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WO2010065531A1 - Criblage de protéine à molécule unique - Google Patents

Criblage de protéine à molécule unique Download PDF

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Publication number
WO2010065531A1
WO2010065531A1 PCT/US2009/066236 US2009066236W WO2010065531A1 WO 2010065531 A1 WO2010065531 A1 WO 2010065531A1 US 2009066236 W US2009066236 W US 2009066236W WO 2010065531 A1 WO2010065531 A1 WO 2010065531A1
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substrate
labeled
molecule
molecules
protein
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Robi David Mitra
Lee Aaron Tessler
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    • 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
    • G01N33/54306—Solid-phase reaction mechanisms
    • 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
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding

Definitions

  • SMD single molecule detection
  • SMD methods for proteins affixed to a surface could enable highly multiplexed immunoassays. For example, by creating -20 overlapping pools of labeled antibodies using a logarithmic pooling strategy like the one used to decode bead-based random microarrays (Gunderson K. L. et al., Genome Res. 2004, 14, 870-877), a single assay could detect the protein targets of all 6,000 nonredundant human proteome antibodies (Berglund L. et al., MoI. Cell. Proteomics 2008, 7, 2019-2027) with only -20 binding rounds.
  • Antibody-based methods are routinely used for protein quantification. Highly sensitive sandwich ELISAs can quantify the abundance of a single protein down to zeptomolar quantities. Since sandwich ELISAs platforms are not amenable to scaling up to multiple targets, antibody microarrays have aimed to fill this gap. Yet they do not obtain the sensitivity of sandwich ELISAs. Promising new alternatives exist in the field of protein quantification. Proximity ligation and bead arrays provide sensitivity comparable sandwich ELISAs and can be multiplexed. However, due to the need for two antibodies per target protein, and for specialized reagents (ligation oligos and bead labeling, respectively) they may not be scalable to proteome-wide throughput.
  • the present invention provides, at least in part, methods for improving single molecule analysis (e.g., detection) of proteins from a sample.
  • the invention provides methods for analyzing proteins (e.g., biomarkers) in a sample, e.g., identifying one or more proteins and/or measuring the expression levels of one or more proteins, e.g., to diagnose diseases (e.g., cancer), e.g., by protein end sequencing.
  • the invention features methods for analyzing (e.g., measuring) the expression levels of proteins (e.g., biomarkers) in a sample, e.g., using pooled molecules (e.g., antibodies).
  • methods useful for improving single molecule protein analysis are provided herein.
  • the invention features a method for single molecule protein analysis, e.g., Digital Analysis of Proteins by End Sequencing (DAPES).
  • the method includes the steps of: (a) preparing a substrate (e.g., glass, fused silica, or glass or silica deposited with a metal film, such as, but not restricted to titanium, gold, or aluminum, e.g., etched to make an array) with a first molecule; (b) providing a sample (e.g., a biological sample, e.g., from a patient) comprising one or more analyte proteins; (c) fragmenting analyte proteins into peptides, e.g., utilizing a protease (e.g., proteinase K) or cyanogen bromide; (d) attaching the peptides directly to the substrate or indirectly to the substrate through the first molecule; (e) modifying the N-terminal amino acid of the fragments, e.
  • the invention features a method for single molecule protein analysis, e.g., Digital Analysis of Proteins by End Sequencing (DAPES).
  • the method includes the steps of: (a) preparing a substrate (e.g., glass, fused silica, or glass or silica deposited with a metal film, such as, but not restricted to titanium, gold, or aluminum, e.g., etched to make an array) with a first molecule; (b) providing a sample (e.g., a biological sample, e.g., from a patient) comprising one or more analyte proteins; (c) fragmenting analyte proteins into peptides; (d) attaching directly or indirectly peptides to the substrate or the first molecule; (e) incubating substrate with a first group of one or more labeled molecules (e.g., antibodies, peptide aptamers, RNA aptamers, DNA aptamers, or engineered proteins (e.g., fused
  • Embodiments of the aforesaid methods may include one or more of the following features.
  • the preparing step comprises coating the substrate with a protein, e.g., Bovine Serum Albumin (BSA) (e.g., acetylated BSA).
  • BSA Bovine Serum Albumin
  • the coating additionally comprises gelatin.
  • the preparing step comprises coating the substrate with a cross-linked polyethylene glycol (PEG), e.g., a multiarm PEG.
  • PEG polyethylene glycol
  • the coating of the substrate can be covalent.
  • the coating can be coupled to a thiol moiety and/or an epoxide moiety on the substrate.
  • the preparing step comprises coating the substrate with a self- assembled monolayer.
  • the first molecule is labeled.
  • the label can be a fluorophore.
  • the first molecule label interacts with the second molecule label, e.g., a fluorophore, quantum dot, or nanoparticle.
  • the detecting step produces an image, e.g., a fluorescence image (e.g., acquired using Fluorescence Resonance Energy Transfer (FRET), Total Internal Reflection Fluorescence (TIRF), or Zero Mode Waveguide (ZMW)).
  • a fluorescence image e.g., acquired using Fluorescence Resonance Energy Transfer (FRET), Total Internal Reflection Fluorescence (TIRF), or Zero Mode Waveguide (ZMW)
  • FRET Fluorescence Resonance Energy Transfer
  • TIRF Total Internal Reflection Fluorescence
  • ZMW Zero Mode Waveguide
  • the compilation of the images makes a digital profile, e.g., a digital profile that identifies the analyte proteins.
  • the preparing step comprises preparing the substrate with aminoethyl or aminopropyl modification.
  • the modifying step comprises modifying N-terminal amino acid with phenylisothiocyanate (PTC).
  • PTC phenylisothiocyanate
  • the antibody specifically recognizes PTC modified amino acids or generally recognizes groups of PTC modified amino acids.
  • the groups of labeled molecules can distinguish hydrophobic terminal amino acids, positively charged amino acids, negatively charged amino acids, and small amino acids.
  • the labeled molecule is removed prior to step of (h).
  • the fragmenting step (c) is omitted (i.e. the analyte proteins are not fragmented but attached directly to the substrate).
  • the invention features a method for single molecule protein analysis, e.g., Digital Analysis of Proteins Using Pooled Antibodies (DAPPA).
  • the method includes the steps of: (a) preparing a substrate (e.g., glass, fused silica, or glass or silica deposited with a metal film, such as, but not restricted to titanium, gold, or aluminum, e.g., etched to make an array) with a first molecule (e.g., an antibody); (b) providing a sample (e.g., a biological sample, e.g., from a patient) comprising one or more analyte proteins; (c) attaching analyte proteins (e.g., antibodies and/or peptides) directly or indirectly to the substrate or the first molecule; (d) incubating substrate with a first group of one or more labeled second molecules (e.g., antibodies, peptide aptamers, RNA aptamers, DNA aptamers
  • the preparing step comprises coating the substrate with a protein, e.g., Bovine Serum Albumin (BSA) (e.g., acetylated BSA).
  • BSA Bovine Serum Albumin
  • the coating additionally comprises gelatin.
  • the preparing step comprises coating the substrate with a crosslinked PEG, e.g., a multiarm PEG.
  • the coating of the substrate can be covalent.
  • the coating can be coupled to a thiol moiety and/or an epoxide moiety on the substrate.
  • the preparing step comprises coating the substrate with a self-assembled monolayer.
  • the first molecule is labeled.
  • the label can be fluorophore.
  • the first molecule label interacts with the second molecule label, e.g., a fluorophore, quantum dot, or nanoparticle.
  • the detecting is an image, e.g., a fluorescence image (e.g., acquired using Fluorescence Resonance Energy Transfer (FRET), Total Internal Reflection Fluorescence (TIRF), or Zero Mode Waveguide (ZMW)).
  • FRET Fluorescence Resonance Energy Transfer
  • TIRF Total Internal Reflection Fluorescence
  • ZMW Zero Mode Waveguide
  • the compilation of the images makes a digital profile, e.g., a digital profile that identifies the analyte proteins.
  • High throughput screening can be enabled using pools of labeled molecules (e.g., antibodies) to identify and quantitate individual protein analytes in a biological sample.
  • labeled molecules e.g., antibodies
  • a plurality of samples is analyzed in the high throughput screening.
  • a plurality of labeled molecules is used for detection in the high throughput screening.
  • FIGURE 1 is a table showing the dramatic improvement of survival rates by early detection of cancer.
  • Source Omenn and American Cancer Society.
  • FIGURES 2A-2C depict the Digital Analysis of Protein by End Sequencing (DAPES) Protocol.
  • FIGURE 2A depicts the addition of phenylisothiocyanate to the immobilized peptides on the slide.
  • FIGURE 2B shows that phenylisothiocyanate reacts with the N-termini of the immobilized peptide to form a phenylthiocarbamoyl derivative.
  • FIGURE 2C depicts the removal of the terminal amino acid by lowering the pH and heating the slide. The cycle is repeated to sequence the next amino acid.
  • FIGURE 3 depicts the off -rate of antibodies bound to single protein molecules. Cy5 labeled antibodies were bound to Cy3 labeled proteins. The slide was kept under constant flow and imaged at various time points to observe dissociation of the complex. After 60 hours, the antibodies were stripped from the slide.
  • FIGURE 4 depicts the ELISA results for a polyclonal antibody titrated against various dipeptide motifs.
  • the ED motif is the only one that shows significant reactivity (shown as triangles). Motifs that showed no reactivity were KK, RR, EE, KR, KE, KD, RE, RD, RK, EK, DK, ER, DR, DE, and DD.
  • FIGURE 5 depicts an example of the substrate coating used to reduce nonspecific binding.
  • FIGURE 6 depicts an example of the Digital Analysis of Proteins Using Pooled Antibodies (DAPPA) strategy.
  • FIGURE 8A is a schematic illustration of the single molecule immunoassay.
  • a chemically adsorbed BSA surface was prepared by reacting BSA with an epoxide-coated glass slide within a flow cell. Unreacted epoxides were quenched, and the BSA was activated for sample immobilization by EDC/NHS. The protein sample (circles) was immobilized to the BSA surface, and unreacted sites were passivated. The flow cell was probed with fluorescently labeled antibody and imaged.
  • FIGURE 8C depicts the image processing by standard, single value thresholding allowed only a small portion of the raw image (the brightest spots) to be used for molecule identification.
  • FIGURE 8D depicts the image processing by iterative thresholding allowed for most of the raw image (regardless of intensity) to be used for molecule identification.
  • FIGURE 8E depicts nonspecific adsorption of antibodies onto 12 surface protocols. Molecules were counted in 5 x 1,000 ⁇ m images, and units were converted to picograms per cm assuming a 155 kDa molecular weight. The chemically adsorbed BSA surfaces suppressed nonspecific adsorption the most.
  • FIGURES 10A-10B depicts the attachment efficiency.
  • the EDC/NHS heterobifunctional crosslinking system can effectively activate BSA molecules on the surface to immobilize target proteins.
  • FIGURE 1OA depicts the number of protein molecules attached to the surface per 2,000 ⁇ m with and without EDC/NHS surface activation.
  • FIGURE 11 depicts the determination of protein accessibility (detection efficiency).
  • the image series illustrates target immobilization, antibody binding, and correlation detection.
  • Each frame is an image of the same position in the flow cell (scale bar ) 2 ⁇ m) and shows -21 of the ⁇ 10 targets analyzed in each binding experiment, (i)
  • FIGURE 12 depicts the negative control for binding.
  • the correlogram analysis shows a random distribution of correlations, indicating no specific binding.
  • FIGURE 13 depicts the protein accessibility (detection efficiency) as a function of antibody concentration. Specific binding (solid) was calculated by subtracting the nonspecific binding (dotted) from total binding (dashed). As much as ⁇ 70% of the target molecules can be specifically bound, enabling efficient protein detection.
  • FIGURE 14 depicts the insignificant dissociation of surface-bound antibody: target complexes over 48 hours. Antibody binding onto immobilized Cy3- targets was performed and the number of antibody: target complexes was counted. The flow cell was washed over 48 hours and the number of complexes was analyzed every 8 hours. The number of complexes was plotted over time. A decay of the number of antibody: target complexes over time was not observed, so there is likely an antibody- surface interaction.
  • FIGURE 16 depicts single molecule protein quantification.
  • Dashed line accurate quantification was achieved in a complex protein sample. Detection of target protein spiked into undiluted rabbit serum produces a quantification curve that deviates only slightly from quantification of the purified sample. No increase in background was observed when detecting in serum.
  • FIGURE 17 depicts quantification of endogenous IgG in serum.
  • the single molecule protein quantification the total IgG levels of a rabbit were measured at various time points after immunization.
  • FIGURE 18 depicts an efficient strategy for multiplexed protein detection.
  • Protein biomarkers are proteins whose expression levels can be used to detect the presence of disease, predict the future onset of disease, diagnose the severity of disease, or monitor disease progression.
  • PSA prostate-specific antigen
  • elevated levels of PSA are a marker for prostate cancer.
  • PSA-based tests are also useful to monitor for prostate cancer recurrence.
  • elevated levels of Alpha Fetoprotein (AFP) and CA- 125 are indicators for hepatocellular carcinoma and ovarian cancer, respectively.
  • biomarkers have not made a major impact on health care because tests based on PSA or CA- 125 are limited by their low specificities, and hepatocellular carcinoma is so rare that routine screening is not cost effective.
  • these examples hint at what is possible if better biomarkers can be found, e.g., a simple blood test that can be used by primary-care physicians to routinely screen the general population and detect cancer at its earliest stages.
  • DAPES Digital Analysis of Proteins by End Sequencing
  • step (j) repeating steps (e) through (i) two or more times using at least a second group of one or more second molecules, wherein at least a partial amino acid sequence of said peptide is determined.
  • a large number (-1O 9 ) of protein molecules are denatured and cleaved into peptides. These peptides are covalently attached to a glass surface and their amino acid sequences are determined in parallel using a method related to Edman Degradation.
  • the immobilized peptide molecules are covered with a solution containing phenylisothiocyanate, which reacts with the N-terminus of each peptide to form a stable phenylthiocarbamoyl derivative (PTC-amino acid).
  • PTC-amino acid a stable phenylthiocarbamoyl derivative
  • the slide is washed and the identity of the terminal amino acid of each peptide molecule is determined through the single molecule detection of antibodies that specifically bind the different PTC-amino acid derivatives.
  • the terminal amino acid is then removed by raising the temperature and lowering pH, and the cycle is repeated to sequence 5-15 amino acids from each peptide on the slide.
  • the absolute concentration of every protein in the original sample can then be calculated based on the number of different peptide sequences observed.
  • DAPES quantifies protein levels by sequencing the N-termini of millions of immobilized protein molecules in parallel.
  • the following steps are performed (FIGURES 2A-2C): 1) The protein sample is cleaved into peptides by enzymatic or chemical treatment, and these peptides are immobilized on the surface of a microscope slide. 2) Phenylisothiocyanate (PITC), the reagent used in Edman Degradation, is added to the slide and this reacts with the N-terminal amino acid of each peptide to form a phenylthiocarbamoyl derivative (PTC-amino acid, FIGURE 2B). This reaction product is stable at neutral pH.
  • PITC Phenylisothiocyanate
  • the identity of the N-terminal amino acid of each peptide is determined by performing, for example, 20 rounds of antibody binding, detection, and stripping.
  • dye-labeled antibodies that specifically bind both the phenyl group of the phenylthiocarbamoyl derivative and the side chain of one amino acid (e.g., arginine) are used. Because this antibody binds the bulky phenyl group as well as the arginine side chain, it will not bind any internal arginines. Therefore, any protein on the slide that is bound must have an arginine at its N-terminus.
  • the order of the modifying step and the detection step can be reversed.
  • step (c), above Since the majority of proteins are blocked at their amino-termini, it is important to fragment the sample into peptides before performing DAPES (step (c), above).
  • DAPES DAPES
  • One method of choice is a partial digestion of the sample using a protease with broad substrate specificity (e.g., proteinase K), followed by cleavage with cyanogen bromide. Cyanogen bromide cleaves polypeptides at methionines, leaving a C-terminal homoserine lactone group which can be covalently attached to aminoethyl or amionopropyl-derivatized slides.
  • a protease with broad substrate specificity e.g., proteinase K
  • Cyanogen bromide cleaves polypeptides at methionines, leaving a C-terminal homoserine lactone group which can be covalently attached to aminoethyl or amionopropyl-derivatized slides.
  • DAPES utilizes 20 unique antibodies that recognize each of the 20 PTC-amino acids derivatives.
  • DAPES can achieve excellent results with only 4 antibodies that can distinguish hydrophobic terminal amino acids, positively charged amino acids, negatively charged amino acids, and small amino acids. In this case, about 7 more cycles of sequencing are performed.
  • the PTC-amino acid moiety bears a strong resemblance to a dipeptide motif - the phenyl group looks approximately like one side chain, and the terminal amino acid provides the other.
  • FIGURE 4 an ELISA curve of a polyclonal serum that binds to X-X-X-E-D-X-X-X, but does not bind any of the other 15 two amino acid combinations tested, including the closely related motif X-X-X-E-E-X-X-X, which differs from the target ligand by a single carbon group.
  • Polyclonal antibodies against ER, DE, and KD dipeptides were also highly specific.
  • DAPES technologies described herein can be used as a discovery tool to find new disease biomarkers and to analyze the abundance levels of all human proteins. Once good biomarkers have been found, it will be important to cost-effectively measure the expression levels of a smaller number (100-1000) of selected proteins to diagnose disease. Towards this goal, a related technology, Digital Analysis of Proteins Using Pooled Antibodies (DAPPA) to measure the expression levels of -1000 pre-selected proteins for about $10-$20 dollars, is described herein.
  • DAPPA Digital Analysis of Proteins Using Pooled Antibodies
  • DAPPA works by taking antibodies that have been raised against individual proteins (e.g., any commercially available monoclonal or polyclonal antibody), labeling them with a fluorescent dye, such as Cy5, and using these to detect single protein molecules attached to a solid surface, for example see FIGURE 5.
  • a fluorescent dye such as Cy5
  • HO ⁇ - albumin is used to block the surface.
  • ethanolamine Nh2 is used to cap the remaining reactive sites.
  • EDC cross-linker attaches IgG and anti-goat antibody captures the protein.
  • Many decode methods can be used in DAPPA, so that 1000 biomarkers can be quantified with only 10 rounds of antibody binding, imaging, and removal.
  • the DAPPA method may comprise:
  • Procedures for quantifying single protein molecules affixed to a surface by counting bound antibodies are described herein.
  • key parameters, image acquisition and processing, nonspecific antibody adsorption, sample immobilization, sample accessibility, and surface dissociation were optimized in a systematic way to enable a single molecule detection of surface-immobilized proteins, e.g., a quantitative immunoassay.
  • a chemically adsorbed bovine serum albumin (BSA) surface was found to facilitate the efficient detection of single target molecules with fluorescent antibodies, and these antibodies bound for lengths of time sufficient for imaging billions of individual protein molecules.
  • BSA bovine serum albumin
  • Endogenous protein abundance was accurately quantified in serum samples by counting bound antibody molecules.
  • the procedures described herein allowed for single, surface-immobilized protein molecules to be detected with high sensitivity and accurately quantified by counting bound antibody molecules. Further, flow cells could be probed multiple times with antibodies, suggesting the feasibility to perform multiplexed single molecule immunoassays.
  • EXAMPLE 1 Detection of Cancer Biomarkers by DAPPA
  • DAPPA can be used to detect 10 known cancer biomarkers (CA- 125, PSA, B-HCG, AFP, VEGF, IL-4, IL-10, IL-I alpha, TNF alpha, and IL-7) using four rounds of antibody binding (see FIGURE 6).
  • a fluorescent dye e.g., Cy5
  • Each antibody is then assigned a number based on the protein that it binds. For example, anti-CA-125 is assigned the number 1, anti-PSA is assigned the number 2, and so on, for each of the 10 antibodies.
  • the second pool consists of antibodies with a " 1 " in the second column from the left in their binary representation, and so on.
  • a blood sample (or urine, etc) is pipetted onto an activated slide, so that the proteins become covalently attached to the surface.
  • the first pool of antibodies is added to the slide and bound proteins are imaged using a fluorescence microscope (see FIGURE 7). The antibodies are removed and the procedure is repeated with the other 3 pools.
  • Each of the 10 kinds of target proteins will bind an antibody in at least one of the four rounds of binding.
  • the images are analyzed and a "1" is assigned to the protein if it binds an antibody in that round, and a "0" is assigned if it does not. In this fashion, each protein molecule will produce a binary number that gives its identity.
  • sensitivity 99.96% + 0.07%
  • specificity 98.47% + 0.76%
  • the flow cell was washed to remove unbound cross-linker and then exposed to Cy3-labeled protein to immobilize the proteins via their primary amines.
  • the flow cell was washed again to remove unbound protein molecules, unreacted cross-linking sites were quenched, and the flow cell was imaged.
  • Cross-linking proteins to the BSA surface allowed for a 10-fold increase in the number of protein molecules affixed to the surface compared to the surface without EDC/NHS activation (950% + 52%). Also, the proteins were able to be attached at over 1,000 molecules per field of view: a density that allows for highthroughput single- molecule sampling (FIGURE 10). Thus, the EDC/NHS system was able to effectively activate the BSA surface and attach a protein sample. The chemically adsorbed BSA surface with EDC/NHS sample immobilization provided the surface chemistry for all subsequent experiments (FIGURE 8A).
  • Protein sample attachment is enabled by generating peptide bonds between the solvent-accessible carboxyl groups of the BSA and the primary amine groups of the target proteins.
  • the method described herein does not rely on prelabeling samples by biotinylation, instead taking advantage of endogenous lysine residues present on most proteins. Therefore this approach may provide a more universal way of attaching heterogeneous biological samples.
  • the accuracy of a single molecule immunoassay depends on the accessibility of target molecules to antibodies; inaccessible ligands will not be detected or counted.
  • Steric, electrodynamic, and thermodynamic variables can hinder binding when repulsive forces of the surface overcome the attractive forces of the antibody-protein complex.
  • Kinetics can also hinder binding if a free energy barrier is sufficiently high to prevent docking on relevant time scales (Heyes C. D. et ah, MoI. Biosyst. 2007, 3, 419-430). To determine what degree these variables affect the accessibility of target molecules to antibodies in the system described herein, the following experiments were performed.
  • FIG. 11 To analyze the binding of target molecules by antibodies, a dual-color, single molecule protein accessibility assay was performed (FIGURE 11).
  • the target proteins were labeled with Cy3 and the antibodies were labeled with Cy5.
  • a BSA surface was prepared within a flow cell, and the target proteins were immobilized on the surface. The reactive cross-linking sites were capped, and a preantibody image was acquired. Then, the surface was probed with antibodies, washed away unbound antibodies, and an image was taken. The positions of the antibodies were compared with the positions of the proteins imaged beforehand by overlaying their locations. To verify that the colocalization of proteins and antibodies was a result of specific binding, the correlation between protein and antibody positions was measured and the correlation for randomness was tested (see EXAMPLE 12).
  • FIGURE 11 The correlogram in FIGURE 11 indicates that antibody binding was specific and not due to chance correlation. (To confirm the specificity of binding, the protein accessibility assay was also performed using a nonspecific target protein with which the antibodies should have had no affinity and a correlogram showing no significant correlations was observed (FIGURE 12).)
  • the accessibility curve follows the behavior of fractional occupancy that is expected from binding theory. When 1 ⁇ g/mL antibody is used, -70% of the target molecules were specifically bound by antibodies. From these results, single protein molecules can be efficiently detected by counting bound antibody molecules.
  • This example is to determine whether the ligand molecules that failed to be detected in the protein accessibility experiments described above were not detected because they were never bound by antibodies or if they were initially bound by antibodies but the complexes dissociated before imaging.
  • Antibody-ligand interactions are known to have dissociation half-lives in solution ranging from minutes to several hours. However, the surface dissociation rate may be slower due to surface-antibody interactions that stabilize the complex. Therefore, an experiment was designed to measure the surface dissociation rate of antibodies bound to single ligand molecules.
  • antibodies were allowed to bind to target proteins that were immobilized on the surface of the flow cell, as previously described.
  • the surface was imaged to determine the starting number of antibody-ligand complexes and then a continual wash was performed to remove unbound antibodies from the flow cell.
  • the surface was imaged every 8 h over a 48 h period. At each time-point the number of antibody-ligand complexes that were lost relative to the starting time point was quantified, and from this the surface dissociation of the antibodies was measured.
  • Ligand rebinding in successive binding rounds could be used to increase detection specificity or to enable efficient sample multiplexing (Gunderson K.L. et al. , Genome Research 2004, 14, 870-877).
  • it was difficult to remove bound antibodies from the surface This was true even after washing using with a low pH buffer as well as various antibody eluting reagents (data not shown). Therefore, the possibility of rebinding ligands was explored by "erasing" antibodies from the surface via photobleaching. Rebinding after photobleaching might be possible because the antibodies used was polyclonal and could theoretically bind multiple epitopes on a single ligand.
  • the solid line in FIGURE 16 illustrates the relationship between number of antibody molecules and number of protein molecules affixed to the surface.
  • LOD lower limit of detection
  • the Cy3 -labeled target protein was also quantified in the presence of serum.
  • Cy3-labeled target protein was spiked at varying concentrations into neat rabbit serum.
  • the complex mixture including target and nontarget proteins, was immobilized to the BSA surface.
  • the surface was probed with fluorescently labeled antibody and the number of target proteins versus the number of antibodies on the surface was quantified. Similar results to the purified protein detection curve were obtained, demonstrating the robustness of the method in the presence of a complex biological fluid (FIGURE 16, dashed line).
  • the LOD in serum was 390 molecules per 1,000 ⁇ m 2 (10 pg cm "2 ) corresponding to a target starting concentration of 1 ⁇ g/mL.
  • the total concentration of the serum was 74 mg/mL (by dry weight). Therefore, despite the overabundance of serum proteins, the serum introduced almost no background. This indicates that single antibody, direct binding can be used to make specific detection measurements in a highly complex biological fluid.
  • the amount of total IgG in blood of a rabbit was quantified at various time points after immunization. Serum samples were diluted in PBS, immobilized to flow cell surfaces, and probed with anti-rabbit IgG Cy5-antibody. Then the antibodies remaining on the flow cell surface were quantified after washing.
  • a photobleaching step after the first round of binding was used to erase surface-associated fluorescence prior to the second hybridization.
  • Photobleaching was used because the rate at which specifically bound antibodies dissociated from the surface was low enough that it was difficult to completely remove them from the flow cell in a reasonable amount of time.
  • the majority (87%) of proteins that were expected to be bound in two binding rounds were in fact bound twice, indicating that competitive binding by the bleached, surface-bound antibodies was minimal. This lends support to the feasibility of multiple rounds of antibody binding and detection, with each round separated by a photobleaching step.
  • a cleavable linker between the antibody and fluorophore which would enable dye removal by exposure to a reducing agent or to light (Mitra R. D. et aL, Anal. Biochem. 2003, 320, 55-65).
  • washing with surfactants and denaturants may allow for better removal of bound antibodies from their targets. For example, it was demonstrated that the efficient stripping of antibodies from Western blots without disrupting protein attachment (Yeung Y. G. and Stanley E. R. Anal. Biochem. 2009, 389, 89-91). To develop such a protocol in a single- molecule setting will require a low-background surface that is also surfactant-compatible (the surfaces described here are not).
  • One type of low background surfactant-compatible surfaces can be surfaces that utilize multiarm PEG nanogels (Tessler L. A. et aL, 2009, in preparation)
  • Some obstacles for developing a multiplexed single molecule immunoassay might include, e.g., the need to characterize each antibody- Ii gand pair beforehand in order to ensure that the concentration of antibody used in the immunoassay is high enough to ensure maximal binding to its immobilized ligand since each antibody-ligand pair might have variable affinities.
  • antibody production and characterization becomes more standardized, it will become possible to obtain large numbers of well-characterized antibodies.
  • the Human Antibody Initiative has already generated and curated antibodies against over 6,000 human proteins, and they aim to expand the collection to the entire human proteome within the decade (Berglund L. et aL, MoL Cell. Proteomics 2008, 7, 2019-2027). Solid phase single-molecule immunoassays could provide a way to leverage such antibody collections toward high-throughput proteomic applications.
  • each laser beam passed through a band-pass filter: HQ545/30 for the green laser and D635/30 for the red laser (Chroma, Brattleboro, VT).
  • Objective type total internal reflection was achieved through a 6Ox TIRF oil objective with index of refraction 1.49 (Nikon, Melville, NY). The chemistry of the assay was performed in a flow cell (see Fluidics) mounted onto the microscope stage.
  • TIRF allows for the excitation of only surface-bound fluorophore-labeled antibodies and therefore reduces the overall fluorescence background.
  • the emitted photons from the labeled antibodies were collected by the objective and passed through a dichroic mirror (custom Cy3/Cy5, Semrock, Rochester, NY) and an emission filter for either the green channel (HQ610/75, Chroma, Brattleboro, VT) or the red channel (LP02-647RU-25, Semrock, Rochester, NY). Light was then detected by a charge coupled device (CoolSnap ED, Roper Scientific, Arlington, AZ) which imaged a 140 ⁇ m by 100 ⁇ m (1,400 pixels x 1,000 pixels) region of the surface.
  • the flow cell was washed with 600 ⁇ L of PBS and loaded with 600 ⁇ L of oxygenscavenger and blink-reduction system32 to prevent dyes from photobleaching and blinking. Then images were acquired in the red and green fluorescence channels at five different positions across the length of the flow cell, with 0.5 s exposure. Custom software written in Metamorph (Molecular Devices, Sunnyvale, CA) and Matlab (Mathworks, Natick, MA) was used to analyze the locations and intensities of the fluorescent molecules.
  • the analysis substrate was a 40 mm diameter, no. 1.5 glass slide (Erie Scientific, Waltham, MA).
  • the substrate was epoxide-derivatized by the vendor unless otherwise specified in Preparation of Surfaces.
  • the slide was loaded into a flow cell (FSC2, Bioptechs, Butler, PA) fitted with perfusion ports to allow for reagents to be passed over the surface. Reagents were flowed through by a custom-made negative pressure vacuum pump.
  • Target Proteins were polyclonal goat IgG molecules labeled with an average of eight Cy3 dyes per molecule.
  • the nonspecific target proteins used as a negative control in the target binding accessibility assay were polyclonal rabbit IgG molecules labeled with Cy3. Proteins were obtained from Abeam (Cambridge, MA).
  • Serum Samples The serum sample used for the spike-in quantification experiment was obtained from rabbit.
  • the serum samples used for the endogenous protein quantification experiment were from preimmunized, week 4, and week 5 rabbits in an antibody production protocol (for an unrelated study) during which rabbits were immunized with antigen and adjuvant. All serum samples were obtained from 21st Century Biochemicals (Marlboro, MA).
  • Antibodies The antibodies used in all experiments with the exception of the endogenous protein quantification experiment were polyclonal anti-goat, labeled with Cy5. The antibodies used to detect endogenous rabbit IgG were polyclonal anti -rabbit, labeled with Cy5. All antibodies were obtained from Abeam (Cambridge, MA).
  • the epoxide-coated glass was loaded into the flow cell and washed in 600 ⁇ L of phosphate buffered saline pH 7.3 (PBS).
  • PBS phosphate buffered saline pH 7.3
  • the glass was reacted with one of the following solutions in PBS for 1 h at room temperature: 1% bovine serum albumin (BSA) (Fisher Scientific, Pittsburgh, PA), 1% BSA/0.1% cold water fish skin gelatin (Aurion, The Netherlands), 1 M glucose, 10% linear polyacrylamide (LPA) MW 1500 Da, 10% LPA MW 10 kDa, 10% LPA MW 1 MDa, 100 mg/mL amino-PEG (Sigma- Aldrich, St.
  • BSA bovine serum albumin
  • LPA linear polyacrylamide
  • a flow cell containing the surface to be tested was loaded with 600 ⁇ L, 100 ng/mL Cy5 antibody. The surface was exposed to the antibody in the dark for 25 min at room temperature. Then, unbound antibodies were removed with a 600 ⁇ L PBS wash, and the flow cell was imaged as described above.
  • a chemically adsorbed BSA surface was formed as described above, and the surface was activated with 0.2 M l-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.05 M N- hydroxysuccinimide (NHS) (Pierce, Rockford, IL) in sodium phosphate buffer pH 5.8 (SPB) for 10 min. Free EDC and NHS was washed away with 600 ⁇ L of SPB.
  • EDC l-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride
  • NHS N- hydroxysuccinimide
  • the attachment of the protein sample of interest to the activated surface was as follows.
  • 100 ng/mL (unless otherwise specified) of target protein in PBS was loaded into the flow cell.
  • dilutions of target protein in PBS were loaded into the flow cell.
  • dilutions of target protein were spiked-in to whole rabbit serum, and the spiked-in serum was loaded into the flow cell.
  • whole rabbit serum was diluted 1:105 in PBS and loaded into the flow cell.
  • Proteins samples that were loaded into the flow cell were allowed to react with the surface for 10 min at room temperature, in the dark. Then, unbound proteins were removed with a 600 ⁇ L PBS wash, and unreacted EDC-NHS sites on the BSA surface were quenched with 1 M Tris pH 8.0 for 20 min.
  • the strong single peak in the correlogram indicates that the correlation is significant. Had the correlation obtained been by chance occurance of overlapping molecules, many peaks of similar height to the highest peak should be expected.
  • the starting point for the intensity threshold was zero and the intensity threshold increased by 50 intensity units throughout the entire spectrum of intensities (0 - 4096).
  • objects were identified by contiguity of pixels and objects that were too large (> 20 pixels) or too small ⁇ 2 pixels) to be single antibodies were removed. The coordinates of the centroid of each of the objects that passed the filter were saved. During the iterations in which the intensity threshold was low, this method identified the dimmest objects, which were located at the periphery of illumination.
  • the algorithm used the locations obtained during the iterative thresholding to generate an output image that has each of the fluorescent objects represented by equally sized objects of 4 x 4 pixels.
  • R and G are matrices of ones and zeros, representing the binary image of size 316 x 316 pixel .
  • the Cy5-Cy3 image pair was allowed to be offset with respect to each other in order to find the alignment that produced the maximum correlation (the true alignment). Once the true alignment was found, the software counted the number of proteins that overlapped antibodies and divided that by the total number of proteins. This ratio was defined as the fractional accessibility or binding efficiency.
  • level surfaces correspond to the background distribution of correlation values, and peaks correspond to correlations that are significantly nonrandom.
  • a high peak was seen around the offset (0, 0). Therefore correlation for the true alignment was nonrandom, and binding was specific.
  • no peak appeared (FIGURE 12), indicating randomness between Cy3 and Cy5 channels (and no specific binding).
  • the total number of antibodies remaining on the surface after washing was used to estimate the frequency of antibody-ligand correlations that occurred merely by chance overlap of molecules - the false positive (FP) rate.
  • the FP rate was defined as the probability that a randomly chosen pixel will be within a radius 2.5 pixels from an antibody pixel. This probability follows a Poisson process, where the parameter lambda is the frequency of antibody pixels out of the total number of pixels. Therefore, for the number of antibodies on the surface A, and total pixel area of the image T,
  • the three rabbit serum samples were used as coating antigens.
  • the detection antibody was polyclonal anti- rabbit antibody conjugated to alkaline-phosphatase (Abeam, Cambridge, MA).
  • Polystyrene microtiter plates (Immulon 2HB) were obtained from Thermo Fisher Scientific (Waltham, MA). Washes were performed using Labsystems Multidrop 384 (Beckman Coulter, Fullerton, CA). Detection of the fluorogenic substrate, (4- methylumbelliferyl phosphate, Sigma Aldrich, St. Louis) was performed on the microtiter plate flourimeter Synergy HT (Biotek, Winooski, VT).
  • ELISA enzyme-linked immunosorbent assay
  • each protein is assigned a unique digital signature.
  • fluorescent antibodies for each protein are pooled into combinations that are determined by the columns of the digital signatures. (In the example above, the three columns of the signatures dictate the compositions of the three "antibody pools”.)
  • immobilized proteins are probed by one antibody pool per binding round. In each binding round, proteins of different species are bound and detected. In between binding rounds, antibodies are stripped. After probing with all of the antibody pools (three in this example), the history of binding at each position on the slide is analyzed. In this manner, each position on the flow cell becomes represented by a binding signature.
  • the pre-assigned digital signatures are used to decode the flow cell positions into protein identities.
  • the number of occurrences of each signature is counted to determine protein abundance.
  • the binding history 1-0-0 i.e. bound in round 1, unbound in round 2, and unbound in round 3.
  • This signature corresponds to Protein 4, so the number of instances of that signature on the flow cell (two), indicates the abundance of Protein 4.

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Abstract

La présente invention concerne des procédés pour améliorer l’analyse de protéine à molécule unique. Ces procédés peuvent être utilisés pour la découverte de nouveaux biomarqueurs, la quantification, et le criblage à rendement élevé. Dans le contexte de cette invention, des peptides liés à la surface peuvent être directement séquencés en utilisant une dégradation d’Edman modifiée suivie par une détection, par exemple, une détection d’anticorps marqué. Le criblage à rendement élevé est effectué en utilisant des groupes de molécules (par exemple, des anticorps marqués) pour identifier et quantifier des analytes protéiques individuels dans un échantillon biologique.
PCT/US2009/066236 2008-12-01 2009-12-01 Criblage de protéine à molécule unique Ceased WO2010065531A1 (fr)

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WO2013112745A1 (fr) * 2012-01-24 2013-08-01 The Regents Of The University Of Colorado, A Body Corporate Identification et séquençage de peptides par la détection d'une seule molécule de peptides subissant une dégradation
WO2014014347A1 (fr) 2012-07-16 2014-01-23 Technische Universiteit Delft Séquençage d'une protéine à molécule unique
KR101415166B1 (ko) * 2013-06-05 2014-07-07 한국과학기술원 전반사 형광 시스템에 사용하는 비특이적 결합방지 기판, 이의 제조방법 및 이를 이용한 단일 분자 수준의 분석 시스템
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