cleaning" up a sample prior to its introduction to the mass analysis.
- Mass spectra can often be crowded or have overlapping signals for larger molecular weight materials such as but not limited to biomaterials. This is especially true for complex samples from proteomics analyses, patient samples, pre-clinical samples from animals, whole cell lysates and the like. Removal of specific materials by the sensor (e.g., by immobilization of specific materials in a sample to the sensor and the use of the solution comprising the unbound specific material in MS analysis) can allow the mass spectra to become more easily determined for other masses, m an additional approach, the mass spectra can confirm the specific removal or selection of a material by the sensor. This could be accomplished by mass analysis of the material that did not adhere to the sensor.
- One embodiment of the present invention has a significant benefit over previously described methods of combining a sensor with mass analysis.
- the present invention can be practiced with a static (meaning non-flow-based system) well based-sensor and as such offers significant advantage over other systems.
- the static based system is able to capture more transient interactions. Molecular interactions are described by rates for the amount of time that it takes for two molecules to form a pair and the amount of time that it takes for the two molecules to dissociate. When the dissociation rate is in a faster regime, the identification and analysis of the association is significantly challenging.
- a static system that is allowed to come to equilibrium has a far greater chance of capturing the interaction and preserving it for analysis than does any flow-based system described to date.
- the present invention provides the mass analyst more sample to work with as a benefit to having any sample to work with at all.
- Another embodiment of the invention provides a system where the colorimetric reflectance optical sensor is coupled to a flow system.
- the flow system can provide for high throughput screening of molecules.
- Another embodiment of the current invention provides a method for the screening of individual or pools of small molecules.
- the world of small molecules for drug discovery is composed of large libraries of compounds many of which are thought to be pure or of substantially a single component while other parts of these libraries are composed of extracts from exotic organisms that have survived the rigors of environmental challenges.
- Many methods have been tried for the identification of specific binders of high affinity to human and veterinary disease targets.
- the present invention allows the detection and identification of members of the library that are binding with high affinity and specificity to said drug targets.
- Another embodiment of the current invention provides a method for the screening of individual or pools of proteins. Following the great focus on human and pathogen genomics, the forefront of academic and pharmaceutical research has spent considerable time and effort studying proteomics or the presence, activity, and interaction of the vast number of proteins prescribed by the animal genome. The present invention provides methods for the study of the animal proteome by various techniques described herein.
- Another embodiment of the invention provides a method for quantifying molecules.
- the molecules can be quantified by colorimetric resonant reflectance optical sensor, MS, or both colorimetric resonant reflectance optical sensor and MS.
- Molecules are quantified by colorimetric resonant reflectance optical sensor by immobilizing a ligand molecule to the colorimetric resonant reflectance optical sensor surface, and then detecting the PWV shift of the quantified molecule when it is exposed to the sensor+ligand surface. If the user had previously developed a calibration curve (PWV shift versus concentration of molecule), then the molecule concentration can be quantified by colorimetric resonant reflectance optical sensor.
- the PWV shift may be generated by a mixture of molecules.
- the bound molecules may be removed from the surface of the colorimetric resonant reflectance optical sensor and suspended in a solution. IfMS analysis is performed on the solution, then the analytes that had been previously bound to the sensor can be quantified.
- a binding constant is determined for one or more molecules.
- Samples comprising a range of analyte concentrations can be exposed to an immobilized ligand on the surface of a colorimetric resonant reflectance optical sensor.
- the binding constant for the ligand+analyte combination is determined.
- the affinity of the analytes can be compared against each other by the magnitude of the sensor PWV signal.
- the bound material can be eluted from the sensor surface. The magnitude of the MS signal from the eluted solution can be measured to determine a binding constant.
- Another embodiment of the invention provides method for preparing a Matrix Assisted Laser Desorption and Ionization/MS (MALDI/MS) matrix.
- the matrix can comprise colorimetric resonant reflectance optical sensor with one or more specific binding substances immobilized on the surface of the sensor.
- the one or more specific binding substances can be bound to their respective binding partners.
- the one or more specific binding substances can be arranged in an array on the sensor surface.
- Colorimetric resonant reflectance optical sensors can comprise a subwavelength structured surface (SWS).
- SWS can create a sharp optical resonant reflection at a particular wavelength that can be used to track with high sensitivity the interaction of materials, including for example, specific binding substances or binding partners or both.
- a SWS acts as a surface binding platform for specific binding substances.
- SWSs are an unconventional type of diffractive optic that can mimic the effect of thin-film coatings.
- a SWS structure comprises a surface-relief grating, such as a one-dimensional, two-dimensional, or three dimensional grating in which the grating period is small compared to the wavelength of incident light.
- the reflected or transmitted color of this structure can be modulated by the addition of molecules such as specific binding substances, binding partners, or both, or inorganic molecules to the upper surface of the cover layer or the grating surface.
- molecules such as specific binding substances, binding partners, or both, or inorganic molecules to the upper surface of the cover layer or the grating surface.
- the dielectric susceptibility of the added molecules result in a modification of the wavelength at which maximum reflectance or transmittance will occur.
- a sensor when illuminated with white light, is designed to reflect only a single wavelength or a narrow band of wavelengths.
- specific binding substances or binding partners or both are attached or immobilized to the surface of the sensor, the reflected wavelength (color) is shifted.
- complementary binding partner molecules can be detected without the use of any kind of fluorescent probe, or particle label or any other type of label.
- one or more labels or indicator molecules can also be used.
- a label molecule can be a dye, a fluorescent molecule, a bioluminescent molecule, and the like.
- An indicator molecule can be a biological or immuno-derived molecule of equal, greater, or lesser molecular mass of the one or more molecules immobilized to the colorimetric resonant reflectance optical sensor, e.g., a protein, peptide, nucleic acid, peptide nucleic acid, locked nucleic acid, and the like.
- the detection technique is capable of resolving changes of, for example, -0.1 nm thickness of protein binding, and can be performed with the sensor surface either immersed in fluid or dried.
- a detection system consists of, for example, a light source that illuminates a small spot of a sensor at normal incidence through, for example, a fiber optic probe, and a spectrometer that collects the reflected light through, for example, a second fiber optic probe also at normal incidence. Because no physical contact occurs between the excitation/detection system and the sensor surface, no special coupling prisms are required and the sensor can be easily adapted to any commonly used assay platform including, for example, microtiter plates and microarray slides. A single spectrometer reading can be performed in several milliseconds, thus it is possible to quickly measure a large number of molecular interactions taking place in parallel upon a sensor surface, and to monitor reaction kinetics in real time.
- the grating period is selected to achieve resonance at a desired wavelength.
- a sensor comprises an optical grating comprised of a high refractive index material, a substrate layer that supports the grating, and one or more specific binding substances immobilized on the surface of the grating opposite of the substrate layer.
- a cover layer covers the grating surface.
- An optical grating made according to the invention is coated with or comprises a high refractive index dielectric film which can be comprised of a material that includes, for example, zinc sulfide, titanium dioxide, tantalum oxide, and silicon nitride.
- a sensor of the invention can also comprise an optical grating comprised of, for example, plastic or epoxy, which is coated with a high refractive index material.
- Linear gratings (i.e., one dimensional gratings) have resonant characteristics where the illuminating light polarization is oriented perpendicular to the grating period.
- a schematic diagram of one embodiment a linear grating structure with an optional cover layer is shown in Figure 7.
- a colorimetric resonant reflectance optical sensor can also comprise, for example, a two-dimensional grating.
- a cross-sectional profile of a grating with optical features can comprise any periodically repeating function, for example, a "square-wave.”
- An optical grating can also comprise a repeating pattern of shapes selected from the group consisting of lines, squares, circles, ellipses, triangles, trapezoids, sinusoidal waves, ovals, rectangles, and hexagons.
- a linear grating has the same pitch (i.e. distance between regions of high and low refractive index), period, layer thicknesses, and material properties as a two-dimensional grating.
- pitch i.e. distance between regions of high and low refractive index
- period i.e. distance between regions of high and low refractive index
- layer thicknesses i.e. thickness between regions of high and low refractive index
- material properties as a two-dimensional grating.
- light must be polarized perpendicular to the grating lines in order to be resonantly coupled into the optical structure in a manner that results in the most sharp resonant peak. Therefore, a polarizing filter oriented with its polarization axis perpendicular to the linear grating must be inserted between the illumination source and the sensor surface.
- An optical grating can also comprise, for example, a "stepped" profile, in which high refractive index regions of a single, fixed height are embedded within a lower refractive index cover layer.
- the high refractive index material is not stepped, but which varies with lateral position.
- the high refractive index material of the two-dimensional grating, n, 2 is sinusoidally varying in height.
- the period of the sinusoid is identical to the period of an equivalent stepped structure.
- the resonant operation of the sinusoidally varying structure and its functionality as a sensor has been verified using GSOLVER (Grating Solver Development Company, Allen, Texas, USA) computer models.
- a sensor of the invention can further comprise a cover layer on the surface of an optical grating opposite of a substrate layer. Where a cover layer is present, the one or more specific binding substances are immobilized on the surface of the cover layer opposite of the grating.
- a cover layer comprises a material that has a lower refractive index than a material that comprises the grating.
- a cover layer can be comprised of, for example, glass (including spin-on glass (SOG)), epoxy, or plastic.
- SOG can be used due to its favorable refractive index, ease of handling, and readiness of being activated with specific binding substances using the wealth of glass surface activation techniques.
- a grating structure of SiN/glass can directly be used as the sensing surface, the activation of which can be done using the same means as on a glass surface.
- a sensor can comprise a substrate coated with a structured thin film layer of high refractive index material. Without the use of a planarizing cover layer, the surrounding medium (such as air or water) fills the grating. Therefore, specific binding substances are immobilized to the sensor on all surfaces of an optical grating exposed to the specific binding substances, rather than only on an upper surface.
- a specific binding substance can be, for example, an organic or inorganic molecule, a nucleic acid, peptide, protein solutions, peptide solutions, single or double stranded DNA solutions, RNA solutions, RNA-DNA hybrid solutions, solutions containing compounds from a combinatorial chemical library, purified or mixtures of small molecule test compounds such as those used in the pharmaceutical industry to develop drug leads, individual or pools of proteins mixtures from various sources such as bacterial, viral, human, or other animal sources for proteome analyses, man-made, synthetic, or animal derived periplasmic extracts, antigen, polyclonal antibody, monoclonal antibody, single chain antibody (scFv), F(ab) fragment, F(ab') 2 fragment, Fv fragment, purified or mixtures of immunobodies (e.g., scFv, sFab, F(ab), whole antibodies),
- immunobodies e.g., scFv, sFab, F(ab), whole antibodies
- a biological sample can be for example, blood, plasma, serum, gastrointestinal secretions, homogenates of tissues or tumors, synovial fluid, feces, saliva, sputum, cyst fluid, amniotic fluid, cerebrospinal fluid, peritoneal fluid, lung lavage fluid, semen, lymphatic fluid, tears, or prostatic fluid.
- a polymer can be selected from the group of long chain molecules with multiple active sites per molecule consisting of hydrogel, dextran, poly-amino acids and derivatives thereof, including poly-lysine (comprising poly-1-lysine and poly-d-lysine), poly-phe-lysine and poly-glu-lysine.
- one or more specific binding substances are arranged in an array of one or more distinct locations on a sensor.
- An array of specific binding substances comprises one or more specific binding substances on a surface of a sensor of the invention' such that a surface contains many distinct locations, each with a different specific binding substance or with a different amount of a specific binding substance.
- an array can comprise 1, 10, 100, 1,000, 10,000 or 100,000 distinct locations.
- Such a sensor surface is called an array because one or more specific binding substances are typically laid out in a regular grid pattern in x-y coordinates.
- an array can comprise one or more specific binding substance laid out in any type of regular or irregular pattern.
- distinct locations can define an array of spots of one or more specific binding substances.
- An array spot can be about 50 to about 500 microns in diameter.
- An array spot can also be about 150 to about 200 microns in diameter.
- One or more specific binding substances can be bound to their specific binding partners.
- An array on a sensor of the invention can be created by placing microdroplets of one or more specific binding substances onto, for example, an x-y grid of locations on a grating or cover layer surface.
- the binding partners will be preferentially attracted to distinct locations on the microarray that comprise specific binding substances that have high affinity for the binding partners. Some of the distinct locations will gather binding partners onto their surface, while other locations will not.
- a specific binding substance specifically binds to a binding partner that is added to the surface of a sensor of the invention.
- a specific binding substance specifically binds to its binding partner, but does not substantially bind other binding partners added to the surface of a sensor.
- the specific binding substance is an antibody and its binding partner is a particular antigen, the antibody specifically binds to the particular antigen, but does not substantially bind other antigens.
- a binding partner can be, for example, an inorganic molecule, an organic molecule, a nucleic acid, peptide, protein solutions, peptide solutions, single or double stranded DNA solutions, RNA solutions, RNA-DNA hybrid solutions, solutions containing compounds from a combinatorial chemical library, purified or mixtures of small molecule test compounds such as those used in the pharmaceutical industry to develop drug leads, individual or pools of proteins mixtures from various sources such as bacterial, viral, human, or other animal sources for proteome analyses, man-made, synthetic, or animal derived periplasmic extracts, antigen, polyclonal antibody, monoclonal antibody, single chain antibody (scFv), F(ab) fragment, F(ab') 2 fragment, Fv fragment, purified or mixtures of immunobodies (e.g., scFv, sFab, F(ab), whole antibodies), small organic molecule, cell, virus, bacteria, polymer, TiO, RaM avidin, biotin, protein A, hybrids of protein A
- a biological sample can be, for example, blood, plasma, serum, gastrointestinal secretions, homogenates of tissues or tumors, synovial fluid, feces, saliva, sputum, cyst fluid, amniotic fluid, cerebrospinal fluid, peritoneal fluid, lung lavage fluid, semen, lymphatic fluid, tears, and prostatic fluid.
- an array of the invention is a nucleic acid array, in which each distinct location within the array contains a different nucleic acid molecule.
- the spots within the nucleic acid microarray detect complementary chemical binding with an opposing strand of a nucleic acid in a test sample.
- microarrays are increasingly seen as a means for maximizing the number of biochemical interactions that can be measured at one time while minimizing the volume of precious reagents.
- specific binding substances By application of specific binding substances with a microarray spotter onto a sensor of the invention, specific binding substance densities of 10,000 specific binding substances/in 2 can be obtained.
- a sensor By focusing an illumination beam to interrogate a single microarray location, a sensor can be used as a microarray readout system.
- a sensor surface can be, for example, an internal surface of a microtiter plate.
- both the microarray and microtiter plate embodiments can be combined such that one or more specific binding substances are arranged in an array of one or more distinct locations on the sensor surface, said surface residing within one or more wells of the microtiter plate and comprising one or more surfaces of the microtiter plate, preferably the bottom surface.
- the array of specific binding substances comprises one or more specific binding substances on the sensor surface within a microtiter plate well such that a surface contains one or more distinct locations, each with a different specific binding substance or with a different amount of a specific binding substance.
- an array can comprise 1, 10, 100, 1,000, 10,000 or 100,000 distinct locations.
- each well of the microtiter plate embodiment can have within it an array of one or more distinct locations separate from the other wells of the microtiter plate embodiment, which allows multiple different samples to be processed on one microtiter plate of the invention, one or more samples for each separate well.
- the array or arrays within any one well can be the same or different than the array or arrays found in any other microtiter wells of the same microtiter plate.
- One or more specific binding substances can be immobilized to a sensor using methods well know in the art. Additionally, specific binding substances or binding partners of both can be eluted from the surface of a sensor using methods well known in the art.
- Test samples can be analyzed by mass spectrometry using any type of mass spectrometer and any type of mass spectrometry methodologies. Additionally, mass spectrometers connected to other devices such as gas chromatographs, liquid cliromatographs, super critical fluid chromatographs, and capillary electrophoresis devices can be used in the methods of the invention.
- Fourier transform ion cyclotron resonance (FT-ICR) spectrometer and time-of-flight (TOF) mass spectrometer, ElectroSpray Ionization Mass Spectrometry (ESI-MS), sector, and quadrupole devices can be used be used in the methods of the invention.
- Selected ion monitoring (SIM) can be used for quantitative analysis if desired.
- Mass spectrometry/mass spectrometry (MS/MS), isotope mass spectrometry, and elemental mass spectrometry techniques can also be used in the methods of the invention. Methods of the Invention.
- Combination of colorimetric resonant reflectance optical sensor assays with mass spectrometry techniques can be used to provide functional and structural characterization of organic and inorganic molecules, for example, proteins from serum. Additionally, the methods of the invention can be used in deorphaning of receptor analysis and protein identification by epitope tagging.
- a colorimetric resonant reflectance optical sensor/MS system is a multidimensional analytical approach that provides complementary information on, for example, protein function and structure, for example, post-translational alterations in a protein, in a simple, high throughput, highly sensitive platform.
- the binding affinities of an immobilized ligand for analytes in, for example, 384 test samples are monitored simultaneously in real time.
- the analyte selectively retained or selectively not retained on the colorimetric resonant reflectance optical sensor microplate wells can be eluted (for the selectively retained molecules) and subsequently analyzed by mass spectrometry, such as MALDI-TOF or ESI-MS, which can confirm the identity of the affinity-retained analyte and detects multiple affinity- retrieved analytes.
- mass spectrometry such as MALDI-TOF or ESI-MS
- colorimetric resonant reflectance optical sensor/MS integration is a cost effective approach for bringing new functionality to MS system users, and for differentiating MS systems from competing platforms.
- Ligand fishing or “deorphaning” is the process by which these receptors are screened against a multitude of compounds or cell/tissue extracts to identify possible ligands for the receptor. See, Williams, Biotechnology match making: screening orphan ligands and receptors. Current Opinion in Biotechnology 2000, 11 :42-46.
- Any well containing a high affinity binder for the orphan will register as a positive shift in peak wavelength value (PWV) in the colorimetric resonant reflectance optical sensor reader, registering a "hit.” Only the wells meeting the hit threshold are eluted for identification of the bound protein by MS.
- PWV peak wavelength value
- Colorimetric resonant reflectance optical sensor/MS can be used with gene- tagging techniques for protein identification. See e.g., Nelson et at, Analytical Chemistry 1999, 71:2858-2865.
- a tag is fused into nominally unknown genes for the purpose of tracking proteins throughput expression and for selectively isolating protein from the expression system (such as E. coli).
- the expression system such as E. coli.
- the sensor is used to affinity isolate, detect, and quantify the tagged polypeptide retrieved from the expression system.
- the masses of the tagged polypeptides are accurately determined by, for example, MALDI TOF analysis, which in turn are used for protein structural characterization such as sequence verification through a database search.
- the colorimetric resonant reflectance optical sensor assay system can be easily interfaced with MS -based analysis systems to concurrently provide, for example, protein affinity and protein identification information. Deorphaning of targets and epitope mapping are other applications that would take advantage of this unique capability, given the sensitivity, throughput, and cost advantages inherent in the colorimetric resonant reflectance optical sensor system that have not previously been available. Preliminary experiments have been performed to confirm the basic approach for off-line colorimetric resonant reflectance optical sensor + MALDI-TOF analysis of a target Fab fragment. (See Examples). Due to the simplicity and low cost of colorimetric resonant reflectance optical sensor reader instruments, colorimetric resonant reflectance optical sensor/MS integration is a cost effective approach for bringing new functionality to MS system users, and for differentiating MS systems from competing platforms.
- Another embodiment of the invention provides a method of analyzing or identifying one or more molecules comprising: contacting a sample comprising one or more molecules with a flow based surface plasmon resonance sensor such that one or more of the one or more molecules become immobilized to the sensor.
- the immobilized molecules can be eluted from the sensor or the non-immobilized molecules can be collected. These molecules are then subjected to mass spectrometry analysis.
- the one or more molecules immobilized to the sensor can be detected.
- the one or more molecules can be quantified.
- Figure 1 shows the protocol used to combine the colorimetric resonant reflectance optical sensor technique with a MALDI type mass spectroscopy experiment.
- the process comprises adsorb antibodies on a colorimetric resonant reflectance optical sensor plate (0.1 mg/ml) and adding Anti-human IgG.
- the control is chicken IgY.
- the antigen eluted with glycine (10 mM, pEN2.0).
- the solution is removed from the colorimetric resonant reflectance optical sensor plate.
- a ZipTip® pipette tip is used to desalt the solution that is removed to facilitate MS analysis.
- MALDI-MS data is obtained on Biogen Idec's ABI/Voyager system.
- MALDI-MS data is obtained on Biogen Idec's ABI/Voyager system.
- MALDI-MS data is obtained on Biogen Idec's ABI/Voyager system.
- a colorimetric resonant reflectance optical sensor TiO sensor was pre-rinsed with PBS 3 times and left at room temperature for 30 min. A baseline reading was taken for a few min and 10 ul of 1 mg/ml of human IgG or chicken IgY was diluted into 90 ul PBS already in the well for a final concentration of the antibodies of 100ug/mL. The protein was put into the well and allowed to bind to the TiO surface for 90 min. Unbound protein solution was removed from the well and the wells were rinsed 3 times each with 200 ul of PBS.
- Another baseline reading was taken for a few minutes and then 1 ul of 1 mg/ml of anti-human IgG (Fab)2 was put into the wells, which were coated with either hlgG (Red) or clgY (Yellow) and allowed to incubate for 60 min. AU the unbound protein solution was removed from the wells and the wells were rinsed 3 times with PBS. The binding signal was monitored for stability for few min. Any retained Fab, bound by hlgG, was eluted with 30 ul of 10 mM Glycine pH 2 buffer in each well. The elution process was monitored on the sensor instrument and any eluted protein was collected for mass spectroscopic analyses.
- Fab anti-human IgG
- Figure 3A shows the data from a control solution containing the Fab that was applied to the MS prior to exposure to the sensor surface.
- the primary peak is at 22300, the other two peaks are signature peaks related to the parent molecular mass
- Figure 3B shows the MALDI-MS data from the solution that was eluted from the sensor surface.
- the mass spectra is identical to the control spectra shown in Figure 3 A.
- Example 4
- Antibodies were adsorbed onto CHO colorimetric resonant reflectance optical sensor plate (low density aldehyde plate) (0.1 mg/mL).
- the antibody is a proprietary Human anti-Ag A antibody.
- the antigen protein was removed as evidenced by the differential signals determined by the sensor analysis.
- the MS data show definitive capture and mass determination of the specific antigen protein material that was eluted from the sensor.
- Antigen A binds at the 42 minute tirnepoint (abscissa values) to Antibody A that was immobilized onto CHO colorimetric resonant reflectance optical sensor.
- Figure 4 shows the raw mass spectra from the ESI-MS experiment for the injection of 6uL of the material eluted from a single 6mm sensor well.
- Figure 5 shows the mass analysis from the ESI-MS experiment for the injection of 6uL of the material eluted from a single 6mm sensor well.
- the parent peak is at the expected mass for the molecule of interest.
- the peak at 18052 mw is a signature peak related to the glycosylation of the primary peak.
- Figure 5 demonstrates the ability of the sensor to "clean" up a sample thus providing a more simplified mass analysis experiment.
- the sensor was initially coated with protein A in order to capture a human monoclonal Ab in preclinical experiments. Following the addition of the mAb to the sensor surface, 100% human serum was added to the sensor.
- the Examples demonstrate two extremes of the amount of materials a colorimetric resonant reflectance optical sensor is capable of providing for mass spectroscopic analyses, hi addition, similar ESI-MS results have been collected using 50% serum samples on a colorimetric resonant reflectance optical sensor system.
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002572459A CA2572459A1 (en) | 2004-06-28 | 2005-06-28 | Integration of direct binding sensors with mass spectrometry for functional and structural characterization of molecules |
AU2005327173A AU2005327173A1 (en) | 2004-06-28 | 2005-06-28 | Integration of direct binding sensors with mass spectrometry for functional and structural characterization of molecules |
EP05857481A EP1769230A2 (en) | 2004-06-28 | 2005-06-28 | Integration of direct binding sensors with mass spectrometry for functional and structural characterization of molecules |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US58356004P | 2004-06-28 | 2004-06-28 | |
US60/583,560 | 2004-06-28 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2006085958A2 true WO2006085958A2 (en) | 2006-08-17 |
WO2006085958A3 WO2006085958A3 (en) | 2006-09-14 |
Family
ID=36649743
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2005/022719 WO2006085958A2 (en) | 2004-06-28 | 2005-06-28 | Integration of direct binding sensors with mass spectrometry for functional and structural characterization of molecules |
Country Status (6)
Country | Link |
---|---|
US (1) | US20060003372A1 (en) |
EP (1) | EP1769230A2 (en) |
CN (1) | CN101057131A (en) |
AU (1) | AU2005327173A1 (en) |
CA (1) | CA2572459A1 (en) |
WO (1) | WO2006085958A2 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102676494A (en) * | 2012-05-03 | 2012-09-19 | 天津大学 | Core-shell structure immobilized enzyme particle and preparation method thereof |
Families Citing this family (27)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7167615B1 (en) | 1999-11-05 | 2007-01-23 | Board Of Regents, The University Of Texas System | Resonant waveguide-grating filters and sensors and methods for making and using same |
US8111401B2 (en) * | 1999-11-05 | 2012-02-07 | Robert Magnusson | Guided-mode resonance sensors employing angular, spectral, modal, and polarization diversity for high-precision sensing in compact formats |
US7118710B2 (en) | 2000-10-30 | 2006-10-10 | Sru Biosystems, Inc. | Label-free high-throughput optical technique for detecting biomolecular interactions |
US7371562B2 (en) * | 2000-10-30 | 2008-05-13 | Sru Biosystems, Inc. | Guided mode resonant filter biosensor using a linear grating surface structure |
US7575939B2 (en) | 2000-10-30 | 2009-08-18 | Sru Biosystems, Inc. | Optical detection of label-free biomolecular interactions using microreplicated plastic sensor elements |
US7524625B2 (en) * | 2000-10-30 | 2009-04-28 | Sru Biosystems, Inc. | Real time binding analysis of antigens on a biosensor surface |
US7927822B2 (en) | 2002-09-09 | 2011-04-19 | Sru Biosystems, Inc. | Methods for screening cells and antibodies |
US7309614B1 (en) | 2002-12-04 | 2007-12-18 | Sru Biosystems, Inc. | Self-referencing biodetection method and patterned bioassays |
US8298780B2 (en) | 2003-09-22 | 2012-10-30 | X-Body, Inc. | Methods of detection of changes in cells |
US20060065622A1 (en) * | 2004-09-27 | 2006-03-30 | Floyd Philip D | Method and system for xenon fluoride etching with enhanced efficiency |
DE602006015793D1 (en) * | 2005-04-12 | 2010-09-09 | Sru Biosystems Inc | PROTEOLIPIDE MEMBRANE AND LIPID MEMBRANE BIOSENSOR |
WO2008131314A1 (en) * | 2007-04-19 | 2008-10-30 | Sru Biosystems, Inc. | Method for employing a biosensor to detect small molecules that bind directly to immobilized targets |
US9134307B2 (en) | 2007-07-11 | 2015-09-15 | X-Body, Inc. | Method for determining ion channel modulating properties of a test reagent |
CN101802611A (en) | 2007-07-11 | 2010-08-11 | Sru生物系统公司 | Differentiate the method for ion channel modulators |
US8257936B2 (en) | 2008-04-09 | 2012-09-04 | X-Body Inc. | High resolution label free analysis of cellular properties |
WO2010014903A1 (en) * | 2008-07-31 | 2010-02-04 | Massachusetts Institute Of Technology | Multiplexed olfactory receptor-based microsurface plasmon polariton detector |
US20100273185A1 (en) * | 2009-04-27 | 2010-10-28 | Sru Biosystems, Inc. | Detection of Biased Agonist Activation |
CN102460171A (en) * | 2009-05-15 | 2012-05-16 | Sru生物系统公司 | Detection of changes in cell populations and mixed cell populations |
JP5479314B2 (en) * | 2010-12-10 | 2014-04-23 | 日東電工株式会社 | SPR sensor cell and SPR sensor |
JP2013061301A (en) * | 2011-09-15 | 2013-04-04 | Nitto Denko Corp | Spr sensor cell and spr sensor |
US20130330761A1 (en) | 2012-06-12 | 2013-12-12 | Celcuity, LLC | Whole cell assays and methods |
JP6029899B2 (en) * | 2012-09-07 | 2016-11-24 | 日東電工株式会社 | SPR sensor cell and SPR sensor |
WO2015089380A2 (en) | 2013-12-12 | 2015-06-18 | Celcuity Llc | Assays and methods for determining the responsiveness of an individual subject to a therapeutic agent |
CN103940752A (en) * | 2014-04-28 | 2014-07-23 | 上海理工大学 | Method for detecting antigen-antibody in 96-microplate in backside manner |
JP2016218963A (en) * | 2015-05-26 | 2016-12-22 | 富士通株式会社 | Information processing apparatus, input control program, and input control method |
KR20190126070A (en) | 2017-03-20 | 2019-11-08 | 셀퀴티 인크. | How to Measure Signaling Pathway Activity for Therapeutic Selection |
EP3914914A1 (en) * | 2019-01-25 | 2021-12-01 | Regeneron Pharmaceuticals, Inc. | Protein a chromatography - electrospray ionization mass spectrometer |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998039632A1 (en) * | 1997-03-03 | 1998-09-11 | The Regents Of The University Of California | Direct colorimetric detection of biocatalysts |
WO2003064995A2 (en) * | 2002-01-28 | 2003-08-07 | Sru Biosystems, Llc | A guided mode resonant filter biosensor using a linear grating surface structure |
WO2004046164A2 (en) * | 2002-05-10 | 2004-06-03 | Engeneos, Inc. | Unique recognition sequences and methods of use thereof in protein analysis |
-
2005
- 2005-06-28 US US11/168,155 patent/US20060003372A1/en not_active Abandoned
- 2005-06-28 EP EP05857481A patent/EP1769230A2/en not_active Withdrawn
- 2005-06-28 CN CNA2005800218763A patent/CN101057131A/en active Pending
- 2005-06-28 CA CA002572459A patent/CA2572459A1/en not_active Abandoned
- 2005-06-28 WO PCT/US2005/022719 patent/WO2006085958A2/en active Application Filing
- 2005-06-28 AU AU2005327173A patent/AU2005327173A1/en not_active Abandoned
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998039632A1 (en) * | 1997-03-03 | 1998-09-11 | The Regents Of The University Of California | Direct colorimetric detection of biocatalysts |
WO2003064995A2 (en) * | 2002-01-28 | 2003-08-07 | Sru Biosystems, Llc | A guided mode resonant filter biosensor using a linear grating surface structure |
WO2004046164A2 (en) * | 2002-05-10 | 2004-06-03 | Engeneos, Inc. | Unique recognition sequences and methods of use thereof in protein analysis |
Non-Patent Citations (1)
Title |
---|
CUNNINGHAM B ET AL: "Colorimetric resonant reflection as a direct biochemical assay technique" SENSORS AND ACTUATORS B, ELSEVIER SEQUOIA S.A., LAUSANNE, CH, vol. 81, no. 2-3, 5 January 2002 (2002-01-05), pages 316-328, XP004329915 ISSN: 0925-4005 cited in the application * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102676494A (en) * | 2012-05-03 | 2012-09-19 | 天津大学 | Core-shell structure immobilized enzyme particle and preparation method thereof |
CN102676494B (en) * | 2012-05-03 | 2013-10-30 | 天津大学 | Core-shell structure immobilized enzyme particle and preparation method thereof |
Also Published As
Publication number | Publication date |
---|---|
WO2006085958A3 (en) | 2006-09-14 |
CN101057131A (en) | 2007-10-17 |
CA2572459A1 (en) | 2006-08-17 |
EP1769230A2 (en) | 2007-04-04 |
US20060003372A1 (en) | 2006-01-05 |
AU2005327173A1 (en) | 2006-08-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20060003372A1 (en) | Integration of direct binding label-free biosensors with mass spectrometry for functional and structural characterization of molecules | |
MacBeath | Protein microarrays and proteomics | |
US7534578B1 (en) | Self-referencing biodetection method and patterned bioassays | |
US7524625B2 (en) | Real time binding analysis of antigens on a biosensor surface | |
Weinberger et al. | Recent trends in protein biochip technology | |
US7396676B2 (en) | Evanescent wave sensor with attached ligand | |
US7175980B2 (en) | Method of making a plastic colorimetric resonant biosensor device with liquid handling capabilities | |
Barry et al. | Quantitative protein profiling using antibody arrays | |
US7429492B2 (en) | Multiwell plates with integrated biosensors and membranes | |
Yuk et al. | Proteomic applications of surface plasmon resonance biosensors: analysis of protein arrays | |
US20080240543A1 (en) | Calibration and normalization method for biosensors | |
US20050227374A1 (en) | Label-free high-throughput optical technique for detecting biomolecular interactions | |
CA2539187A1 (en) | Label-free methods for performing assays using a colorimetric resonant reflectance optical biosensor | |
JP2009503447A (en) | Label-free high-throughput biomolecule screening system and method | |
EP1602917A2 (en) | Evanescent wave sensor containing nanostructures and methods of using the same | |
EP1674855A2 (en) | Addressable recovery of bound analytes from an evanescent wave sensor | |
US20100112722A1 (en) | Immunoassays and Characterization of Biomolecular Interactions Using Self-Assembled Monolayers | |
Lausted et al. | SPR imaging for high throughput, label-free interaction analysis | |
US20040263853A1 (en) | SPR sensor surface support | |
Sage | Product Review: Protein biochips go high tech | |
US20090081694A1 (en) | Modified well plates for molecular binding studies | |
Bonnel et al. | Label‐Free Biosensor Affinity Analysis Coupled to Mass Spectrometry | |
Beusink | Label-free biomolecular interaction sensing on microarrays using surface plasmon resonance imaging |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
WWE | Wipo information: entry into national phase |
Ref document number: 200580021876.3 Country of ref document: CN Ref document number: 2572459 Country of ref document: CA |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWW | Wipo information: withdrawn in national office |
Country of ref document: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2005327173 Country of ref document: AU |
|
WWE | Wipo information: entry into national phase |
Ref document number: 552572 Country of ref document: NZ |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2005857481 Country of ref document: EP |
|
ENP | Entry into the national phase |
Ref document number: 2005327173 Country of ref document: AU Date of ref document: 20050628 Kind code of ref document: A |
|
WWP | Wipo information: published in national office |
Ref document number: 2005327173 Country of ref document: AU |
|
WWP | Wipo information: published in national office |
Ref document number: 2005857481 Country of ref document: EP |