Patent Application
20070224644
The present invention relates to the analysis and monitoring of ocular fluids for determining the physiological state of an organism, e.g., to monitor drug efficacy and dynamics, for early disease or other physiological state detection, as well as to monitor/quantitate certain molecular markers and fingerprints identified in such analysis.
This invention relates to, e.g., a method of characterizing the physiological state of the eye, comprising detecting the presence or absence in vitreous fluid of one or more polypeptides, or fragments thereof; a method of characterizing the physiological state of the eye, comprising detecting the presence or absence in vitreous fluid of one or more biomarker attractant-associated polypeptides, or fragments thereof; a method of characterizing the physiological state of a living system, comprising detecting the presence or absence in a vitreous fluid of one or more polypeptides, or fragments thereof; a method of monitoring the efficacy of a tyrosine kinase inhibitor or other drug in a subject to whom said inhibitor or drug has been administered, comprising measuring the presence of a phosphorylated polypeptide in the case of said inhibitor or a polypeptide in the case of said drug in a vitreous fluid sample extracted from a subject, wherein the subject has been administered a tyrosine kinase inhibitor, drug, or a biological, chemical, protein, antibody, or other therapeutic agent.
The vitreous actively participates in the development of pathologic conditions and contains proteins that may correlate with specific retinal pathologies. These proteins have been implicated in angiogenesis, mechanical traction via increased osmolarity and aging. The proteins retained in the vitreous provide a record of the state of ocular tissues.
Vitreous fluid contains proteins that can correlate with specific retinal pathologies, such as diabetic retinopathy. Diabetic retinopathy (DR) is the most prevalent cause of vision loss in working adults. Most patients with type 1 diabetes mellitus and over 60% of those with type 2 diabetes eventually develop retinal vascular abnormalities. 20% to 30% of these patients advance to active proliferative diabetic retinopathy (PDR) and/or diabetic macular edema. Increased retinal vascular permeability (RVP) is a primary cause of diabetic macular edema and a characteristic finding in PDR. While photocoagulation surgery and vitrectomy are highly effective in reducing vision loss, early diagnosis and preventative treatments for these disorders remain a major unmet clinical need. The following discusses additional disease applications for vitreous proteomics discovery and vitreous diagnostic testing.
Wet Age-related Macular Degeneration (AMD) is the leading cause of blindness in people over the age of 55. Most severe vision loss occurs in people who develop the wet form of the disease. Treatments for and early detection of wet AMD are being developed at an unprecedented rate. Study of these new drugs has clearly established that the best chance for maintaining functional vision depends upon treatment in the earliest stages of the transition from dry to wet AMD. Furthermore, dry AMD often persists for widely varying durations (with a range of decades) prior to progressing to the wet form. For now there are only rough indicators available through subjective testing and angiography. There is a great need for a method to detect and predict the progression of AMD that could be used to augment the subjective and imaging indicators already in use.
Very little is known about the biochemical signals that modulate the exudative process that causes the severe degradation of vision in patients with wet AMD. There is some data implicating VEGF (Vascular endothelial growth factor) in this process, but almost no data regarding possible other factors. New drugs that block VEGF in the vitreous, retina and choroid have been able to slow the course of the disease but there is need for developing methods for permanently halting the progression of the disease and ultimately reversing some of its effects. Understanding the retinal proteome in wet AMD will produce opportunities for major advances in the treatment of this blinding disorder.
Retinal vein occlusion is the leading cause of vision loss after diabetic retinopathy and macular degeneration. The natural history of these diseases vary significantly. The only predictive parameter is a crude measure of retinal vascular perfusion. There is a need to stage the severity of the retinal damage and look for parameters that will predict the visual outcome. These parameters will provide improved guidance for the treating physician. In addition, these tests will be crucial in developing new treatment methods in a disease that currently has only limited treatment options.
Cystoid Macular Edema (CME) is a type of edema of the macula that causes retinal damage and occurs in a wide variety of ocular disorders. There is intense interest among practitioners and pharmaceutical companies to develop treatments for CME. Understanding the ocular fluid proteome profile of patients with CME will provide new opportunities in prevention and treatment.
Although cataracts affect millions of people per year in the USA alone, very little is know about the factors that control cataract development. Our recent findings show that lens proteins called crystallins are found in the vitreous cavity. It is also known that typical senile type cataracts form within months of removal of the gel portion (vitreous humor) of the vitreous body. Tracking the proteome of the vitreous gel and the remaining fluid that accumulates following removal of the gel may lead to the identification of new factors that could prevent cataract formation. In many ways this has been the holy grail of ophthalmology.
Tools are needed to track the pharmacokinetics of intraocular drugs delivered directly or indirectly and track ocular/systemic drug partition between vitreous and serum. Thereby drug development and modification will be greatly facilitated providing significant value to the rapidly growing industry of developing drugs for retinal disorders.
This invention provides techniques and methods which have value for such problems.
Any ocular or eye-related fluid can be analyzed in accordance with the present invention, including, e.g., vitreous fluids; aqueous fluids; retinal blood, such as blood present in the choroid; and tears, including tears extracted from the lacrimal sac. Fluids can be extracted routinely, e.g., by surgical vitrectomy procedures. In some cases the state of specific diseases as reflected in ocular fluids can be measured by fluorescent, magnetic, or radio nucleotide imaging.
The present invention provides a proteomic fingerprint of an ocular fluid sample, comprising at least one polypeptide or other molecule present in the sample. Polypeptides (also referred to as. “biomarkers”) can be isolated using any suitable technology. For example, biomarkers can be harvested from low molecular weight fractions in which a biomarker attractant is associated with a polypeptide or other biomolecules (“biomarkers”). Methods of isolating biomarker attractant-associated biomolecules are described in WO05036180, which is hereby incorporated by reference in its entirety.
The term “biomarker attractant molecule,” or “BAM,” refers to a molecule, or other substance to which biomarkers in a biological fluid adhere. In particular examples, biomarkers adhere to a BAM with a low binding affinity (for example, a binding affinity of less than 10−3, 10−4, 10−5, 10−6, 10−7 or 10−8 L/mol-min). An antibody may be a BAM to the extent that it binds biomarkers, other than through the specific antigen antibody interaction that results from the immune response that stimulated its production. For example, biomarker binding to an antibody BAM may occur outside of the complementarity defining region (CDR), or outside of the variable region altogether, for example by binding to the Fc portion of the antibody. However, in certain embodiments of the disclosed methods, the BAM is not an antibody. Although a particular BAM may selectively bind a class of biomarkers, the binding affinities of the biomarkers in a particular class do not differ as significantly as the binding affinities of an antigen to a particular antibody compared to other non-recognized molecules. The less specific nature of biomarker binding may be illustrated in certain examples of the BAM in which more than one biomarker binds to the BAM, for example, at least 2, at least 5, at least 10, at least 20, or even 50 or more biomarkers bind to the BAM.
Typically, BAMs have a half-life of existence in a particular biological fluid (for example in the body) that is longer than the half-life of biomarkers that become adhered to the BAMs and thereby concentrate the biomarker in the biological fluid. For example, BAMs can have a half-life of greater than about 1 day, such as greater than 2, 5, 10, 20 or 50 days. In particular examples, the BAM has size and/or shape such that it is not substantially filtered from the blood by the kidneys. In other particular examples, the BAM has a molecular weight of greater than 25 kDa, for example, greater than 30, 50, 75, 100, 150, 200 or 300 kDa. In yet other particular examples, the BAM molecule has a molecular weight falling within a particular range, for example between 30 and 50 kDa, between 50 and 75 kDa, between 75 and 100 kDa, between 100 and 150 kDa, between 150 and 200 kDa, between 200 and 300 kDa, or any other range between 30 kDa and 300 kDa. Biomarkers may adsorb to the surface or be absorbed into the interior of the BAM, or both.
Examples of BAMs include proteins (including natural and engineered proteins such as chimeric proteins, proteins with modified amino acid composition, proteins modified postranslationally, nucleic acids, carbohydrate decorated molecules, and organic polymers), dendrimers and particles (such as microparticles and nanoparticles, including silica, metal, ceramic and carbohydrate microparticles and nanoparticles), and cellular microparticles (see, for example, Diamant et al, Eur J Clin Invest. 34: 392-401, 2004).
BAMs may be produced or derivatized to provide ionic groups (such as carboxylate, protonated amine, quaternary ammonium, and sulfate groups), hydrogen-bond acceptors or hydrogen-bond donors, electron donors or electron acceptors, polar groups (such as amino, hydroxyl, ester, sulfhydryl and nitrile groups), hydrophobic groups (such as alkyl, alkenyl and alkynyl groups or groups with specific partition coefficients), peptides, proteins, nucleic acids, carbohydrates, lipids or any combination thereof, on their surfaces or in their interiors. Where the BAM is a protein, such as a naturally occurring protein, it may also be referred to as a “carrier protein” to reflect its role in collecting and concentrating LMM biomarkers from biological fluids. Examples of carrier proteins include albumin, iron binding proteins (such as transferrin), fibrinogen, alpha-2-macroglobulin, immunoglobulins (such as IgA, IgE and IgG), complement, haptoglobulin, lipoproteins, prealbumin, alpha-l-acid glycoprotein, fibronectin, and ceruloplasmin, and fragments, combinations and chemical derivatives thereof.
A proteomic fingerprint can comprise as few as one polypeptide, or it can comprise more than one polypeptide (i.e., a plurality). Any method of analyzing ocular fluid content can be utilized. For example, biomarker attractants (also referred to as a biomarker attractant molecules or “BAM”) present in an ocular fluid can be utilized without limitation for purification purposes, including albumin, proteoglycans, glucosaminglycans, and heparan sulfates. The polypeptides can be present as intact proteins, or as fragments. Such fragments can be naturally-occurring, or can be produced during processing of a sample, either by inadvertent or deliberate proteolysis (e.g., contacting a sample with a proteolytic enzyme or a chemical cleavage agent).
Examples of biomarkers that have been isolated from ocular fluids are shown in Table 2 to Table 13. These were obtained by running a sample of ocular fluid on an SDS-PAGE gel, and then digesting the entire gel lane from high to low protein molecular weight with trypsin, followed by MS/MS analysis.
The set of polypeptides detected in accordance with the present invention can be described as a “fingerprint” in that they are a distinctive pattern of polypeptides present in the ocular fluid. Fingerprints can be prepared using the BAM techniques described above, or using other technologies or purification processes, e.g., characterizing polypeptides present in the ocular fluids without a BAM-enrichment step (See Table 2 to Table 13 for a representative example of such polypeptides).
Just as with a fingerprint, the set of polypeptides can be used as a unique identifier to characterize the fluid, as well as the physiological status of the subject. The ocular fingerprint can be viewed as a snapshot of the elements (e.g., polypeptides) that are involved in, or a product of, the physiological processes that are occurring in the body. Examples of physiological states that can be characterized in accordance with the present invention include without limitation, diseases states (e.g., cancer, retinopathy, diabetes, macular degeneration, venous occlusive disease, cataracts, and other disorders mentioned herein); therapeutic states (e.g., for monitoring drug efficacy and adverse events); organ function (e.g., to monitor normal organ function, such as brain, kidney, and liver functions); toxicological states (e.g., to detect toxins or perturbations caused by toxins); etc. Thus, an ocular fluid fingerprint can be used for a variety of medical, diagnostic, and therapeutic purposes, including, for example: to detect the risk of cataract formation (see below); to monitor blood-ocular breakdown; to detect age-related macular degeneration; to detect therapeutic efficacy of kinase inhibitors and other drugs; etc.
For example, ocular fluids can be removed from a patient using a whole-bore vitrectomy cannula or cutter containing agents that inhibit polypeptide degradation, and then subjecting the fluid to analysis for the presence of biomarkers. Such biomarkers can be used to determine the risk of cataracts (e.g., when crystallins are elevated); the integrity of the blood-ocular barrier; and other retinal conditions and diseases. This can be especially useful in patients who are at risk for an ocular disease, e.g., subjects with diabetes, aging subjects, or subjects who have been identified as a carrier of a gene defect associated with an ocular disorder.
Other viable polypeptide candidates which can be routinely analyzed for the assessment of the physiological state of an individual comprise proteins that are pathologically correlated with certain physiological state. For example, retinol binding protein-4 (RBP4) is known to contribute to the development of diabetes by blocking the action of insulin. Ocular detection of this protein may lead to important insights into the initiation and/or progression of diabetes in a subject. Other examples of these polypeptide candidates include, but are not limited to, Secreted Protein Acidic and Rich in Cysteine (SPARC). It is known that SPARC is upregulated after injury and modulates cell adhesion and proliferation and by releasing the KGHK peptide, which stimulates angiogenesis. SPARC binds VEGF, inhibits its interaction with extracellular surface, and also inhibits activation of downstream effectors (e.g., ERK1/2) and VEGF-induced DNA synthesis. It has been thought that SPARC not only modulates angiogenesis but, moreover, regulation of SPARC levels appears to be the key to control angiogenesis in macular degeneration. A third example of a protein that may be utilized as a diagnostic marker of a disease state (for example, cancer) comprises detection of phosphorylation status of Akt. Akt is activated by growth factors or cytokines in a PI3K-dependent manner, and phosphorylation of two residues by PDK1 (T308) and PDK2 (S473) is required for its full activation. The instant method comprises detecting the phosphorylation status of one or more amino acid residues of Akt in normal subject and a patient, and comparing the status with, for example, progression of cancer in the patient. Other cancer biomarkers, for e.g., VEGFR, EGFR, Bcr-Abl, Her2-Neu (erbB2), TGFR, etc. may also be routinely analyzed.
Therefore, in addition to the ocular disorders, the ocular fluids can also be used generally to monitor a subject's health and physiological status. The ocular fluid is in communication with other body compartments, and thus is useful to monitor extra-ocular compartments, including the brain, kidney, liver, etc. Since developmentally the eye is an extension of the brain the state of the molecular composition of ocular fluids can provide information about diseases in the brain. With regard to more distant organs, molecules derived from these organs may enter the ocular fluids through the circulation, or the ocular fluid markers may reflect a systemic body-wide process that effects the distant organ.
The present invention also relates to methods of monitoring the physiological status of a subject, comprising: measuring the presence of a post-translationally modified polypeptide (e.g., phosphorylation) in a vitreous fluid sample extracted from a subject. In certain claims, signaling pathways can be monitored. Signaling pathways include any pathway in the body that involves generating a chemical event (e.g., phosphorylation) that modulates a cellular activity (e.g., indicating receptor occupancy, site-directed protein-protein binding, and triggering a cascade of enzymatic reactions that culminates in gene expression). For example, phosphorylation is a key post-translational modification event in many biological pathways involved in cell growth, cell death, gene expression, and cellular responses to stimuli. In addition, aberrant phosphorylation patterns may be associated with diseases, such as cancer and other hyper-proliferation disorders.
Further examples include, e.g., G-protein receptor mediated pathways, especially receptors for tyrosine kinases, such as vascular growth factor receptors (e.g., VEGFR-1, VEGFR-2), epidermal growth factor receptors (EGFR), HER2, adrenergic receptors (e.g., alpha- and beta-types); hormone mediated receptors; etc. Examples of receptors include, VEGFR-2 (e.g., including phosphorylation sites Y951, Y996, Y1054, Y1059, Y1175, Y1214); PDGFR-beta (e.g., including phosphorylation sites Y740, Y751, and Y771), and EGFR (e.g., including phosphorylation sites Y1173, Y1148, Y1068, Y845, and Y992).
These methods can also be utilized to measure or monitor the efficacy of a drug, especially a drug which is utilized to modulate a kinase, such as a tyrosine kinase, or a biological based therapeutic, such as an autologous platelet concentrate. A variety of therapeutic agents are being used to treat diseases or disorders associated with aberrant or increased kinase activity, including cancers and angiogenesis. Targets include, but are not limited to, e.g., raf, PDGFR-alpha, PDGFR-beta, EGFR, VEGFR, VEGFR1, VEGFR2, VEGFR3, HER-2, KIT, FLT3, c-MET, FGFR, FGFR1, FGFR3, c-FMS, RET, ABL, ALK, ARG, NTRK1, NTRK3, JAK2, ROS, etc. Other signaling targets include, e.g., ERK, AKT, PYK2, etc.
Examples of kinase effecting drugs, include, but are not limited to, e.g., avastin (bevacizumab), cetuximab, erlotinib (tarceva or OSI774), everolimus (RAD0001), fasudil, FK506, gefitinib (ZD1839), imatinib mesylate (STI57 or Gleevec), lapatinib ditosylate (GSK572016), rapamycin, sorafinib, sirolimus, sunitinib (sutent), trastuzumab (Herceptin), serafanib and wortmannin.
One goal of such drug therapy is to reduce the amount of phosphorylation of a target polypeptide. For example, several anti-cancer drugs are being utilized to block angiogenesis by blocking the phosphorylation of VEGFR-2. The efficacy of such drugs can be monitored by detecting the appearance of shed phosphorylated receptor into the vitreous fluid. As shown in the attached example, phosphorylated VEGFR-2 and PDGF-R polypeptide fragments were detected in vitreous fluid using reverse phase assays.
Reverse phase protein microarray is a technique that is routinely used for efficient and accurate detection of proteins in a sample. The proteins extracted from a single sample are immobilized on the substratum. The captured analytes are detected with a primary antibody directed toward the protein/polypeptide of interest and a second tagged molecule is incorporated for the detection strategy. Each spot on the array corresponds to a different sample. Total lysates of different samples are immobilized on the array and incubated with one antibody. Each spot on the array corresponds to a different sample (up to 640 lysates per array). Reverse phase microarray allows for probing into the networking and cross-talk between proteins involved in intracellular signaling. Uses of reverse phase microarray techniques in, for example, microarray printing, protein detection, and/or protein quantification are all commensurate with the scope of the instant invention.
Detection of polypeptides can be made by any suitable technique. Polypeptide backbone can be detected, as well as post-translational modifications of it, such as glycosylation and phosphorylation. Antibodies can be used routinely, e.g., which are generated to amino acid epitopes of the target polypeptide; phosphorylated amino acids, etc. Reverse phase assay can be used to detect ocular polypeptides, where the array is comprised of ocular fluid immobilized to a substrate such as nitrocellulose, and binding partners (such as antibodies) are applied that specifically bind the target of interest. These can be rapidly used to characterize the contents of the fluid and generate disease biomarkers, including proteomic fingerprints. See, e.g., Grubb et al., Proteomics, 3:2142-2146, 2003. Mass spectroscopy and other conventional proteomic methods can also be used.
In the instant invention, there is provided a method for detecting macular diseases, retinal detachment, inflammation of the eye, diabetic retinopathy and many other diseases comprising comparing a profile of shed receptors or signal transduction molecules and/or their phosphorylated forms, for e.g., VEGFR, PDGFR, EGFR, RBP4 in a healthy subject with that of a patient. Of note is that these receptor proteins are known to be existing drug targets for existing drugs such as Gleevac, Iressa, and Avastin, demonstrating that this information could be used to tailor therapy for the patient. With regard to cataracts the instant invention relates to identification of a series of crystallins in vitreous samples of patients who have had a vitrectomy for retinal detachment. Such patients have virtually a certain chance of immediately developing cataracts. With regard to macular hole or macular detachment therapy or macular vascular leak, therapy can include administration of natural autologous protein such as platelet extracts.
Given the correlations described in this application, for a particular patient, the presence of a disease will be measured by, for example, extracting vitreous fluid from the patient and determining the content of the particular polypeptide or fragment of interest using fully conventional methods such as (immunologic techniques, antibody diagnostics, radioimmunoassays, mass spectrometry, microarrays, western blotting, gel electrophoresis, and labeled or enzyme amplified diagnostic technologies.
Using the technique described previously, one of ordinary skill in the art, using routine methods may develop correlations that cater to, or are specific towards the detecting and diagnosis of a particular disease. For instance, the method provides a means for characterizing the identity and/or content of vitreous fluid with respect to the levels or amounts of particular peptides which will be indicative of disease. Peptides that are unique to a disease, wherein the presence of any amount of such peptides will indicate the likelihood of the disease being present are described. One of ordinary skill in the art could utilize existing knowledge of peptide biomarkers which correlate with a particular disease or a physiological state, and screen for said peptide(s) using the method described by the instant invention. In some cases the presence or absence of the molecule above background may be diagnostic of the disease, because that molecule may not be expected otherwise. An example is molecules associated with vascular leakage during wet macular degeneration. In other cases the level of the molecule concentration or the level of the phosphorylated molecules (phosphorylation on one or more specific residues) may be quantitatively related to the severity of the disease or the amount of disease suppression produced by a drug administered to the patient. An example is a method for detecting the phosphorylation status of the VEGFR, (which may have no correlation with the amount of total receptor protein) as a predictor of (a) requirement for an angiogenesis inhibitor, and (b) whether or not an angiogenesis inhibitor is working to suppress the VEGF ligand from triggering its receptor. If the receptor is active or engaged with ligand then and only then will it be phosphorylated.
The instant invention relates to the use of the vitreous fluid as a reservoir of important biological markers. As is explained in the tables, samples may be isolated from a live specimen or from a cadaver. Tables 2-13 provide a representative list of peptides which are present in the vitreous fluid of the eye.
The instant invention also provides a method for identification of novel proteins/peptides which are potential biomarkers of diseases and/or physiological state in subject. Representative examples of such peptides in relation to ocular diseases (for e.g., macular hole, retinal degeneration, or a combination of macular hole and retinal degeneration) are provided in the tables (Tables 5-13). Polypeptides that are specifically associated with a disease, for e.g., when compared to a different disease or a control sample, are highlighted/underlined.
Additionally, the present invention relates to a method for detecting proteins in the vitreous fluid of the eye comprising isolation of the protein, enzymatic hydrolysis (for e.g., using trypsin), HPLC separation, resolved using mass spectrometric analysis, and the retrieved fragments are searched a database of candidate polypeptides. Routine methods for HPLC analysis of peptides are known in the art, and may involve utilization of separation columns and/or buffers of interest (for e.g., modified C-18 column). Techniques for mass-spectrometric analysis of peptides are also known, and may involve, for e.g., nano-spray/linear Ion Trap mass spectrometric analysis.
The present invention also provides an improved hollow bore cannula or cutter for performing a vitrectomy, wherein the improvement comprises a reservoir in said cannula or cutter that comprises at least one chemical to protect polypeptide integrity. Chemicals that can be included in the reservoir include, e.g., protease inhibitors; phosphatase inhibitors; etc. Specific examples include, serine protease inhibitors, cysteine protease inhibitors, aspartic protease inhibitors, and metalloprotease inhibitors. Examples of these include, AEBSF, aprotinin, E-64, EDTA, leupeptin, bestatin, O-phenanthroline, cathepsin, etc.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the following invention to its fullest extent. The following specific preferred claims are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
The entire disclosures of all applications, patents and publications, cited above and below, are hereby incorporated by reference.
The invention will be explained below with reference to the following non-limiting examples.
In the forgoing and in the following examples, all temperatures are set forth uncorrected in degrees Celsius (° C.) and, all parts and percentages are by weight, unless otherwise indicated.
Experimental Procedure
Vitreous Sampling:
Pars Plana Vitrectomy
All vitreous samples were obtained prior to the vitrectomy portion of the surgery. The surgery would have been done regardless of participation in the study. The patient was prepped and draped in the usual sterile fashion. Prior to the vitrectomy portion of the study, a minute amount of vitreous (approximately 0.1 ml) was obtained in a sterile TB syringe through the pars plana. The vitreous sample was then frozen at −20° C. or −80° C. for storage and subsequent analysis of the vitreous proteome. There was no additional risk to the patient in addition to that incurred from the surgery alone.
Autopsy Study
At an unrestricted consented autopsy, 2-8 ml of clear vitreous gel were extracted by inserting in the lateral palpebral fissure a 22 g needle attached to a 10 ml tuberculin. The samples were immediately frozen at −80° C.
Approximately 5-10 cc of vitreous was collected from each eye. Nine separate autopsy samples were procured. The samples were stored at −80° C. until the analysis could be performed.
Samples From Patients Undergoing a Total Vitrectomy
The patients were prepared accordingly to the anesthetic/surgical protocols prescribed for the specific pathology which the patients were suffering from. Vitrectomy was carried out using a surgical microscope and external lenses designed to provide a clear image of the back of the eye. Using the sclerotome, tree tiny incisions just a few millimeters in length were made on the sclera, then the retinal surgeon inserted the following instruments: 1) a fiber optic light source to illuminate inside the eye; 2) the infusion line to maintain the eye's shape and tone during surgery and 3) the vitrectome to cut and remove the vitreous. The total vitreous was aspirated by the vitrectome and diluted 5-8 times with Ringer-lactate buffer solution kept at room temperature during the surgical session, depending on the length of the surgical procedure, on whether additional procedures were required and on the overall health of the eye. Immediately after the vitreous removal, the cassette containing the diluted vitreous was placed at 4° C., and within 60 min gently aspirated, then diluted 1:1 with cold Ringer-lactate buffer solution (at 4° C.). Then the suspension was carefully mixed five times, then passed through a sterile pipette with narrow tip (20 passages) until any macroscopic material was completely dissolved. Finally, the re-suspended material was divided into small aliquots (1 ml) into plastic tubes previously labeled, immediately frozen with liquid nitrogen and stored at −80° C. within 15 min. Smaller aliquots were also frozen to carry out subsequently the protein quantification, using a commercial Bradford assay (Biorad). All the procedures were carried out using sterile plastic ware.
To develop a reproducible procedure to analyze the proteins contained in eye tissues, preliminary experiments were carried out with bovine eyeballs obtained from a local slaughterhouse. The eyeballs were maintained at 4° C. until the vitreous was extracted, i.e. 6-8 hours after the death of the animal. The eyeballs were cut 3 mm posterior to the limbus and the whole vitreous and the whole lens were removed as described (Facchiano et al, 1996). Then, samples were re-suspended by mechanical dissociation of the material using cold saline buffer. Protein concentration and stability in the eye tissues homogenates was checked by SDS-PAGE analysis and silver staining procedure.
Nanoflow Reversed-Phase Liquid Chromatography Tandem Mass Spectrometry
Vitreous samples were digested by trypsin and peptides were purified by Zip-tip (Waters). The peptides were then analyzed by reversed-phase liquid chromatography nanospray tandem mass spectrometry using a linear ion-trap mass spectrometer (LTQ, ThermoElectron, San Jose, Calif.). Reverse phase column was slurry-packed in-house with 5 μm, 200 Å pore size C18 resin (Michrom BioResources, CA) in 100 μm i.d.×10 cm long fused silica capillary (Polymicro Technologies, Phoenix, Ariz.) with a laser-pulled tip. After sample injection, the column was washed for 5 min with mobile phase A (0.1% formic acid) and peptides were eluted using a linear gradient of 0% mobile phase B (0.1% formic acid, 80% acetonitrile) to 50% mobile phase B in 30 min at 250 nl/min, then to 100% B in an additional 5 min. The LTQ mass spectrometer was operated in a data-dependent mode in which each full MS scan was followed by five MS/MS scans where the five most abundant molecular ions were dynamically selected and fragmented by collision-induced dissociation (CID) using a normalized collision energy of 35%.
Bioinformatic Analysis
Tandem mass spectra were matched against Swiss-Prot human database through the Sequest Bioworks Browser (ThermoFinnigan) using tryptic cleavage constraints and static cysteine alkylation by iodoacetamide. For a peptide to be considered legitimately identified, it had to achieve cross correlation scores of 1.5 for [M+H]1+, 2.0 for [M+2H]2+, 2.5 for [M+3H]3+, ΔCn>0.1, and a maximum probabilities of randomized identification of 0.01.
The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.
From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention and, without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.
It is believed that one skilled in the art, using the preceding information and information available in the art, can utilize the present invention to its fullest extent. It should be apparent to one of ordinary skill in the art that changes and modifications can be made to this invention without departing from the spirit or scope of the invention as it is set forth herein. The topic headings set forth above and below are meant as guidance where certain information can be found in the application, but are not intended to be the only source in the application where information on such topic can be found. All publications and patents cited above are incorporated herein by reference.
tide
tot ids in
8.0 ul vitreous/patient
8.0 × 5 = 40 ul/patient for study
tide
s in 10
LGIHC-
GIHCA-
GLHCDEA
EABFGH
FIHGB-
ECDBFG
ILHGA-
LGIAE-
GLDEA
This application claims the benefit of earlier-filed U.S. Provisional Application Ser. No. 60/762,499, filed Jan. 27, 2006, which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60762499 | Jan 2006 | US |
