US20230324407A1 - Identification and use of glycopeptides as biomarkers for diagnosis and treatment monitoring - Google Patents
Identification and use of glycopeptides as biomarkers for diagnosis and treatment monitoring Download PDFInfo
- Publication number
- US20230324407A1 US20230324407A1 US18/180,789 US202318180789A US2023324407A1 US 20230324407 A1 US20230324407 A1 US 20230324407A1 US 202318180789 A US202318180789 A US 202318180789A US 2023324407 A1 US2023324407 A1 US 2023324407A1
- Authority
- US
- United States
- Prior art keywords
- disease
- cancer
- samples
- glycopeptides
- biological samples
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
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
- G01N33/6854—Immunoglobulins
- G01N33/6857—Antibody fragments
-
- 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
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
-
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57415—Specifically defined cancers of breast
-
- 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
- G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
-
- 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
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
- G16B40/10—Signal processing, e.g. from mass spectrometry [MS] or from PCR
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
- G16B40/20—Supervised data analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2560/00—Chemical aspects of mass spectrometric analysis of biological material
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/08—Hepato-biliairy disorders other than hepatitis
Definitions
- the present disclosure relates generally to the field of multi-omics, in particular, glycomics and glycoproteomics, advanced instrumentation big data, machine learning and artificial intelligence to identify biomarkers for disease diagnosis and treatment monitoring.
- Protein glycosylation and other post-translational modifications play vital structural and functional role in all aspects of human growth and development. Defective protein glycosylation accompanies several diseases. Identifying altered glycosylation at early disease stages provides opportunities for early detection, intervention and greater chance of survival in subjects affected.
- biomarkers that can detect early cancer and discriminate a certain type of cancer from other diseases. Those methods include proteomics, peptidomics, metabolics, proteoglycomics and glvcomics using mass spectrometry (MS).
- the present disclosure relates to methods of identifying biomarkers for various diseases.
- the biomarkers are the glycosylated peptide fragments obtained via fragmentation of glycosylated proteins from biological samples.
- the methods of identifying the biomarkers rely upon the use of advanced mass spectrometry techniques that allow for the accurate mass measurements of the glycosylated peptide fragments as well as the site-specific glycosylation analysis.
- the mass spectroscopy methods of the present disclosure are advantageously useful in analyzing a large number of glycosylated proteins from the biological samples at a time.
- the present disclosure provides a method for identifying glycosylated peptide fragments as potential biomarkers, comprising:
- the method comprises the subjects having a disease or a condition and subjects not having the disease or the condition.
- the subjects comprise subjects receiving a treatment for a disease or a condition and subjects having the disease or the condition but not receiving a treatment.
- the methods of the present disclosure are applicable to any disease or condition that can be detected by analyzing the glycosylated peptide fragments from the biological samples of a subject.
- the disease is cancer.
- the disease is an autoimmune disease.
- the methods of the present disclosure provide glycosylated peptide fragments that are 0-glycosylated or N-glycosylated.
- the methods of the present disclosure provide glycosylated peptide fragments having an average length of from 5 to 50 amino acid residues.
- the methods of the present disclosure employ glycosylated proteins that are one or more of alpha-1-acid glycoprotein, alpha-1-antitrypsin, alpha-1B-glycoprotein, alpha-2-HS-glycoprotein, alpha-2-macroglobulin, antithrombin-III, apolipoprotein B-100, apolipoprotein D, apolipoprotein F, beta-2-glycoprotein 1, ceruloplasmin, fetuin, fibrinogen, immunoglobulin (Ig) A, IgG, IgM, haptoglobin, hemopexin, histidine-rich glycoprotein, kininogen-1, serotransferrin, transferrin, vitronectin and zinc-alpha-2-glycoprotein.
- glycosylated proteins that are one or more of alpha-1-acid glycoprotein, alpha-1-antitrypsin, alpha-1B-glycoprotein, alpha-2-HS-glycoprotein, alpha
- the methods of the present disclosure comprise fragmentation of the glycosylated proteins using at least two proteases.
- the methods of the present disclosure employ LC-MS techniques using multiple reaction monitoring mass spectrometry (MRM-MS).
- the present disclosure provides methods for identifying glycosylated peptide fragments as potential biomarkers for various diseases as described herein, wherein the biological sample is body tissue, saliva, tears, sputum, spinal fluid, urine, synovial fluid, whole blood, serum or plasma obtained from the subjects.
- the subjects are mammals. In another embodiment, the subjects are humans.
- the present disclosure provides a method for identifying glycosylated peptide fragments as potential biomarkers, comprising:
- the present disclosure provides methods for identifying glycosylated peptide fragments as potential biomarkers for various diseases as described herein, wherein the analysis further comprises genomic data, proteomics, metabolics, lipidomics data, or a combination thereof.
- FIG. 1 is a Schematic diagram showing the integration of Glycomics, LC/MS and machine learning that can further be combined with protemomics, genomic, lipidomics and metabolics;
- FIG. 2 shows changes in immunoglobulin G (IgG) glycopeptide ratios in plasma samples from breast cancer patients versus controls;
- FIG. 3 shows changes in IgG glycopeptide ratios in plasma samples from primary sclerosing cholangitis (PSC) and primary biliary cirrhosis (PBC) samples versus healthy donors;
- FIG. 4 shows the separate discriminant analysis data for IgG, IgA and IgM glycopeptides in plasma samples from PSC and PBC samples versus healthy donors;
- FIG. 5 shows the combined discriminant analysis data for IgG, IgA and IgM glycopeptides in plasma samples from PSC and PBC patients versus healthy donors.
- biological sample refers to mean any biological fluid, cell, tissue, organ or a portion thereof. It also includes, but is not limited to, a tissue section obtained by biopsy, or cells that are placed in or adapted to tissue culture. It further includes, but is not limited to, saliva, tears, sputum, sweat, mucous, fecal material, gastric fluid, abdominal fluid, amniotic fluid, cyst fluid, peritoneal fluid, spinal fluid, urine, synovial fluid, whole blood, serum, plasma, pancreatic juice, breast milk, lung lavage, marrow, and the like.
- biomarker refers to a distinctive biological or biologically-derived indicator of a process, event or a condition.
- a biomarker is also indicative of a certain biological state, such as presence of a disease or a condition or risk of a disease or a condition. It includes a biological molecule, or a fragment of a biological molecule, the change or detection of which can be correlated with a particular physical state or a condition.
- biomarkers include, but are not limited to, biological molecules comprising nucleotides, amino acids, fatty acids, steroid, antibodies, hormones, steroids, peptides, proteins, carbohydrates, and the like. Further examples include glycosylated peptide fragments, lipoproteins, and the like.
- compositions and methods include the recited methods, but do not exclude others.
- glycocan refers to the carbohydrate portion of a glycoconjugate, such as a glycopeptide, glycoprotein, glycolipid or proteoglycan.
- glycoform refers to a unique primary, secondary, tertiary and quaternary structure of a protein with an attached glycan of a specific structure.
- glycosylated peptide fragment refers to a glycosylated peptide (or glycopeptide) having an amino acid sequence that is the same as part but not all of the amino acid sequence of the glycosylated protein from which the glycosylated peptide is obtained via fragmentation, e.g., with one or more proteases.
- MRM-MS multiple reaction monitoring mass spectrometry
- protease refers to an enzyme that performs proteolysis or breakdown of proteins into smaller polypeptides or amino acids.
- examples of a protease include, serine protease, threonine protease, cysteine protease, aspartate protease, glutamic acid protease, metalloprotease, asparagine peptide lyase and a combination thereof.
- subject refers to a mammal.
- the non-liming examples of a mammal include a human, non-human primate, mouse, rat, dog, cat, horse, or cow, and the like. Mammals other than humans can be advantageously used as subjects that represent animal models of disease, pre-disease, or a pre-disease condition.
- a subject can be male or female.
- a subject can be one who has been previously identified as having a disease or a condition, and optionally has already undergone, or is undergoing, a therapeutic intervention for the disease or condition. Alternatively, a subject can also be one who has not been previously diagnosed as having a disease or a condition.
- a subject can be one who exhibits one or more risk factors for a disease or a condition, or a subject who does not exhibit disease risk factors, or a subject who is asymptomatic for a disease or a condition.
- a subject can also be one who is suffering from or at risk of developing a disease or a condition.
- treatment means any treatment of a disease or condition in a subject, such as a mammal, including: 1) preventing or protecting against the disease or condition, that is, causing the clinical symptoms not to develop; 2) inhibiting the disease or condition, that is, arresting or suppressing the development of clinical symptoms; and/or 3) relieving the disease or condition that is, causing the regression of clinical symptoms.
- the present disclosure in some embodiments, relates to glycoproteomics glycoproteomics for biomarker discovery, target discovery and validation using advanced LC/MS instrumentation.
- the disclosure utilizes machine learning methods to process the molecular data.
- the analysis further comprises utilizing genomic data, proteomics, metabolics, lipidomics data, or a combination thereof in discovering new biomarkers for various diseases.
- the general schematic for the methods of this disclosure is shown in FIG. 1 .
- the present disclosure provides methods for site-specific glycosylation analysis that leads to identification of new types of biomarkers with higher sensitivity and specificity.
- the method comprises quantitation of glycosylated peptides, thus facilitating differential analysis of distinct glycoforms associated with specific proteins at distinct sites.
- the method provides information regarding the amount of protein and the site-specific glycosylation profile, thus providing insight into whether the altered glycosylation profile is due to the change in protein glycosylation or it is due to a change in protein concentration.
- the site specific glycosylation analysis in combination with machine learning method(s) provide identification of new biomarkers for various diseases or conditions.
- the quantitative glycoproteomics methods of the disclosure are used to discover biomarkers of various diseases.
- the methods are based on the fact that specific glycoforms are elevated and others are down regulated in several diseases and the LC/MS methods of the present disclosure differentiate between the disease versus no disease by analyzing the significant glycosylation changes.
- the site-specific glycosylation analysis comprises identifying the glycoproteins of interest, the sites of modification, what the modification is and then measuring the relative abundance of each modification.
- the disease is cancer.
- the disease is an autoimmune disease.
- the biological samples from thousands of subjects are digitized to generate tremendous amount of data that undergoes deep machine learning analysis to discover new targets for various diseases.
- deep learning is used to compare clustering of known and unknown peptides and their glycosylation signatures as seen by LC/MS in disease versus control states.
- Such discriminant analysis of the glycosylated peptides leads to the identification of the disease biomarkers.
- the identification of the biomarkers and their corresponding features such as their expression level are then used for developing diagnostic test methods for a disease or a condition, wherein the methods rely upon, at least in part, on measuring one or more of the selected biomarkers and analyzing the result for an association with the disease or the condition.
- the methods can further be used in selecting one or more therapies, determining a treatment regimen or to monitor response to a therapy for the particular disease or condition.
- the present disclosure provides methods for prevention, diagnosis, therapy, monitoring and prognosis of a disease or a condition.
- the methods are useful in discriminating between the subjects having a disease or a condition and healthy subjects.
- the methods are useful in discriminating between subjects having cancer and healthy subjects.
- the methods are useful in aiding the diagnosis of cancer or for monitoring cancer.
- the biomarker discovery methods of the present disclosure employ both targeted and/or non-targeted approaches.
- the methods typically comprise three different phases, namely, discovery phase, pre-validation phase and validation phase.
- the targeted approach comprises identifying and monitoring the known glycoproteins with their known glycoforms in the biological samples of subjects.
- the glycosylation changes of the biomarkers are tumor-specific and are useful in identifying a possible risk of the disease or a disease stage.
- the targeted approach focuses on the known glycoproteins and their glycoforms that are chemically characterized and biologically annotated with the established biological importance at the start of the study before data acquisition is performed. Quantification is performed through the use of internal standards and authentic chemical standards.
- the site-specific glycosylation analysis is conducted in biological samples from case-control study of a number of subjects having a disease or condition and equal number of matched control subjects not having the disease or condition.
- the glycoprotein of interest such as a disease related glycoprotein or a glycoprotein with a biological activity, is first identified in the biological sample. It is then analyzed using LC/MS for the site of modification, nature of modification, identity of the modification and the relative abundance of each modification, leading to identification and quantification of the peptide fragments.
- This approach uses triple quadrupole (QQQ) mass spectrometer for the quantification of the glycosylated peptide fragments which are then analyzed for its relation to the classification of the subjects.
- the non-targeted approach comprises learning the glycosylation patterns of known as well as unknown peptide fragments to provide more information on changes in glycosylation patterns that is useful in identifying the classification of the subjects.
- the non-targeted approach is based on relative quantitation technique that provides “up or down regulation” of the glycoproteins. Specifically, the up or down regulation of the glycoproteins is monitored in relation to the classification of the subjects. For example, the glycoprotein fragments are monitored for subjects having a disease or a condition versus subjects not having a disease or a condition. This approach does not know the chemical identity of each glycoprotein fragment before the data is acquired.
- the non-targeted approach uses quadrupole time-of-flight (qTOF) mass spectrometer for the analysis of the glycosylated peptide fragments.
- qTOF quadrupole time-of-flight
- the candidates differently expressed between the groups are selected for further evaluation, using machine learning methods to allow for the prediction of classification with feature selection techniques with important clinical characteristics.
- Performance is evaluated using internal cross validation in which features are selected and models are constructed using the training set. The resulting models are evaluated on the test set that was not used in the construction of the model.
- the false positive rate is controlled by using the false discovery rate (FDR) approach introduced by Benjamin and Hochberg.
- the candidate biomarkers thus identified in the discovery phase are then tested in an independent test set of biological samples obtained from a number of subjects having a disease or a condition and their matched controls not having the disease or condition, to determine the performance of the candidate biomarkers.
- the selected biomarker, its ranking, together with any parameter estimation of the models developed in the discovery phase are all part of the modelling and are tested with this independent pre-validation phase.
- a diagnostic test classifies the biological samples into two groups: those with a disease and those without a disease. The test is then assessed for its usefulness based on positive predictive value, negative predictive value, specificity and sensitivity.
- the diagnostic performance is evaluated using receiver operating characteristic (ROC) curves to test which biomarkers or a combination of multiple biomarkers are statistically better diagnostic tests for a disease or condition.
- ROC receiver operating characteristic
- the individual biomarkers that are successfully validated are examined for subsequent inclusion to form a panel of composite markers.
- the composite markers are constructed by weighted multi-variable logistic regression or other classification algorithms.
- the candidate biomarkers retained in the pre-validation phase are then validated through independent validations using independent blinded biological samples from a number of subjects.
- the purpose of this phase is to assess the diagnostic precision of the selected biomarkers.
- the biomarker discovery method is applied to biological samples obtained from subjects having cancer.
- biological samples from at least 20, at least 40, at least 60, at least 80 or at least 100 subjects are analyzed in each group (i.e. a group having cancer or a group not having cancer).
- both targeted and/or non-targeted approaches along with the machine learning methods as described herein, provide new diagnostic methods for identifying possible risk and/or early stage detection of various diseases.
- this disclosure provides the methods of identification of biomarkers that are based on the convergence of targeted and non-targeted approaches in combination with the machine learning method.
- the biomarkers identified by the methods of the present disclosure are useful in methods of diagnosis, methods of prognosis assessment, monitoring results of therapy, identifying subjects likely to respond to a particular treatment, drug screening, and the like.
- the present disclosure provides a method for identifying glycosylated peptide fragments as potential biomarkers, comprising:
- the present disclosure provides the method as described herein, wherein subjects comprise subjects having a disease or condition and subjects not having the disease or condition. In a further embodiment, the subjects comprise subjects receiving a treatment for a disease and subjects having the disease but not receiving a treatment for the disease.
- the methods of the present disclosure are applicable to any disease or condition that can be detected by analyzing the glycosylated peptide fragments from the biological samples of a subject.
- the disease is cancer.
- the cancer selected from breast cancer, cervical cancer or ovarian cancer.
- the disease is an autoimmune disease.
- the autoimmune disease is HIV, primary sclerosing cholangitis, primary biliary cirrhosis or psoriasis.
- the present disclosure provides the methods as described herein, wherein the glycosylated proteins are one or more of alpha-1-acid glycoprotein, alpha-1-antitrypsin, alpha-1B-glycoprotein, alpha-2-HS-glycoprotein, alpha-2-macroglobulin, antithrombin-III, apolipoprotein B-100, apolipoprotein D, apolipoprotein F, beta-2-glycoprotein 1, ceruloplasmin, fetuin, fibrinogen, immunoglobulin (Ig) A, IgG, IgM, haptoglobin, hemopexin, histidine-rich glycoprotein, kininogen-1, serotransferrin, transferrin, vitronectin and zinc-alpha-2-glycoprotein.
- the glycosylated proteins are one or more of alpha-1-acid glycoprotein, immunoglobulin (Ig) A, IgG or IgM.
- the present disclosure provides the methods as described herein, wherein the glycosylated peptide fragment is N-glycosylated or O-glycosylated.
- the present disclosure provides the methods as described herein, wherein the glycosylated peptide fragments have an average length of from about 5 to about 50 amino acid residues.
- the glycosylated peptide fragments have an average length of from about 5 to about 45, or from about 5 to about 40, or from about 5 to about 35, or from about 5 to about 30, or about from 5 to about 25, or from about 5 to about 20, or from about 5 to about 15, or from about 5 to about 10, or from about 10 to about 50, or from about 10 to about 45, or from about 10 to about 40, or from about 10 to about 35, or from about 10 to about 30, or from about 10 to about 25, or from about 10 to about 20, or from about 10 to about 15, or from about 15 to about 45, or from about 15 to about 40, or from about 15 to about 35, or from about 15 to about 30, or about from 15 to about 25 or from about 15 to about 20 amino acid residues.
- the glycosylated peptide fragments have an average length of about 15 amino acid residues. In another embodiment, the glycosylated peptide fragments have an average length of about 10 amino acid residues. In another embodiment, the glycosylated peptide fragments have an average length of about 5 amino acid residues.
- the present disclosure provides the methods as described herein, wherein the one or more proteases comprise any protease that is used for fragmenting proteins.
- the protease is a serine protease, threonine protease, cysteine protease, aspartate protease, glutamic acid protease, metalloprotease, asparagine peptide lyase or a combination thereof.
- protease examples include, but are not limited to, trypsin, chymotrypsin, endoproteinase, Asp-N, Arg-C, Glu-C, Lys-C, pepsin, thermolysin, ealastase, papain, proteinase K, subtilisin, clostripain, carboxypeptidase and the like.
- the present disclosure provides the methods as described herein, wherein the one or more proteases comprise at least two proteases.
- the one of more biomarkers are useful to discriminate between the pre-disease state from a disease state, or a disease state from a normal state.
- Other non-disease specific health states can also be determined.
- changes of the biomarker can be assayed at different times: in a subject with a disease, to monitor the progress of the disease; in a subject undergoing treatment, to monitor the effect of the treatment and in a subject post-treatment, to monitor a possible relapse.
- the levels of a specific amount of biomarker also may allow for choosing the course of treatment of the disease.
- a biological sample can be provided from a subject undergoing treatment regimens for a disease.
- Such treatment regimens can include, but are not limited to, exercise regimens, dietary supplementation, weight loss, surgical intervention, device implantation, and treatment with therapeutics or prophylactics used in subjects diagnosed or identified with a disease or condition.
- changes in glycopeptide ratios in a plurality of glycoproteins can be associated with a certain disease sate or absence of a disease.
- presence of a plurality of particular glycopeptides in a biological sample may indicate absence of a disease
- presence of a plurality of other specific glycopeptides in a biological sample may indicate presence of the disease.
- various glycopeptide profiles or panels of glycopeptide biomarkers can be correlated with various states of a disease.
- Example 2 shows quantitation results of changes in IgG1, IgG0 and IgG2 glycopeptides in plasma samples from breast cancer patients versus the controls.
- FIG. 2 illustrates that the levels of glycopeptides A1 and A2 were elevated as compared to the control, whereas the levels of glycopeptides A8, A9 and A10 were reduced as compared to the control in all stages of breast cancer studied in this experiment, thus indicating that glycopeptides A1, A2, A8, A9 and A10 are potential biomarkers for breast cancer.
- Example 3 shows quantification results of changes in IgG, IgM and IgA glycopeptides in plasma samples from patients having PSC and patients having PSC.
- FIG. 3 illustrates that glycopeptide A was elevated as compared to the healthy donors in plasma samples of patients having PBC and PSC, whereas glycopeptides H, I and J were reduced as compared to the healthy donors in plasma samples of patients having PBC and PSC.
- glycopeptides A, H, I and J are potential biomarkers for PBC and PSC.
- the separate and combined discriminant analysis results are provided in FIG. 4 and FIG. 5 respectively indicating an accuracy of 88% for predicting the disease state in the combined discriminant analysis.
- the present disclosure provides methods, wherein the number of biomarkers that are detected and analyzed are 1, or more than 1, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23, 24, 25, 26, 27, 28, 30 or more.
- the disclosure also provides a panel of biomarkers that is useful in the diagnosis of a disease or condition.
- the present disclosure provides methods as described herein that comprise quantitating the glycosylated peptide fragments by using a mass spectrometer.
- the methods employ a technique called “multiple reaction monitoring (MRM).” This technique is often coupled with liquid chromatography (LC/MRM-MS) and allows the quantitation of hundreds of glycosylated peptide fragments (and their parent proteins) in a single LC/MRM-MS analysis.
- MRM multiple reaction monitoring
- LC/MRM-MS liquid chromatography
- the advanced mass spectroscopy techniques of the present disclosure provide effective ion sources, higher resolution, faster separations and detectors with higher dynamic ranges that allow for broad untargeted measurements that also retain the benefits of targeted measurements.
- the mass spectroscopy methods of the present disclosure are applicable to several glycosylated proteins at a time. For example, at least more than 50, or at least more than 60 or at least more than 70, or at least more than 80, or at least more than 90, or at least more than 100, or at least more than 110 or at least more than 120 glycosylated proteins can be analyzed at a time using the mass spectrometer.
- the mass spectroscopy methods of the present disclosure employ QQQ or qTOF mass spectrometer. In another embodiment, the mass spectroscopy methods of the present disclosure provide data with high mass accuracy of 10 ppm or better; or 5 ppm or better; or 2 ppm or better; or 1 ppm or better; or 0.5 ppm or better; or 0.2 ppm or better or 0.1 ppm or better at a resolving power of 5,000 or better; or 10,000 or better; or 25,000 or better; or 50,000 or better or 100,000 or better.
- the biological samples are one or more clinical samples collected in the past, thus reducing the resources and time that must be committed to identifying new biomarkers.
- the biological samples are from one or more past studies that occurred over a span of 1 to 50 years or more.
- the studies are accompanied by various other clinical parameters and previously known information such as the subject's age, height, weight, ethnicity, medical history, and the like. Such additional information can be useful in associating the subject with a disease or a condition.
- the biological samples are one or more clinical samples collected prospectively from the subjects.
- the present disclosure provides the methods as described herein, wherein the biological sample isolated from the subjects is one or more of saliva, tears, sputum, sweat, mucous, fecal material, gastric fluid, abdominal fluid, amniotic fluid, cyst fluid, peritoneal fluid, spinal fluid, urine, synovial fluid, whole blood, serum, plasma, pancreatic juice, breast milk, lung lavage, marrow.
- the biological sample isolated from the subjects is body tissue, saliva, tears, sputum, spinal fluid, urine, synovial fluid, whole blood, serum or plasma.
- the biological sample isolated from the subjects is whole blood, serum or plasma.
- the subjects are mammals. In other embodiments, the subject are humans.
- the methods of the present disclosure are applicable to any disease or condition that can be detected by analyzing the glycosylated peptide fragments from the biological samples of a subject.
- the disease or condition is cancer.
- the cancer is acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical cancer, anal cancer, bladder cancer, blood cancer, bone cancer, brain tumor, breast cancer, cancer of the female genital system, cancer of the male genital system, central nervous system lymphoma, cervical cancer, childhood rhabdomyosarcoma, childhood sarcoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CIVIL), colon and rectal cancer, colon cancer, endometrial cancer, endometrial sarcoma, esophageal cancer, eye cancer, gallbladder cancer, gastric cancer, gastrointestinal tract cancer, hairy cell leukemia, head and neck cancer, hepatocellular cancer,
- ALL acute lymph
- the disease is an autoimmune disease.
- the autoimmune disease is acute disseminated encephalomyelitis, Addison's disease, agammaglobulinemia, age-related macular degeneration, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, antisynthetase syndrome, atopic allergy, atopic dermatitis, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune uticaria, autoimmune uveitis, Balo disease/Balo concentric sclerosis, Behcet's disease, Berger's disease, Bi
- the biological samples are obtained from thousands of subjects which are then used for digitizing with the intention of deep mining for and validating previously undiscovered markers.
- the biological samples are tumor samples or blood samples. They are digitized using LC/MS instruments to generate tremendous amount of data that undergoes deep machine learning analysis to discover new targets for various diseases.
- the disease is cancer or autoimmune disease.
- the present disclosure provides a method for identifying glycosylated peptide fragments as potential biomarkers, comprising:
- the glycoprotein of interest is first identified in the biological sample and then analyzed using LC/MS for the site of modification, nature of modification, identity of the modification and the relative abundance of each modification, leading to identification and quantification of the peptide fragments.
- This approach uses triple quadrupole (QQQ) mass spectrometer for the quantification of the glycosylated peptide fragments which is then analyzed for its relation to the classification of the subjects.
- the glycosylation patterns of all peptide fragments are analyzed to information on changes in glycosylation patterns in various subjects.
- the up or down regulation of the glycoproteins is monitored in relation to the classification of the subjects.
- the glycoprotein fragments are monitored for subjects having a disease or a condition versus subjects not having a disease or a condition.
- This approach uses quadrupole time-of-flight (qTOF) mass spectrometer for the analysis of the glycosylated peptide fragments.
- qTOF quadrupole time-of-flight
- Plasma samples from breast cancer patients having various stages of cancer and their aged matched controls were analyzed for the IgG1, IgG0 and IgG2 glycopeptides and the changes in their ratios were compared. Specifically, 20 samples in Tis stage, 50 samples in EC1 stage, samples in EC2 stage, 25 samples in EC3 stage, 9 samples in EC4 stage and their 73 age matched control samples were subjected to MRM quantitative analysis on a QQQ mass spectrometer. As can be seen from the quantitative results in FIG. 2 , the levels of certain IgG1 glycopeptides were elevated as compared to the controls, whereas the levels of certain IgG1 glycopeptides were reduced as compared to the controls in all stages of breast cancer studied in this experiment. See for example, IgG1 glycopeptides named as
- glycopeptides A1, A2, A8, A9 and A10 are potential biomarkers for breast cancer.
- Plasma samples from patients having primary sclerosing cholangitis (PSC), patients having primary biliary cirrhosis (PBC) and plasma samples from healthy donors were analyzed for IgG1 and IgG2 glycopeptides and the changes in their glycopepide ratios were compared.
- 100 PBC plasma samples, 76 PSC plasma samples and plasma samples from 49 healthy donors were subjected to MRM quantitative analysis on a QQQ mass spectrometer.
- certain IgG1 glycopeptides were elevated as compared to the healthy donors, whereas certain IgG1 glycopeptides were reduced as compared to the controls in plasma samples of patients having PBC and PSC.
- glycopeptide A was elevated as compared to the healthy donors in patients having PBC and PSC, whereas glycopeptides H, I and J were reduced as compared to the healthy donors in plasma samples of patients having PBC and PSC.
- glycopeptides A, H, I and J are potential biomarkers for PBC and PSC.
Abstract
Provided herein are methods for identifying new biomarkers for various diseases using proteomics, peptidomics, metabolics, proteoglycomics, glvcomics, mass spectrometry and machine learning. The present disclosure also provides glycopeptides as biomarkers for various diseases such as cancer and autoimmune diseases.
Description
- This application claims priority to U.S. Provisional Patent Application No. 62/553,676, filed Sep. 1, 2017, which is incorporated by reference herein.
- The present disclosure relates generally to the field of multi-omics, in particular, glycomics and glycoproteomics, advanced instrumentation big data, machine learning and artificial intelligence to identify biomarkers for disease diagnosis and treatment monitoring.
- Protein glycosylation and other post-translational modifications play vital structural and functional role in all aspects of human growth and development. Defective protein glycosylation accompanies several diseases. Identifying altered glycosylation at early disease stages provides opportunities for early detection, intervention and greater chance of survival in subjects affected. Currently, there are methods to identify biomarkers that can detect early cancer and discriminate a certain type of cancer from other diseases. Those methods include proteomics, peptidomics, metabolics, proteoglycomics and glvcomics using mass spectrometry (MS).
- Although protein glycosylation provides useful information about cancer and other diseases, one drawback of the method is that the glycan cannot be traced back to the protein site of origin. To gain more knowledge about cancer biology and an early detection of cancer, it is important not only to identify the glycan, but also its site of attachment within the protein. Glycoprotein analysis is challenging in general due to several reasons. For example, a single glycan composition in a peptide may contain a large number of isomeric structures because of different glycosidic linkages, branching and many monosaccharides having the same mass. Further, the presence of multiple glycans that share the same peptide backbone causes the MS signal to split into various glycoforms, lowering their individual abundances compared to the peptides that are not glycosylated. Therefore, it has been challenging to develop algorithms that can identify glycans and their peptides from the tandem MS data. It is also challenging to obtain comprehensive fragmentation for both the glycan and the peptide as they have different fragmentation efficiencies.
- Thus, there is a need to provide a method for site-specific glycoprotein analysis to obtain crucial and detailed information about protein glycosylation patterns that provide precise quantitative information about the glycosylation site heterogeneity in diseased cells, tissues or bio-fluids compared with the non-diseased ones. Such a method will lead to identify disease biomarkers, particularly for diseases such as cancer. There is also a need to reduce the time in identifying new biomarkers by combining the site-specific glycoprotein analysis data with deep learning and advanced LC/MS instrumentation to identify and validate new disease targets, such as glycan-based drug targets, for diseases such as cancer.
- The present disclosure relates to methods of identifying biomarkers for various diseases. The biomarkers are the glycosylated peptide fragments obtained via fragmentation of glycosylated proteins from biological samples. The methods of identifying the biomarkers rely upon the use of advanced mass spectrometry techniques that allow for the accurate mass measurements of the glycosylated peptide fragments as well as the site-specific glycosylation analysis. The mass spectroscopy methods of the present disclosure are advantageously useful in analyzing a large number of glycosylated proteins from the biological samples at a time.
- In one embodiment, the present disclosure provides a method for identifying glycosylated peptide fragments as potential biomarkers, comprising:
-
- fragmenting glycosylated proteins in each of a plurality of biological samples isolated from subjects, with one or more proteases, to produce glycosylated peptide fragments;
- quantitating the glycosylated peptide fragments with liquid chromatography and mass spectrometry (LC-MS) to provide quantitation results;
- analyzing the quantitation results along with classification of the subjects with a machine learning method to select glycosylated peptide fragments useful for predicting the classification; and
- determining the identity of glycosylated peptide fragments.
- In another embodiment, the method comprises the subjects having a disease or a condition and subjects not having the disease or the condition. In a further embodiment, the subjects comprise subjects receiving a treatment for a disease or a condition and subjects having the disease or the condition but not receiving a treatment.
- In another embodiment, the methods of the present disclosure are applicable to any disease or condition that can be detected by analyzing the glycosylated peptide fragments from the biological samples of a subject. In one embodiment, the disease is cancer. In another embodiment, the disease is an autoimmune disease. In another embodiment, the methods of the present disclosure provide glycosylated peptide fragments that are 0-glycosylated or N-glycosylated. In another embodiment, the methods of the present disclosure provide glycosylated peptide fragments having an average length of from 5 to 50 amino acid residues.
- In another embodiment, the methods of the present disclosure employ glycosylated proteins that are one or more of alpha-1-acid glycoprotein, alpha-1-antitrypsin, alpha-1B-glycoprotein, alpha-2-HS-glycoprotein, alpha-2-macroglobulin, antithrombin-III, apolipoprotein B-100, apolipoprotein D, apolipoprotein F, beta-2-
glycoprotein 1, ceruloplasmin, fetuin, fibrinogen, immunoglobulin (Ig) A, IgG, IgM, haptoglobin, hemopexin, histidine-rich glycoprotein, kininogen-1, serotransferrin, transferrin, vitronectin and zinc-alpha-2-glycoprotein. - In another embodiment, the methods of the present disclosure comprise fragmentation of the glycosylated proteins using at least two proteases. In another embodiment, the methods of the present disclosure employ LC-MS techniques using multiple reaction monitoring mass spectrometry (MRM-MS).
- In another embodiment, the present disclosure provides methods for identifying glycosylated peptide fragments as potential biomarkers for various diseases as described herein, wherein the biological sample is body tissue, saliva, tears, sputum, spinal fluid, urine, synovial fluid, whole blood, serum or plasma obtained from the subjects. In one embodiment, the subjects are mammals. In another embodiment, the subjects are humans.
- In another embodiment, the present disclosure provides a method for identifying glycosylated peptide fragments as potential biomarkers, comprising:
-
- fragmenting glycosylated proteins in each of a plurality of biological samples isolated from subjects, with one or more proteases, to produce glycosylated peptide fragments;
- quantitating the glycosylated peptide fragments with liquid chromatography and mass spectrometry (LC-MS) to provide quantitation results;
- analyzing the quantitation results along with classification of the subjects with a machine learning method to select glycosylated peptide fragments useful for predicting the classification; and
- determining the identity of glycosylated peptide fragments, wherein the machine learning approach is deep learning, neural network, linear discriminant analysis, quadratic discriminant analysis, support vector machine, random forest, nearest neighbor or a combination thereof. In another embodiment, the machine learning approach is deep learning, neural network or a combination thereof.
- In another embodiment, the present disclosure provides methods for identifying glycosylated peptide fragments as potential biomarkers for various diseases as described herein, wherein the analysis further comprises genomic data, proteomics, metabolics, lipidomics data, or a combination thereof.
-
FIG. 1 is a Schematic diagram showing the integration of Glycomics, LC/MS and machine learning that can further be combined with protemomics, genomic, lipidomics and metabolics; -
FIG. 2 shows changes in immunoglobulin G (IgG) glycopeptide ratios in plasma samples from breast cancer patients versus controls; -
FIG. 3 shows changes in IgG glycopeptide ratios in plasma samples from primary sclerosing cholangitis (PSC) and primary biliary cirrhosis (PBC) samples versus healthy donors; -
FIG. 4 shows the separate discriminant analysis data for IgG, IgA and IgM glycopeptides in plasma samples from PSC and PBC samples versus healthy donors; -
FIG. 5 shows the combined discriminant analysis data for IgG, IgA and IgM glycopeptides in plasma samples from PSC and PBC patients versus healthy donors. - As used in the present specification, the following words and phrases are generally intended to have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise.
- It is to be noted that as used herein and in the claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
- The term “biological sample” refers to mean any biological fluid, cell, tissue, organ or a portion thereof. It also includes, but is not limited to, a tissue section obtained by biopsy, or cells that are placed in or adapted to tissue culture. It further includes, but is not limited to, saliva, tears, sputum, sweat, mucous, fecal material, gastric fluid, abdominal fluid, amniotic fluid, cyst fluid, peritoneal fluid, spinal fluid, urine, synovial fluid, whole blood, serum, plasma, pancreatic juice, breast milk, lung lavage, marrow, and the like.
- The term “biomarker” refers to a distinctive biological or biologically-derived indicator of a process, event or a condition. A biomarker is also indicative of a certain biological state, such as presence of a disease or a condition or risk of a disease or a condition. It includes a biological molecule, or a fragment of a biological molecule, the change or detection of which can be correlated with a particular physical state or a condition. Example of biomarkers include, but are not limited to, biological molecules comprising nucleotides, amino acids, fatty acids, steroid, antibodies, hormones, steroids, peptides, proteins, carbohydrates, and the like. Further examples include glycosylated peptide fragments, lipoproteins, and the like.
- The term “comprising” is intended to mean that the compositions and methods include the recited methods, but do not exclude others.
- The term “glycan” refers to the carbohydrate portion of a glycoconjugate, such as a glycopeptide, glycoprotein, glycolipid or proteoglycan.
- The term “glycoform” refers to a unique primary, secondary, tertiary and quaternary structure of a protein with an attached glycan of a specific structure.
- The term “glycosylated peptide fragment” refers to a glycosylated peptide (or glycopeptide) having an amino acid sequence that is the same as part but not all of the amino acid sequence of the glycosylated protein from which the glycosylated peptide is obtained via fragmentation, e.g., with one or more proteases.
- The term “multiple reaction monitoring mass spectrometry (MRM-MS)” refers to a highly sensitive and selective method for the targeted quantification of protein/peptide in biological samples. Unlike traditional mass spectrometry, MRM-MS is highly selective (targeted), allowing researchers to fine tune an instrument to specifically look for peptides/protein fragments of interest. MRM allows for greater sensitivity, specificity, speed and quantitation of peptides/protein fragments of interest, such as a potential biomarker. MRM-MS involves using a triple quadrupole (QQQ) mass spectrometer or quadrupole time-of-flight (qTOF) mass spectrometer.
- The term “protease” refers to an enzyme that performs proteolysis or breakdown of proteins into smaller polypeptides or amino acids. Examples of a protease include, serine protease, threonine protease, cysteine protease, aspartate protease, glutamic acid protease, metalloprotease, asparagine peptide lyase and a combination thereof.
- The term “subject” refers to a mammal. The non-liming examples of a mammal include a human, non-human primate, mouse, rat, dog, cat, horse, or cow, and the like. Mammals other than humans can be advantageously used as subjects that represent animal models of disease, pre-disease, or a pre-disease condition. A subject can be male or female. A subject can be one who has been previously identified as having a disease or a condition, and optionally has already undergone, or is undergoing, a therapeutic intervention for the disease or condition. Alternatively, a subject can also be one who has not been previously diagnosed as having a disease or a condition. For example, a subject can be one who exhibits one or more risk factors for a disease or a condition, or a subject who does not exhibit disease risk factors, or a subject who is asymptomatic for a disease or a condition. A subject can also be one who is suffering from or at risk of developing a disease or a condition.
- The term “treatment” or “treating” means any treatment of a disease or condition in a subject, such as a mammal, including: 1) preventing or protecting against the disease or condition, that is, causing the clinical symptoms not to develop; 2) inhibiting the disease or condition, that is, arresting or suppressing the development of clinical symptoms; and/or 3) relieving the disease or condition that is, causing the regression of clinical symptoms.
- The present disclosure, in some embodiments, relates to glycoproteomics glycoproteomics for biomarker discovery, target discovery and validation using advanced LC/MS instrumentation. The disclosure utilizes machine learning methods to process the molecular data. The analysis further comprises utilizing genomic data, proteomics, metabolics, lipidomics data, or a combination thereof in discovering new biomarkers for various diseases. The general schematic for the methods of this disclosure is shown in
FIG. 1 . - The present disclosure provides methods for site-specific glycosylation analysis that leads to identification of new types of biomarkers with higher sensitivity and specificity. The method comprises quantitation of glycosylated peptides, thus facilitating differential analysis of distinct glycoforms associated with specific proteins at distinct sites. The method provides information regarding the amount of protein and the site-specific glycosylation profile, thus providing insight into whether the altered glycosylation profile is due to the change in protein glycosylation or it is due to a change in protein concentration. The site specific glycosylation analysis in combination with machine learning method(s) provide identification of new biomarkers for various diseases or conditions.
- The quantitative glycoproteomics methods of the disclosure are used to discover biomarkers of various diseases. The methods are based on the fact that specific glycoforms are elevated and others are down regulated in several diseases and the LC/MS methods of the present disclosure differentiate between the disease versus no disease by analyzing the significant glycosylation changes. In one embodiment, the site-specific glycosylation analysis comprises identifying the glycoproteins of interest, the sites of modification, what the modification is and then measuring the relative abundance of each modification. In some embodiments, the disease is cancer. In other embodiments, the disease is an autoimmune disease.
- Using the methods of this disclosure, the biological samples from thousands of subjects are digitized to generate tremendous amount of data that undergoes deep machine learning analysis to discover new targets for various diseases. Specifically, deep learning is used to compare clustering of known and unknown peptides and their glycosylation signatures as seen by LC/MS in disease versus control states. Such discriminant analysis of the glycosylated peptides leads to the identification of the disease biomarkers.
- The identification of the biomarkers and their corresponding features such as their expression level are then used for developing diagnostic test methods for a disease or a condition, wherein the methods rely upon, at least in part, on measuring one or more of the selected biomarkers and analyzing the result for an association with the disease or the condition. The methods can further be used in selecting one or more therapies, determining a treatment regimen or to monitor response to a therapy for the particular disease or condition. Thus, the present disclosure provides methods for prevention, diagnosis, therapy, monitoring and prognosis of a disease or a condition. In some embodiments, the methods are useful in discriminating between the subjects having a disease or a condition and healthy subjects. In some embodiments, the methods are useful in discriminating between subjects having cancer and healthy subjects. In some embodiments, the methods are useful in aiding the diagnosis of cancer or for monitoring cancer.
- Targeted and Non-Targeted Approaches
- The biomarker discovery methods of the present disclosure employ both targeted and/or non-targeted approaches. The methods typically comprise three different phases, namely, discovery phase, pre-validation phase and validation phase.
- Discovery Phase
- The targeted approach comprises identifying and monitoring the known glycoproteins with their known glycoforms in the biological samples of subjects. There are known FDA approved glycoprotein biomarkers for various diseases and those are monitored using the methods of this disclosure to identify the classification of the subjects. Typically, the glycosylation changes of the biomarkers are tumor-specific and are useful in identifying a possible risk of the disease or a disease stage. The targeted approach focuses on the known glycoproteins and their glycoforms that are chemically characterized and biologically annotated with the established biological importance at the start of the study before data acquisition is performed. Quantification is performed through the use of internal standards and authentic chemical standards.
- Specifically, in the targeted approach, the site-specific glycosylation analysis is conducted in biological samples from case-control study of a number of subjects having a disease or condition and equal number of matched control subjects not having the disease or condition. The glycoprotein of interest, such as a disease related glycoprotein or a glycoprotein with a biological activity, is first identified in the biological sample. It is then analyzed using LC/MS for the site of modification, nature of modification, identity of the modification and the relative abundance of each modification, leading to identification and quantification of the peptide fragments. This approach uses triple quadrupole (QQQ) mass spectrometer for the quantification of the glycosylated peptide fragments which are then analyzed for its relation to the classification of the subjects.
- The non-targeted approach comprises learning the glycosylation patterns of known as well as unknown peptide fragments to provide more information on changes in glycosylation patterns that is useful in identifying the classification of the subjects. The non-targeted approach is based on relative quantitation technique that provides “up or down regulation” of the glycoproteins. Specifically, the up or down regulation of the glycoproteins is monitored in relation to the classification of the subjects. For example, the glycoprotein fragments are monitored for subjects having a disease or a condition versus subjects not having a disease or a condition. This approach does not know the chemical identity of each glycoprotein fragment before the data is acquired. In one embodiment, the non-targeted approach uses quadrupole time-of-flight (qTOF) mass spectrometer for the analysis of the glycosylated peptide fragments. This approach involves using the instrumentation to accurately measure the mass of components in a sample, without any preconceived notion about what those components might be.
- The candidates differently expressed between the groups (disease vs. no disease) are selected for further evaluation, using machine learning methods to allow for the prediction of classification with feature selection techniques with important clinical characteristics. Performance is evaluated using internal cross validation in which features are selected and models are constructed using the training set. The resulting models are evaluated on the test set that was not used in the construction of the model. The false positive rate is controlled by using the false discovery rate (FDR) approach introduced by Benjamin and Hochberg.
- Pre-Validation Phase
- The candidate biomarkers thus identified in the discovery phase are then tested in an independent test set of biological samples obtained from a number of subjects having a disease or a condition and their matched controls not having the disease or condition, to determine the performance of the candidate biomarkers. The selected biomarker, its ranking, together with any parameter estimation of the models developed in the discovery phase are all part of the modelling and are tested with this independent pre-validation phase. According to the signals of candidate biomarkers, a diagnostic test classifies the biological samples into two groups: those with a disease and those without a disease. The test is then assessed for its usefulness based on positive predictive value, negative predictive value, specificity and sensitivity. Also, the diagnostic performance is evaluated using receiver operating characteristic (ROC) curves to test which biomarkers or a combination of multiple biomarkers are statistically better diagnostic tests for a disease or condition. The individual biomarkers that are successfully validated are examined for subsequent inclusion to form a panel of composite markers. The composite markers are constructed by weighted multi-variable logistic regression or other classification algorithms.
- Validation Phase
- The candidate biomarkers retained in the pre-validation phase are then validated through independent validations using independent blinded biological samples from a number of subjects. The purpose of this phase is to assess the diagnostic precision of the selected biomarkers.
- In one embodiment, the biomarker discovery method is applied to biological samples obtained from subjects having cancer. In some embodiments, biological samples from at least 20, at least 40, at least 60, at least 80 or at least 100 subjects are analyzed in each group (i.e. a group having cancer or a group not having cancer).
- Both targeted and/or non-targeted approaches, along with the machine learning methods as described herein, provide new diagnostic methods for identifying possible risk and/or early stage detection of various diseases. In one embodiment, this disclosure provides the methods of identification of biomarkers that are based on the convergence of targeted and non-targeted approaches in combination with the machine learning method. The biomarkers identified by the methods of the present disclosure are useful in methods of diagnosis, methods of prognosis assessment, monitoring results of therapy, identifying subjects likely to respond to a particular treatment, drug screening, and the like.
- In one embodiment, the present disclosure provides a method for identifying glycosylated peptide fragments as potential biomarkers, comprising:
-
- fragmenting glycosylated proteins in each of a plurality of biological samples isolated from subjects, with one or more proteases, to produce glycosylated peptide fragments;
- quantitating the glycosylated peptide fragments with liquid chromatography and mass spectrometry (LC-MS) to provide quantitation results;
- analyzing the quantitation results along with classification of the subjects with a machine learning method to select glycosylated peptide fragments useful for predicting the classification; and
- determining the identity of glycosylated peptide fragments.
- In another embodiment, the present disclosure provides the method as described herein, wherein subjects comprise subjects having a disease or condition and subjects not having the disease or condition. In a further embodiment, the subjects comprise subjects receiving a treatment for a disease and subjects having the disease but not receiving a treatment for the disease.
- The methods of the present disclosure are applicable to any disease or condition that can be detected by analyzing the glycosylated peptide fragments from the biological samples of a subject. In one embodiment, the disease is cancer. In another embodiment, the cancer selected from breast cancer, cervical cancer or ovarian cancer. In another embodiment, the disease is an autoimmune disease. In another embodiment, the autoimmune disease is HIV, primary sclerosing cholangitis, primary biliary cirrhosis or psoriasis.
- In another embodiment, the present disclosure provides the methods as described herein, wherein the glycosylated proteins are one or more of alpha-1-acid glycoprotein, alpha-1-antitrypsin, alpha-1B-glycoprotein, alpha-2-HS-glycoprotein, alpha-2-macroglobulin, antithrombin-III, apolipoprotein B-100, apolipoprotein D, apolipoprotein F, beta-2-
glycoprotein 1, ceruloplasmin, fetuin, fibrinogen, immunoglobulin (Ig) A, IgG, IgM, haptoglobin, hemopexin, histidine-rich glycoprotein, kininogen-1, serotransferrin, transferrin, vitronectin and zinc-alpha-2-glycoprotein. In another embodiment, the glycosylated proteins are one or more of alpha-1-acid glycoprotein, immunoglobulin (Ig) A, IgG or IgM. - In another embodiment, the present disclosure provides the methods as described herein, wherein the glycosylated peptide fragment is N-glycosylated or O-glycosylated.
- In another embodiment, the present disclosure provides the methods as described herein, wherein the glycosylated peptide fragments have an average length of from about 5 to about 50 amino acid residues. In some embodiments, the glycosylated peptide fragments have an average length of from about 5 to about 45, or from about 5 to about 40, or from about 5 to about 35, or from about 5 to about 30, or about from 5 to about 25, or from about 5 to about 20, or from about 5 to about 15, or from about 5 to about 10, or from about 10 to about 50, or from about 10 to about 45, or from about 10 to about 40, or from about 10 to about 35, or from about 10 to about 30, or from about 10 to about 25, or from about 10 to about 20, or from about 10 to about 15, or from about 15 to about 45, or from about 15 to about 40, or from about 15 to about 35, or from about 15 to about 30, or about from 15 to about 25 or from about 15 to about 20 amino acid residues. In one embodiment, the glycosylated peptide fragments have an average length of about 15 amino acid residues. In another embodiment, the glycosylated peptide fragments have an average length of about 10 amino acid residues. In another embodiment, the glycosylated peptide fragments have an average length of about 5 amino acid residues.
- In another embodiment, the present disclosure provides the methods as described herein, wherein the one or more proteases comprise any protease that is used for fragmenting proteins. In one embodiment, the protease is a serine protease, threonine protease, cysteine protease, aspartate protease, glutamic acid protease, metalloprotease, asparagine peptide lyase or a combination thereof. A few representative examples of a protease include, but are not limited to, trypsin, chymotrypsin, endoproteinase, Asp-N, Arg-C, Glu-C, Lys-C, pepsin, thermolysin, ealastase, papain, proteinase K, subtilisin, clostripain, carboxypeptidase and the like. In another embodiment, the present disclosure provides the methods as described herein, wherein the one or more proteases comprise at least two proteases.
- The methods of the present disclosure have several further applications. For example, the one of more biomarkers are useful to discriminate between the pre-disease state from a disease state, or a disease state from a normal state. Other non-disease specific health states can also be determined. For example, changes of the biomarker can be assayed at different times: in a subject with a disease, to monitor the progress of the disease; in a subject undergoing treatment, to monitor the effect of the treatment and in a subject post-treatment, to monitor a possible relapse. Also, the levels of a specific amount of biomarker also may allow for choosing the course of treatment of the disease. For example, a biological sample can be provided from a subject undergoing treatment regimens for a disease. Such treatment regimens can include, but are not limited to, exercise regimens, dietary supplementation, weight loss, surgical intervention, device implantation, and treatment with therapeutics or prophylactics used in subjects diagnosed or identified with a disease or condition.
- Moreover, changes in glycopeptide ratios in a plurality of glycoproteins can be associated with a certain disease sate or absence of a disease. For example, presence of a plurality of particular glycopeptides in a biological sample may indicate absence of a disease, whereas presence of a plurality of other specific glycopeptides in a biological sample may indicate presence of the disease. Thus, various glycopeptide profiles or panels of glycopeptide biomarkers can be correlated with various states of a disease.
- Example 2 shows quantitation results of changes in IgG1, IgG0 and IgG2 glycopeptides in plasma samples from breast cancer patients versus the controls.
FIG. 2 illustrates that the levels of glycopeptides A1 and A2 were elevated as compared to the control, whereas the levels of glycopeptides A8, A9 and A10 were reduced as compared to the control in all stages of breast cancer studied in this experiment, thus indicating that glycopeptides A1, A2, A8, A9 and A10 are potential biomarkers for breast cancer. - Example 3 shows quantification results of changes in IgG, IgM and IgA glycopeptides in plasma samples from patients having PSC and patients having PSC.
FIG. 3 illustrates that glycopeptide A was elevated as compared to the healthy donors in plasma samples of patients having PBC and PSC, whereas glycopeptides H, I and J were reduced as compared to the healthy donors in plasma samples of patients having PBC and PSC. Thus, glycopeptides A, H, I and J are potential biomarkers for PBC and PSC. Further, the separate and combined discriminant analysis results are provided inFIG. 4 andFIG. 5 respectively indicating an accuracy of 88% for predicting the disease state in the combined discriminant analysis. - In some embodiments, the present disclosure provides methods, wherein the number of biomarkers that are detected and analyzed are 1, or more than 1, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23, 24, 25, 26, 27, 28, 30 or more. Thus, the disclosure also provides a panel of biomarkers that is useful in the diagnosis of a disease or condition.
- Mass Spectroscopy
- In one embodiment, the present disclosure provides methods as described herein that comprise quantitating the glycosylated peptide fragments by using a mass spectrometer. In one embodiment, the methods employ a technique called “multiple reaction monitoring (MRM).” This technique is often coupled with liquid chromatography (LC/MRM-MS) and allows the quantitation of hundreds of glycosylated peptide fragments (and their parent proteins) in a single LC/MRM-MS analysis. The advanced mass spectroscopy techniques of the present disclosure provide effective ion sources, higher resolution, faster separations and detectors with higher dynamic ranges that allow for broad untargeted measurements that also retain the benefits of targeted measurements.
- The mass spectroscopy methods of the present disclosure are applicable to several glycosylated proteins at a time. For example, at least more than 50, or at least more than 60 or at least more than 70, or at least more than 80, or at least more than 90, or at least more than 100, or at least more than 110 or at least more than 120 glycosylated proteins can be analyzed at a time using the mass spectrometer.
- In one embodiment, the mass spectroscopy methods of the present disclosure employ QQQ or qTOF mass spectrometer. In another embodiment, the mass spectroscopy methods of the present disclosure provide data with high mass accuracy of 10 ppm or better; or 5 ppm or better; or 2 ppm or better; or 1 ppm or better; or 0.5 ppm or better; or 0.2 ppm or better or 0.1 ppm or better at a resolving power of 5,000 or better; or 10,000 or better; or 25,000 or better; or 50,000 or better or 100,000 or better.
- Biological Samples
- The present disclosure provides methods that are based on quantitating the glycosylated peptide fragments from biological samples. In some embodiments, the biological samples are one or more clinical samples collected in the past, thus reducing the resources and time that must be committed to identifying new biomarkers. In some embodiments, the biological samples are from one or more past studies that occurred over a span of 1 to 50 years or more. In some embodiments, the studies are accompanied by various other clinical parameters and previously known information such as the subject's age, height, weight, ethnicity, medical history, and the like. Such additional information can be useful in associating the subject with a disease or a condition. In some embodiments, the biological samples are one or more clinical samples collected prospectively from the subjects.
- In one embodiment, the present disclosure provides the methods as described herein, wherein the biological sample isolated from the subjects is one or more of saliva, tears, sputum, sweat, mucous, fecal material, gastric fluid, abdominal fluid, amniotic fluid, cyst fluid, peritoneal fluid, spinal fluid, urine, synovial fluid, whole blood, serum, plasma, pancreatic juice, breast milk, lung lavage, marrow. In another embodiment, the biological sample isolated from the subjects is body tissue, saliva, tears, sputum, spinal fluid, urine, synovial fluid, whole blood, serum or plasma. In another embodiment, the biological sample isolated from the subjects is whole blood, serum or plasma. In some embodiments, the subjects are mammals. In other embodiments, the subject are humans.
- The methods of the present disclosure are applicable to any disease or condition that can be detected by analyzing the glycosylated peptide fragments from the biological samples of a subject. In some embodiments, the disease or condition is cancer. In other embodiments, the cancer is acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical cancer, anal cancer, bladder cancer, blood cancer, bone cancer, brain tumor, breast cancer, cancer of the female genital system, cancer of the male genital system, central nervous system lymphoma, cervical cancer, childhood rhabdomyosarcoma, childhood sarcoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CIVIL), colon and rectal cancer, colon cancer, endometrial cancer, endometrial sarcoma, esophageal cancer, eye cancer, gallbladder cancer, gastric cancer, gastrointestinal tract cancer, hairy cell leukemia, head and neck cancer, hepatocellular cancer, Hodgkin's disease, hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, malignant fibrous histiocytoma, malignant thymoma, melanoma, mesothelioma, multiple myeloma, myeloma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, nervous system cancer, neuroblastoma, non-Hodgkin's lymphoma, oral cavity cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary tumor, plasma cell neoplasm, primary CNS lymphoma, prostate cancer, rectal cancer, respiratory system, retinoblastoma, salivary gland cancer, skin cancer, small intestine cancer, soft tissue sarcoma, stomach cancer, testicular cancer, thyroid cancer, urinary system cancer, uterine sarcoma, vaginal cancer, vascular system, Waldenstrom's macroglobulinemia, Wilms' tumor, and the like. In another embodiment, the cancer is breast cancer, cervical cancer or ovarian cancer.
- In another embodiment, the disease is an autoimmune disease. In another embodiment, the autoimmune disease is acute disseminated encephalomyelitis, Addison's disease, agammaglobulinemia, age-related macular degeneration, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, antisynthetase syndrome, atopic allergy, atopic dermatitis, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune uticaria, autoimmune uveitis, Balo disease/Balo concentric sclerosis, Behcet's disease, Berger's disease, Bickerstaff s encephalitis, Blau syndrome, Bullous pemphigoid, cancer, Castleman's disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy, chronic recurrent multifocal osteomyelitis, chronic obstructive pulmonary disease, Churg-Strauss syndrome, cicatricial pemphigoid, Cogan syndrome, cold agglutinin disease, complement component 2 deficiency, contact dermatitis, cranial arteritis, CREST syndrome, Crohn's disease, Cushing's syndrome, cutaneous leukocytoclastic angiitis, Dego's disease, Dercum's disease, dermatitis herpetiformis, dermatomyositis, diabetes mellitus type 1, diffuse cutaneous systemic sclerosis, Dressler's syndrome, drug-induced lupus, discoid lupus erythematosus, eczema, endometriosis, enthesitis-related arthritis, eosinophilic fasciitis, eosinophilic gastroenteritis, epidermolysis bullosa acquisita, erythema nodosum, erythroblastosis fetalis, essential mixed cryoglobulinemia, Evan's syndrome, fibrodysplasia ossificans progressive, fibrosing alveolitis, gastritis, gastrointestinal pemphigoid, glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillan-Barre syndrome, Hashimoto's encephalopathy, Hashimoto's thyroiditis, Henoch-Schonlein purpura, HIV, gestational pemphigoid, hidradenitis suppurativa, Hughes-Stovin syndrome, hypogammaglobulinemia, idiopathic inflammatory demyelinating diseases, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, IgA nephropathy, inclusion body myositis, chronic inflammatory demyelinating polyneuropathy, interstitial cystitis, juvenile idiopathic arthritis, Kawasaki's disease, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, linear IgA disease, lupus erythematosus, Majeed syndrome, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease, morphea, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica, neuromyotonia, ocular cicatricial pemphigoid, opsoclonus myoclonus syndrome, Ord's thyroiditis, palindromic rheumatism, pediatric autoimmune neuropsychiatric disorders associated with streptococcus, paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria, Parry Romberg syndrome, Parsonage-Turner syndrome, Pars planitis, pemphigus vulgaris, pernicious anemia, perivenous encephalomyelitis, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatic, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, progressive inflammatory neuropathy, psoriasis, psoriatic arthritis, pyoderma gangrenosum, pure red cell aplasia, Rasmussen's encephalitis, Raynaud phenomenon, relapsing polychondritis, Reiter's syndrome, restless leg syndrome, retroperitoneal fibrosis, rheumatoid arthritis, rheumatic fever, sarcoidosis, schizophrenia, Schmidt syndrome, Schnitzler syndrome, scleritis, scleroderma, serum sickness, Sjogren's syndrome, spondyloarthropathy, stiff person syndrome, subacute bacterial endocarditis, Susac's syndrome, Sweet's syndrome, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis, thrombocytopenia, Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, urticarial vasculitis, vasculitis, vitiligo and Wegener's granulomatosis, and the like. In another embodiment, the autoimmune disease is HIV, primary sclerosing cholangitis, primary biliary cirrhosis or psoriasis.
- Machine Learning
- The biological samples are obtained from thousands of subjects which are then used for digitizing with the intention of deep mining for and validating previously undiscovered markers. In some embodiments, the biological samples are tumor samples or blood samples. They are digitized using LC/MS instruments to generate tremendous amount of data that undergoes deep machine learning analysis to discover new targets for various diseases. In some embodiments, the disease is cancer or autoimmune disease.
- In one embodiment, the present disclosure provides a method for identifying glycosylated peptide fragments as potential biomarkers, comprising:
-
- fragmenting glycosylated proteins in each of a plurality of biological samples isolated from subjects, with one or more proteases, to produce glycosylated peptide fragments;
- quantitating the glycosylated peptide fragments with liquid chromatography and mass spectrometry (LC-MS) to provide quantitation results;
- analyzing the quantitation results along with classification of the subjects with a machine learning method to select glycosylated peptide fragments useful for predicting the classification; and
- determining the identity of glycosylated peptide fragments, wherein the machine learning approach is deep learning, neural network, linear discriminant analysis, quadratic discriminant analysis, support vector machine, random forest, nearest neighbor or a combination thereof. In some embodiments, the machine learning approach is deep learning, neural network or a combination thereof. The analysis further comprises genomic data, proteomics, metabolics, lipidomics data, or a combination thereof.
FIG. 1 displays a Schematic diagram showing the integration of Glycomics, LC/MS and machine learning that is further combined with protemomics, genomic, lipidomics and metabolics to identify the biomarkers for various diseases.
- In the targeted approach, the glycoprotein of interest, is first identified in the biological sample and then analyzed using LC/MS for the site of modification, nature of modification, identity of the modification and the relative abundance of each modification, leading to identification and quantification of the peptide fragments. This approach uses triple quadrupole (QQQ) mass spectrometer for the quantification of the glycosylated peptide fragments which is then analyzed for its relation to the classification of the subjects.
- In the non-targeted approach, the glycosylation patterns of all peptide fragments (known as well as unknown) are analyzed to information on changes in glycosylation patterns in various subjects. Specifically, the up or down regulation of the glycoproteins is monitored in relation to the classification of the subjects. For example, the glycoprotein fragments are monitored for subjects having a disease or a condition versus subjects not having a disease or a condition. This approach uses quadrupole time-of-flight (qTOF) mass spectrometer for the analysis of the glycosylated peptide fragments.
- Plasma samples from breast cancer patients having various stages of cancer and their aged matched controls were analyzed for the IgG1, IgG0 and IgG2 glycopeptides and the changes in their ratios were compared. Specifically, 20 samples in Tis stage, 50 samples in EC1 stage, samples in EC2 stage, 25 samples in EC3 stage, 9 samples in EC4 stage and their 73 age matched control samples were subjected to MRM quantitative analysis on a QQQ mass spectrometer. As can be seen from the quantitative results in
FIG. 2 , the levels of certain IgG1 glycopeptides were elevated as compared to the controls, whereas the levels of certain IgG1 glycopeptides were reduced as compared to the controls in all stages of breast cancer studied in this experiment. See for example, IgG1 glycopeptides named as - A1-A11, were monitored and it was found that the levels of glycopeptides A1 and A2 were elevated as compared to the control, whereas the levels of glycopeptides A8, A9 and A10 were reduced as compared to the control in all stages of breast cancer studied in this experiment. Thus, glycopeptides A1, A2, A8, A9 and A10 are potential biomarkers for breast cancer.
- Plasma samples from patients having primary sclerosing cholangitis (PSC), patients having primary biliary cirrhosis (PBC) and plasma samples from healthy donors were analyzed for IgG1 and IgG2 glycopeptides and the changes in their glycopepide ratios were compared. Specifically, 100 PBC plasma samples, 76 PSC plasma samples and plasma samples from 49 healthy donors were subjected to MRM quantitative analysis on a QQQ mass spectrometer. As can be seen from the quantitative results in
FIG. 3 , certain IgG1 glycopeptides were elevated as compared to the healthy donors, whereas certain IgG1 glycopeptides were reduced as compared to the controls in plasma samples of patients having PBC and PSC. See for example, glycopeptide A was elevated as compared to the healthy donors in patients having PBC and PSC, whereas glycopeptides H, I and J were reduced as compared to the healthy donors in plasma samples of patients having PBC and PSC. Thus, glycopeptides A, H, I and J are potential biomarkers for PBC and PSC. - Similar analysis was carried out on IgA and IgM glycoproteins in plasma samples of patients having PBC and plasma samples of patients having PSC. The discriminant analysis results are provided in
FIG. 4 which indicate the % accuracy that can be predicted based on the separate data on IgG, IgM and IgA is 59%, 69% and 74% respectively. However, when the results are combined for all IgG, IgM and IgA, the discriminant analysis provides an accuracy of about 88% as shown inFIG. 5 .
Claims (26)
1.-20. (canceled)
21. A method for determining a prediction model for a classification of an unclassified human subject, the method comprising:
(a) subjecting each of a plurality of biological samples to one or more proteases to produce a set of respective protease-processed samples,
wherein each of the plurality of biological samples is from a human subject having a classification assigned based on having a disease or not having the disease, and
wherein the plurality of biological samples comprises samples from human subjects having the disease and human subjects not having the disease;
(b) subjecting each protease-processed sample to a liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM-MS) technique configured to selectively interrogate target species of interest,
wherein the target species of interest comprise a plurality of glycopeptides;
(c) analyzing the information obtained from the LC-MRM-MS technique to produce quantitation results for each protease-processed sample; and
(d) subjecting the quantitation results of each protease-processed sample along with the associated classification to a machine learning method to determine the prediction model.
22. The method of claim 21 , wherein the plurality of glycopeptides is associated with at least more than 50 glycoproteins.
23. The method of claim 21 , wherein the disease is cancer.
24. The method of claim 23 , wherein the cancer is breast cancer.
25. The method of claim 23 , wherein the cancer is breast cancer, cervical cancer, or ovarian cancer.
26. The method of claim 21 , wherein the disease is an autoimmune disease.
27. The method of claim 26 , wherein the autoimmune disease is HIV infection-associated autoimmune disease, primary sclerosing cholangitis, primary biliary cirrhosis, or psoriasis.
28. The method of claim 27 , wherein the autoimmune disease is primary biliary cholangitis or primary biliary cirrhosis.
29. The method of claim 21 , wherein the target species of interest comprise glycopeptides from one or more of alpha-1-acid glycoprotein, alpha-1-antitrypsin, alpha-1B-glycoprotein, alpha-2-HS-glycoprotein, alpha-2-macroglobulin, antithrombin-III, apolipoprotein B-100, apolipoprotein D, apolipoprotein F, beta-2-glycoprotein 1, ceruloplasmin, fetuin, fibrinogen, immunoglobulin (Ig) A, IgG, IgM, haptoglobin, hemopexin, histidine-rich glycoprotein, kininogen-1, serotransferrin, transferrin, and vitronectin zinc-alpha-2-glycoprotein.
30. The method of claim 29 , wherein the target species of interest comprise glycopeptides from one or more of immunoglobulin (Ig) A, IgG, and IgM.
31. The method of claim 30 , wherein the quantitation results comprising information from IgG, IgA, and IgM glycopeptides of each protease-processed sample along with the associated classification are subjected to a machine learning method comprising a combined discriminant analysis.
32. The method of claim 21 , wherein the one or more proteases comprise a serine protease.
33. The method of claim 32 , wherein the serine protease is selected from the group consisting of trypsin, chymotrypsin, endoproteinase, Arg-C, Glu-C, Lys-C, and proteinase K.
34. The method of claim 21 , wherein the plurality of biological samples are each selected from the group consisting of a whole blood sample, serum sample, and plasma sample.
35. The method of claim 34 , wherein the plurality of biological samples are whole blood samples.
36. The method of claim 34 , wherein the plurality of biological samples are serum samples.
37. The method of claim 34 , wherein the plurality of biological samples are plasma samples.
38. The method of claim 21 , wherein the machine learning method comprises a deep learning, neural network, discriminant analysis, support vector machine, random forest, nearest neighbor algorithm, or a combination thereof.
39. The method of claim 38 , wherein the machine learning method comprises the deep learning, neural network algorithm, or a combination thereof.
40. The method of claim 21 , further comprising identifying one or more glycopeptides indicative of the classification of an unclassified human subject.
41. The method of claim 21 , wherein the human subjects not having the disease are healthy donors.
42. The method of claim 21 , wherein the LC-MRM-MS technique is performed on a triple quadrupole mass spectrometer.
43. The method of claim 42 , wherein the triple quadrupole mass spectrometer has a mass accuracy of 10 ppm or better.
44. The method of claim 21 , wherein the plurality of biological samples comprises samples from at least 20 human subjects having the disease and at least 20 human subjects not having the disease.
45. The method of claim 21 , wherein the plurality of biological samples comprises samples from at least 40 human subjects having the disease and at least 40 human subjects not having the disease.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US18/180,789 US20230324407A1 (en) | 2017-09-01 | 2023-03-08 | Identification and use of glycopeptides as biomarkers for diagnosis and treatment monitoring |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201762553676P | 2017-09-01 | 2017-09-01 | |
US16/120,016 US10837970B2 (en) | 2017-09-01 | 2018-08-31 | Identification and use of glycopeptides as biomarkers for diagnosis and treatment monitoring |
US17/067,460 US11624750B2 (en) | 2017-09-01 | 2020-10-09 | Identification and use of glycopeptides as biomarkers for diagnosis and treatment monitoring |
US18/180,789 US20230324407A1 (en) | 2017-09-01 | 2023-03-08 | Identification and use of glycopeptides as biomarkers for diagnosis and treatment monitoring |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/067,460 Continuation US11624750B2 (en) | 2017-09-01 | 2020-10-09 | Identification and use of glycopeptides as biomarkers for diagnosis and treatment monitoring |
Publications (1)
Publication Number | Publication Date |
---|---|
US20230324407A1 true US20230324407A1 (en) | 2023-10-12 |
Family
ID=65527845
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/120,016 Active 2039-01-05 US10837970B2 (en) | 2017-09-01 | 2018-08-31 | Identification and use of glycopeptides as biomarkers for diagnosis and treatment monitoring |
US17/067,460 Active 2038-12-19 US11624750B2 (en) | 2017-09-01 | 2020-10-09 | Identification and use of glycopeptides as biomarkers for diagnosis and treatment monitoring |
US18/180,789 Pending US20230324407A1 (en) | 2017-09-01 | 2023-03-08 | Identification and use of glycopeptides as biomarkers for diagnosis and treatment monitoring |
Family Applications Before (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/120,016 Active 2039-01-05 US10837970B2 (en) | 2017-09-01 | 2018-08-31 | Identification and use of glycopeptides as biomarkers for diagnosis and treatment monitoring |
US17/067,460 Active 2038-12-19 US11624750B2 (en) | 2017-09-01 | 2020-10-09 | Identification and use of glycopeptides as biomarkers for diagnosis and treatment monitoring |
Country Status (7)
Country | Link |
---|---|
US (3) | US10837970B2 (en) |
EP (1) | EP3676393A4 (en) |
JP (2) | JP2020532732A (en) |
KR (2) | KR102633621B1 (en) |
CN (1) | CN111148844A (en) |
AU (1) | AU2018324195A1 (en) |
WO (1) | WO2019046814A1 (en) |
Families Citing this family (20)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR102633621B1 (en) | 2017-09-01 | 2024-02-05 | 벤 바이오사이언시스 코포레이션 | Identification and use of glycopeptides as biomarkers for diagnosis and therapeutic monitoring |
AU2018351147A1 (en) * | 2017-10-18 | 2020-05-07 | Venn Biosciences Corporation | Identification and use of biological parameters for diagnosis and treatment monitoring |
SG11202108327VA (en) | 2019-02-01 | 2021-08-30 | Venn Biosciences Corp | Biomarkers for diagnosing ovarian cancer |
CA3131254A1 (en) | 2019-03-29 | 2020-10-08 | Daniel SERIE | Automated detection of boundaries in mass spectrometry data |
IL295136A (en) | 2020-01-31 | 2022-09-01 | Venn Biosciences Corp | Biomarkers for diagnosing ovarian cancer |
WO2021202620A1 (en) * | 2020-03-31 | 2021-10-07 | The Board Of Trustees Of The Leland Stanford Junior University | Metabolomics approach combined with machine learning to recognize a medical condition |
CN111781292B (en) * | 2020-07-15 | 2022-06-21 | 四川大学华西医院 | Urine proteomics spectrogram data analysis system based on deep learning model |
CA3198807A1 (en) * | 2020-11-25 | 2022-06-02 | Venn Biosciences Corporation | Biomarkers for diagnosing non-alcoholic steatohepatitis (nash) or hepatocellular carcinoma (hcc) |
CN116490772A (en) * | 2020-12-07 | 2023-07-25 | 金伯利-克拉克环球有限公司 | Method and consumer product for detecting metabolites |
CN113009147A (en) * | 2021-02-10 | 2021-06-22 | 中国医学科学院北京协和医院 | Sugar chain marker for diagnosing anti-gp 210 antibody positive and negative PBC patients and application thereof |
CN113009148A (en) * | 2021-02-10 | 2021-06-22 | 中国医学科学院北京协和医院 | Sugar chain marker for diagnosing PBC patients positive and negative to SP100 antibody and application thereof |
CN115112778B (en) * | 2021-03-19 | 2023-08-04 | 复旦大学 | Disease protein biomarker identification method |
CA3227374A1 (en) * | 2021-08-04 | 2023-02-09 | Daniel SERIE | Biomarkers for diagnosing colorectal cancer or advanced adenoma |
WO2023019093A2 (en) * | 2021-08-07 | 2023-02-16 | Venn Biosciences Corporation | Detection of peptide structures for diagnosing and treating sepsis and covid |
WO2023075591A1 (en) * | 2021-10-29 | 2023-05-04 | Venn Biosciences Corporation | Ai-driven glycoproteomics liquid biopsy in nasopharyngeal carcinoma |
KR102380684B1 (en) * | 2021-11-09 | 2022-04-01 | 주식회사 셀키 | Method and apparatus for determining cancer-specific biomarkers through glycopeptide analysis based on mass spectrum based on ai |
CN114200056B (en) * | 2021-12-13 | 2022-09-20 | 中国医学科学院基础医学研究所 | Biomarker for predicting sensitivity of advanced vitiligo to hormone therapy and application thereof |
WO2023154943A1 (en) * | 2022-02-14 | 2023-08-17 | Venn Biosciences Corporation | De novo glycopeptide sequencing |
CN115662500B (en) * | 2022-10-21 | 2023-06-20 | 清华大学 | Method for distinguishing glycan structural isomers by computer simulation replacement of similar mass isotopes |
CN116879558B (en) * | 2023-09-05 | 2023-12-01 | 天津云检医学检验所有限公司 | Ovarian cancer diagnosis marker, detection reagent and detection kit |
Family Cites Families (143)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7118737B2 (en) | 2000-09-08 | 2006-10-10 | Amylin Pharmaceuticals, Inc. | Polymer-modified synthetic proteins |
US20030013120A1 (en) | 2001-07-12 | 2003-01-16 | Patz Edward F. | System and method for differential protein expression and a diagnostic biomarker discovery system and method using same |
US7297556B2 (en) | 2001-08-30 | 2007-11-20 | Vermillion, Inc. | Method of diagnosing nephrotic syndrome |
US20040043436A1 (en) | 2001-09-21 | 2004-03-04 | Antonia Vlahou | Biomarkers of transitional cell carcinoma of the bladder |
WO2004030511A2 (en) | 2002-05-10 | 2004-04-15 | Eastern Virginia Medical School | Prostate cancer biomarkers |
JP4583168B2 (en) | 2002-06-03 | 2010-11-17 | ザ インスティテュート フォー システムズ バイオロジー | Method for quantitative proteome analysis of glycoproteins |
US7031845B2 (en) | 2002-07-19 | 2006-04-18 | University Of Chicago | Method for determining biological expression levels by linear programming |
US7501286B2 (en) | 2002-08-14 | 2009-03-10 | President And Fellows Of Harvard College | Absolute quantification of proteins and modified forms thereof by multistage mass spectrometry |
AU2008202217B2 (en) | 2002-08-16 | 2012-07-26 | Agensys, Inc. | Nucleic acids and corresponding proteins entitled 191PAD12(b) useful in treatment and detection of cancer |
US8163896B1 (en) | 2002-11-14 | 2012-04-24 | Rosetta Genomics Ltd. | Bioinformatically detectable group of novel regulatory genes and uses thereof |
US20070077553A1 (en) | 2003-10-30 | 2007-04-05 | Rosetta Genomics | Bioinformatically detectable group of novel vaccinia regulatory genes and uses thereof |
US7790867B2 (en) | 2002-12-05 | 2010-09-07 | Rosetta Genomics Inc. | Vaccinia virus-related nucleic acids and microRNA |
US20050048547A1 (en) | 2003-07-17 | 2005-03-03 | Hongyu Zhao | Classification of disease states using mass spectrometry data |
US7298474B2 (en) | 2003-10-24 | 2007-11-20 | Purdue Research Foundation | Plasmonic and/or microcavity enhanced optical protein sensing |
CA2832293C (en) | 2003-11-26 | 2015-08-04 | Celera Corporation | Single nucleotide polymorphisms associated with cardiovascular disorders and statin response, methods of detection and uses thereof |
US7608458B2 (en) | 2004-02-05 | 2009-10-27 | Medtronic, Inc. | Identifying patients at risk for life threatening arrhythmias |
US20060127950A1 (en) * | 2004-04-15 | 2006-06-15 | Massachusetts Institute Of Technology | Methods and products related to the improved analysis of carbohydrates |
EP1766412B1 (en) * | 2004-05-21 | 2009-04-01 | The Institute for Systems Biology | Compositions and methods for quantification of serum glycoproteins |
CA2614507A1 (en) | 2004-07-09 | 2006-08-17 | Amaox, Ltd. | Immune cell biosensors and methods of using same |
EP1797425A2 (en) | 2004-07-19 | 2007-06-20 | University of Rochester | Biomarkers of neurodegenerative disease |
JP2008508538A (en) | 2004-08-02 | 2008-03-21 | チルドレンズ・メディカル・センター・コーポレイション | Platelet biomarkers for cancer |
EP1792263A2 (en) | 2004-09-02 | 2007-06-06 | Vialogy Corporation | Detecting events of interest using quantum resonance interferometry |
EP1838732A4 (en) | 2005-01-07 | 2010-07-14 | Univ Johns Hopkins | Biomarkers for melanoma |
EP1696237A1 (en) | 2005-02-23 | 2006-08-30 | Rescom GmbH | Diagnosis of dry-eye syndrome by SELDI analysis of proteins in tears |
EP1869462B1 (en) | 2005-03-11 | 2013-05-08 | Ciphergen Biosystems, Inc. | Biomarkers for ovarian cancer and endometrial cancer: hepcidin |
US9075062B2 (en) | 2005-03-22 | 2015-07-07 | The United States Of America, As Represented By The Secretary, Department Of Health & Human Services | Identification of biomarkers by serum protein profiling |
EP1866818A1 (en) | 2005-03-31 | 2007-12-19 | Koninklijke Philips Electronics N.V. | System and method for collecting evidence pertaining to relationships between biomolecules and diseases |
WO2006110621A2 (en) | 2005-04-11 | 2006-10-19 | Cornell Research Foundation, Inc. | Multiplexed biomarkers for monitoring the alzheimer's disease state of a subject |
BRPI0609302A2 (en) | 2005-04-15 | 2011-10-11 | Becton Dickinson Co | methods for predicting the development of sepsis and for diagnosing sepsis in an individual to be tested, microarray, kit for predicting the development of sepsis in an individual to be tested, computer program product, computer, computer system for determining if an individual is likely to develop sepsis, digital signal embedded in a carrier wave, and, graphical user interface to determine if an individual is likely to develop sepsis |
US20090203055A1 (en) | 2005-04-18 | 2009-08-13 | Massachusetts Institute Of Technology | Compositions and methods for RNA interference with sialidase expression and uses thereof |
EP3578985B1 (en) | 2005-05-05 | 2021-01-13 | Drexel University | Diagnosis of liver pathology through assessment of protein glycosylation |
GB0510511D0 (en) | 2005-05-23 | 2005-06-29 | St Georges Entpr Ltd | Diagnosis of tuberculosis |
EP1904845A4 (en) | 2005-07-07 | 2009-11-25 | David E Kohne | Improved protein expression comparison assay results and applications |
US8759005B2 (en) | 2005-07-11 | 2014-06-24 | Glykos Finland Oy | Tissue carbohydrate compositions and analysis thereof |
FI20055417A0 (en) | 2005-07-20 | 2005-07-20 | Glykos Finland Oy | Cancer-specific glycans and their use |
CN100410663C (en) | 2005-09-11 | 2008-08-13 | 翁炳焕 | Proteomics ante partum diagnosis process |
BRPI0506117A (en) | 2005-10-14 | 2007-07-03 | Fundacao Oswaldo Cruz | diagnostic method based on proteinaceous and / or genetic patterns by support vectors applied to mass spectrometry |
EP1938104A2 (en) | 2005-10-17 | 2008-07-02 | Institute for Systems Biology | Tissue-and serum-derived glycoproteins and methods of their use |
EP1948227A4 (en) | 2005-10-26 | 2010-03-31 | Protelix Inc | Influenza combinatorial antigen vaccine |
CA2627892A1 (en) | 2005-11-01 | 2007-05-10 | Janssen Pharmaceutica N.V. | Substituted dihydroisoindolones as allosteric modulators of glucokinase |
US20090258848A1 (en) | 2005-12-06 | 2009-10-15 | The Johns Hopkins University | Biomarkers for inflammatory bowel disease |
US20090035801A1 (en) | 2005-12-27 | 2009-02-05 | Power3 Medical Products, Inc. | Twelve (12) protein biomarkers for diagnosis and early detection of breast cancer |
EP2008096A2 (en) | 2006-04-03 | 2008-12-31 | Massachusetts Institute of Technology | Glycomic patterns for the detection of disease |
WO2007123976A2 (en) | 2006-04-18 | 2007-11-01 | The Board Of Trustees Of The Leland Stanford Junior University | Antibody profiling for determination of patient responsiveness |
GB0611669D0 (en) | 2006-06-13 | 2006-07-19 | Astrazeneca Uk Ltd | Mass spectrometry biomarker assay |
EP2047257A4 (en) | 2006-06-29 | 2011-11-16 | Suomen Punainen Risti Veripalvelu | Novel cellular glycan compositions |
WO2008108803A2 (en) | 2006-07-13 | 2008-09-12 | Amaox, Ltd. | Immune cell biosensors and methods of using same |
US7899625B2 (en) | 2006-07-27 | 2011-03-01 | International Business Machines Corporation | Method and system for robust classification strategy for cancer detection from mass spectrometry data |
TWI426269B (en) | 2006-08-14 | 2014-02-11 | Academia Sinica | Method of gastric juice protein analysis for stomach cancer diagnosis |
US20080132420A1 (en) | 2006-09-18 | 2008-06-05 | Mariusz Lubomirski | Consolidated approach to analyzing data from protein microarrays |
WO2008047086A2 (en) | 2006-10-16 | 2008-04-24 | The University Of Nottingham | Biomarker |
US8288110B2 (en) | 2006-12-04 | 2012-10-16 | Perkinelmer Health Sciences, Inc. | Biomarkers for detecting cancer |
WO2008085024A1 (en) | 2007-01-12 | 2008-07-17 | Erasmus University Medical Center Rotterdam | Identification and detection of peptides relating to specific disorders |
WO2008144041A1 (en) | 2007-05-21 | 2008-11-27 | The Ohio State University Research Foundation | Hepcidins as biomarkers for impending lupus nephritis flare |
WO2008149088A2 (en) | 2007-06-04 | 2008-12-11 | The Nottingham Trent University | Melanoma assay and antigens |
EP2156191A2 (en) | 2007-06-15 | 2010-02-24 | Smithkline Beecham Corporation | Methods and kits for predicting treatment response in type ii diabetes mellitus patients |
KR101262202B1 (en) | 2007-06-29 | 2013-05-16 | 안국약품 주식회사 | Predictive markers for ovarian cancer |
WO2009006382A1 (en) * | 2007-07-02 | 2009-01-08 | Purdue Research Foundation | Detection of glycopeptides and glycoproteins for medical diagnostics |
JP2009057337A (en) | 2007-08-31 | 2009-03-19 | Dainippon Sumitomo Pharma Co Ltd | Metabolome data analysis method and metabolism-related marker |
WO2009075883A2 (en) | 2007-12-12 | 2009-06-18 | University Of Georgia Research Foundation, Inc. | Glycoprotein cancer biomarker |
JP2009168646A (en) | 2008-01-17 | 2009-07-30 | Fuji Pharmaceutical Co Ltd | Specific biomarker for endometriosis |
WO2009092068A1 (en) | 2008-01-18 | 2009-07-23 | President And Fellows Of Harvard College | Methods of detecting signatures of disease or conditions in bodily fluids |
CA2618163A1 (en) | 2008-02-07 | 2009-08-07 | K. W. Michael Siu | Head and neck cancer biomarkers |
US9684001B2 (en) | 2008-02-07 | 2017-06-20 | Ranju Ralhan | Biomarkers for head-and-neck cancers and precancers |
AU2009258038A1 (en) | 2008-03-14 | 2009-12-17 | Eastern Virginia Medical School | Imaging mass spectrometry for improved prostate cancer diagnostics |
WO2009117666A1 (en) | 2008-03-21 | 2009-09-24 | Indiana University Research And Technology Corporation | Glycan markers of hepatocellular carcinoma |
JP2011518548A (en) | 2008-04-10 | 2011-06-30 | ザ・ユニバーシティ・オブ・ブリティッシュ・コロンビア | Methods for diagnosing chronic cardiac allograft rejection |
EP2281201B1 (en) | 2008-05-14 | 2018-03-28 | ETH Zurich | Method for biomarker and drug-target discovery for prostate cancer diagnosis and treatment as well as biomarker assays determined therewith |
CN102119224A (en) | 2008-05-30 | 2011-07-06 | 不列颠哥伦比亚大学 | Methods of diagnosing rejection of a kidney allograft using genomic or proteomic expression profiling |
US20100029006A1 (en) | 2008-08-04 | 2010-02-04 | Rosenblatt Kevin P | Multiplexed diagnostic test for preterm labor |
US20110224913A1 (en) | 2008-08-08 | 2011-09-15 | Juan Cui | Methods and systems for predicting proteins that can be secreted into bodily fluids |
SI2340506T1 (en) | 2008-09-09 | 2016-02-29 | Somalogic, Inc. | Lung cancer biomarkers and uses thereof |
US20100221752A2 (en) | 2008-10-06 | 2010-09-02 | Somalogic, Inc. | Ovarian Cancer Biomarkers and Uses Thereof |
CA2741566A1 (en) | 2008-11-03 | 2010-06-03 | Schering Corporation | Inflammatory bowel disease biomarkers and related methods of treatment |
CN102301234B (en) | 2008-11-18 | 2015-06-17 | 里奇诊断学股份有限公司 | Metabolic Syndrome And HPA Axis Biomarkers For Major Depressive Disorder |
US8497066B2 (en) | 2008-12-04 | 2013-07-30 | Rush University Medical Center | DNA methylation based test for monitoring efficacy of treatment |
US8535891B2 (en) | 2008-12-30 | 2013-09-17 | Children's Medical Center Corporation | Method of predicting acute appendicitis |
US20110275536A1 (en) | 2009-01-30 | 2011-11-10 | Pronota N.V. | Biomarker for diagnosis, prediction and/or prognosis of acute heart failure and uses thereof |
JP2012517607A (en) * | 2009-02-20 | 2012-08-02 | オンコノム,インコーポレイテッド | Equipment set and method for colorectal cancer diagnosis and prognosis determination |
KR20120034593A (en) | 2009-03-09 | 2012-04-12 | 유니버시티 오브 조지아 리서치 파운데이션 인코퍼레이티드 | Protein markers identification for gastric cancer diagnosis |
CA2757659A1 (en) | 2009-04-06 | 2010-10-14 | Ridge Diagnostics, Inc. | Biomarkers for monitoring treatment of neuropsychiatric diseases |
WO2010118525A1 (en) | 2009-04-14 | 2010-10-21 | Socpra Sciences Santé Et Humaines S.E.C. | Signature of secreted protein isoforms specific to ovarian cancer |
WO2010144794A1 (en) | 2009-06-12 | 2010-12-16 | Vaccine Technologies, Incorporated | Baculovirus-expressed fusion polypeptide vaccines with enhanced immunogenicity and uses thereof |
WO2010144797A2 (en) | 2009-06-12 | 2010-12-16 | Vaccine Technologies, Incorporated | Influenza vaccines with enhanced immunogenicity and uses thereof |
CN102573883A (en) | 2009-06-12 | 2012-07-11 | 疫苗技术公司 | Methods and compositions for promoting a cell-mediated immune response |
CN102625852A (en) * | 2009-07-07 | 2012-08-01 | 南加利福尼亚大学 | Biomarkers for the early detection of autoimmune diseases |
US9752191B2 (en) | 2009-07-09 | 2017-09-05 | The Scripps Research Institute | Gene expression profiles associated with chronic allograft nephropathy |
EP2462164A2 (en) | 2009-08-04 | 2012-06-13 | Biosystems International SAS | Lung cancer biomarkers |
EA201290056A1 (en) | 2009-08-07 | 2012-08-30 | Астьют Медикал, Инк. | METHODS AND COMPOSITIONS FOR DIAGNOSIS AND PREDICTION OF KIDNEY DAMAGE AND RENAL FAILURE |
US9598682B2 (en) | 2009-09-29 | 2017-03-21 | Vib Vzw | Hydrolysis of mannose-1-phospho-6-mannose linkage to phospho-6-mannose |
US20120283123A1 (en) | 2009-11-25 | 2012-11-08 | Sarwal Minnie M | Biomarkers for the Diagnosis of Kidney Graft Rejection |
EP2507393A4 (en) | 2009-11-30 | 2013-05-01 | Caris Life Sciences Luxembourg Holdings | Methods and systems for isolating, storing, and analyzing vesicles |
WO2011082321A1 (en) | 2009-12-31 | 2011-07-07 | Van Andel Research Institute | Methods for diagnosing the malignant potential of pancreatic cystic lesions |
AU2011205230A1 (en) | 2010-01-13 | 2012-08-02 | Caris Life Sciences Switzerland Holdings Gmbh | Detection of gastrointestinal disorders |
KR101556726B1 (en) | 2010-02-24 | 2015-10-02 | 바이오디식스, 인크. | Cancer Patient Selection for Administraionof Therapeutic Agents Using Mass Spectral Analysis |
MX2012011648A (en) | 2010-04-07 | 2012-11-29 | Momenta Pharmaceuticals Inc | High mannose glycans. |
WO2011156594A2 (en) | 2010-06-09 | 2011-12-15 | Vaccine Technologies, Incorporated | Therapeutic immunization in hiv infected subjects receiving stable antiretroviral treatment |
WO2016030888A1 (en) | 2014-08-26 | 2016-03-03 | Compugen Ltd. | Polypeptides and uses thereof as a drug for treatment of autoimmune disorders |
US20120171694A1 (en) | 2010-07-30 | 2012-07-05 | Vermillion, Inc. | Predictive markers and biomarker panels for ovarian cancer |
WO2012016333A1 (en) | 2010-08-06 | 2012-02-09 | University Health Network | Biomarkers for malaria |
GB201014837D0 (en) | 2010-09-07 | 2010-10-20 | Immunovia Ab | Biomarker signatures and uses thereof |
WO2012056008A1 (en) * | 2010-10-28 | 2012-05-03 | Jonas Nilsson | Diagnosis and treatment of alzheimer's disease |
EP2463659A1 (en) * | 2010-12-13 | 2012-06-13 | Université de Liège | Biomarkers for cancer diagnosis |
WO2012112315A2 (en) | 2011-02-20 | 2012-08-23 | The Board Of Trustees Of The Leland Stanford Junior University | Methods for diagnosis of kawasaki disease |
US20140162370A1 (en) | 2011-06-14 | 2014-06-12 | The Board Of Trustees Of The Leland Stanford Junior University | Urine biomarkers for necrotizing enterocolitis and sepsis |
JP2014526682A (en) | 2011-09-12 | 2014-10-06 | クリエイティクス エルエルシー | Non-invasive method for detecting target molecules |
KR101219516B1 (en) | 2012-03-27 | 2013-01-11 | 한국기초과학지원연구원 | Polypeptide markers for the diagnosis of cancers and methods for the diagnosis of cancers using the same |
US9459258B2 (en) | 2012-05-21 | 2016-10-04 | Indiana University Research And Technology Corp. | Identification and quantification of intact glycopeptides in complex samples |
WO2013192530A2 (en) | 2012-06-21 | 2013-12-27 | Children's Medical Center Corporation | Methods and reagents for glycoproteomics |
JP2014027898A (en) | 2012-07-31 | 2014-02-13 | Yamaguchi Univ | Method for judging onset risk of hepatocarcinoma |
WO2014056885A1 (en) | 2012-10-09 | 2014-04-17 | Universiteit Gent | Cooperia vaccine |
MX366272B (en) | 2013-03-14 | 2019-07-04 | Immucor Gti Diagnostics Inc | METHODS and COMPOSITIONS FOR DIAGNOSING PREECLAMPSIA. |
CN103278576B (en) | 2013-05-03 | 2014-12-24 | 中国农业科学院北京畜牧兽医研究所 | Serum metabonomic method for screening biomarkers of transgenic animal |
WO2015006515A1 (en) | 2013-07-09 | 2015-01-15 | Sri International | Biomarker panel for dose assessment of radiation injury and micro plasma filter |
WO2015009907A1 (en) | 2013-07-17 | 2015-01-22 | The Johns Hopkins University | A multi-protein biomarker assay for brain injury detection and outcome |
KR101527283B1 (en) * | 2013-08-13 | 2015-06-10 | 서울대학교산학협력단 | Method for screening cancer marker based on de-glycosylation of glycoproteins and marker for HCC |
GB201322800D0 (en) | 2013-12-20 | 2014-02-05 | Univ Dublin | Prostate cancer biomarkers |
WO2015136298A1 (en) | 2014-03-13 | 2015-09-17 | Isis Innovation Limited | Methods and system for determining the disease status of a subject |
WO2015149030A1 (en) | 2014-03-28 | 2015-10-01 | Applied Proteomics, Inc. | Protein biomarker profiles for detecting colorectal tumors |
US20170052200A1 (en) * | 2014-05-02 | 2017-02-23 | Momenta Pharmaceuticals, Inc. | Methods and compositions for the diagnosis and treatment of kawasaki disease |
US20150376723A1 (en) | 2014-06-27 | 2015-12-31 | University Health Network | Prognostic biomarkers for influenza |
WO2015200898A1 (en) | 2014-06-28 | 2015-12-30 | Relevance Health | System for assessing global wellness |
WO2016004375A2 (en) | 2014-07-02 | 2016-01-07 | Ridge Diagnostics, Inc. | Methods and materials for treating pain and depression |
US20170205427A1 (en) | 2014-07-23 | 2017-07-20 | University Of British Columbia | Biomarkers for anderson-fabry disease |
WO2016036705A1 (en) | 2014-09-03 | 2016-03-10 | Musc Foundation For Research Development | Glycan panels as specific tumor tissue biomarkers |
US20160069884A1 (en) * | 2014-09-09 | 2016-03-10 | The Johns Hopkins University | Biomarkers for distinguishing between aggressive prostate cancer and non-aggressive prostate cancer |
ES2883628T3 (en) | 2014-11-17 | 2021-12-09 | Univ Queensland | Glycoprotein biomarkers for adenocarcinoma of the esophagus and Barrett's esophagus and their uses |
US20180017580A1 (en) | 2014-12-05 | 2018-01-18 | Myriad Genetics, Inc. | Biomarkers for distinguishing mood disorders |
AU2015100100A4 (en) | 2014-12-30 | 2015-03-12 | Macau University Of Science And Technology | N-Glycans on IgG As Biomarkers for Autoimmune Diseases Explored Via Comprehensive Glycomic Approach |
US20180107783A1 (en) | 2015-05-28 | 2018-04-19 | Immunexpress Pty Ltd | Validating biomarker measurement |
WO2017011329A1 (en) | 2015-07-10 | 2017-01-19 | West Virginia University | Markers of stroke and stroke severity |
AU2015101434A4 (en) * | 2015-07-29 | 2015-11-12 | Macau University Of Science And Technology | Use of glycan as biomarkers for autoimmune diseases |
WO2017044850A1 (en) | 2015-09-10 | 2017-03-16 | Alter Galit | Synthesizing vaccines, immunogens, and antibodies |
RU2018127709A (en) | 2016-01-22 | 2020-02-25 | Отрэйсис, Инк. | SYSTEMS AND METHODS FOR IMPROVING DIAGNOSTICS OF DISEASES |
US10513725B2 (en) | 2016-02-29 | 2019-12-24 | eNano Health Limited | Public personalized mobile health sensing system, method and device |
GB201603571D0 (en) | 2016-03-01 | 2016-04-13 | Univ Warwick | Markers for skeletal disorders |
US20190113520A1 (en) | 2016-03-31 | 2019-04-18 | Discerndx, Inc. | Biomarker Database Generation and Use |
WO2017173428A1 (en) | 2016-04-01 | 2017-10-05 | 20/20 Genesystems Inc. | Methods and compositions for aiding in distinguishing between benign and maligannt radiographically apparent pulmonry nodules |
WO2017190218A1 (en) | 2016-05-06 | 2017-11-09 | University Health Network | Liquid-biopsy signatures for prostate cancer |
US20180003706A1 (en) | 2016-06-30 | 2018-01-04 | Sightline Innovation Inc. | System, method, and module for biomarker detection |
KR102633621B1 (en) | 2017-09-01 | 2024-02-05 | 벤 바이오사이언시스 코포레이션 | Identification and use of glycopeptides as biomarkers for diagnosis and therapeutic monitoring |
AU2018351147A1 (en) | 2017-10-18 | 2020-05-07 | Venn Biosciences Corporation | Identification and use of biological parameters for diagnosis and treatment monitoring |
SG11202108327VA (en) | 2019-02-01 | 2021-08-30 | Venn Biosciences Corp | Biomarkers for diagnosing ovarian cancer |
-
2018
- 2018-08-31 KR KR1020207007267A patent/KR102633621B1/en active IP Right Grant
- 2018-08-31 KR KR1020247003609A patent/KR20240019862A/en active Application Filing
- 2018-08-31 EP EP18852234.6A patent/EP3676393A4/en active Pending
- 2018-08-31 US US16/120,016 patent/US10837970B2/en active Active
- 2018-08-31 JP JP2020512809A patent/JP2020532732A/en active Pending
- 2018-08-31 CN CN201880063851.7A patent/CN111148844A/en active Pending
- 2018-08-31 AU AU2018324195A patent/AU2018324195A1/en active Pending
- 2018-08-31 WO PCT/US2018/049256 patent/WO2019046814A1/en unknown
-
2020
- 2020-10-09 US US17/067,460 patent/US11624750B2/en active Active
-
2022
- 2022-12-23 JP JP2022207662A patent/JP2023152669A/en active Pending
-
2023
- 2023-03-08 US US18/180,789 patent/US20230324407A1/en active Pending
Also Published As
Publication number | Publication date |
---|---|
EP3676393A1 (en) | 2020-07-08 |
US11624750B2 (en) | 2023-04-11 |
US20210208159A1 (en) | 2021-07-08 |
KR102633621B1 (en) | 2024-02-05 |
US10837970B2 (en) | 2020-11-17 |
EP3676393A4 (en) | 2021-10-13 |
KR20200046047A (en) | 2020-05-06 |
JP2023152669A (en) | 2023-10-17 |
AU2018324195A1 (en) | 2020-04-02 |
WO2019046814A1 (en) | 2019-03-07 |
JP2020532732A (en) | 2020-11-12 |
KR20240019862A (en) | 2024-02-14 |
US20190101544A1 (en) | 2019-04-04 |
CN111148844A (en) | 2020-05-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11624750B2 (en) | Identification and use of glycopeptides as biomarkers for diagnosis and treatment monitoring | |
US20200240996A1 (en) | Identification and use of biological parameters for diagnosis and treatment monitoring | |
US10365288B2 (en) | Citrullinated brain and neurological proteins as biomarkers of brain injury or neurodegeneration | |
Ercan et al. | Aberrant IgG galactosylation precedes disease onset, correlates with disease activity, and is prevalent in autoantibodies in rheumatoid arthritis | |
Skates et al. | Statistical design for biospecimen cohort size in proteomics-based biomarker discovery and verification studies | |
Vickery et al. | Peanut oral immunotherapy modifies IgE and IgG4 responses to major peanut allergens | |
Ahmed et al. | Biomarkers of early stage osteoarthritis, rheumatoid arthritis and musculoskeletal health | |
Chan et al. | Current application of proteomics in biomarker discovery for inflammatory bowel disease | |
Tin et al. | Novel brain reactive autoantibodies: prevalence in systemic lupus erythematosus and association with psychoses and seizures | |
WO2014040042A2 (en) | Diagnostic assay for alzheimer's disease | |
Chao et al. | Towards proteome standards: the use of absolute quantitation in high-throughput biomarker discovery | |
Sitole et al. | Metabolic profiling of HIV infected individuals on an AZT-based antiretroviral treatment regimen reveals persistent oxidative stress | |
CN105308455B (en) | Method and composition for diagnosing pre-eclampsia | |
CN109690312A (en) | Biomarker characteristic and its purposes | |
DK2671084T3 (en) | Biomarkers for osteoarthritis | |
US10317401B2 (en) | Methods and compositions for the prediction and treatment of focal segmental glomerulosclerosis | |
WO2018133553A1 (en) | Method for establishing quantitative reference range for healthy person urinary proteome and acquiring disease-related urinary protein marker | |
Sohaei | The use of proteomic analyses to identify potential cerebrospinal fluid biomarkers in Multiple Sclerosis | |
Yadav et al. | Synovial Fluid Proteomics and Serum Metabolomics Reveal Molecular and Metabolic Changes in Osteoarthritis | |
Ahmed et al. | Biomarker combination detects early-stage and discriminates osteoarthritis, rheumatoid arthritis and other inflammatory joint disease | |
Gutierrez Reyes et al. | Differential expression of N‐glycopeptides derived from serum glycoproteins in mild cognitive impairment (MCI) patients | |
TW202321695A (en) | Biomarkers for diagnosing non-alcoholic steatohepatitis (nash) or hepatocellular carcinoma (hcc) | |
CN114895018A (en) | Application of product for detecting metabolite level in serum in preparation of preparation for diagnosing various SLE patients and preparation | |
JP2011158261A (en) | Method for detecting progressive degree of osteoarthritis |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
AS | Assignment |
Owner name: VENN BIOSCIENCES CORPORATION, CALIFORNIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:CARRASCOSO, ALDO MARIO EDUARDO SILVA;BERTOZZI, CAROLYN RUTH;LEBRILLA, CARLITO BANGELES;AND OTHERS;SIGNING DATES FROM 20190324 TO 20190506;REEL/FRAME:064926/0879 |