WO2013106851A2 - Syncollin, pancreatic triacylglycerol lipase, and other biomarkers for diabetes - Google Patents

Abstract

The present invention provides biomarkers that are useful in determining whether a subject has type 1 diabetes or is at risk for developing type I diabetes. Specifically, syncollin, pancreatic triacylglycerol lipase, as well as fragments and variants of these proteins can be used to diagnose an individual with type I diabetes or one at risk for developing type I diabetes. Additional type I diabetes biomarkers are also provided.

Description

SYNCOLLIN, PANCREATIC TRIACYLGLYCEROL LIPASE, AND OTHER BIOMARKERS FOR DIABETES RELATED APPLICATION

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S.

Provisional Application No. 61/586,363 filed on Jan. 13, 2012, which is hereby incorporated by reference herein in its entirety.

STATEMENT CONCERNING GOVERNMENT RIGHTS IN FEDERALLY-SPONSORED RESEARCH

[0002] This invention was made with government support under Grant No.

DK55240, of the National Institutes of Health/National Institute of Diabetes and

Digestive and Kidney Diseases. The government has certain rights in this invention.

FIELD OF THE INVENTION

[0003] This invention relates to novel biomarkers for treating, diagnosing, and preventing diabetes. The invention also relates to methods of identifying, characterizing, and using such diabetes biomarkers.

BACKGROUND

[0004] Current technologies measure fasting blood glucose, oral glucose tolerance test, and hemoglobin Ale to diagnose and monitor Type I diabetes. Further, current technologies rely on insulin replacement therapy for treatment. These methods are costly and invasive.

[0005] There is a need for less expensive, less invasive, and more convenient methods for diagnosing, treating, and preventing Type I diabetes, especially in children.

[0006] Type 1 diabetes is a serious debilitating disease where insulin producing beta- cells (β-cells) are the selective target for autoimmune destruction by T-cells (1).

Presently, there is no cure for type 1 diabetes, and diabetics endure lifelong insulin replacement therapy. Novel therapies such as pancreatic transplants, islet β-cell regeneration and multipotent stem cell treatments (2) have been recently developed, however there is no clear strategy for preventing the development of type 1 diabetes (3). Trials of new therapies that modify the natural progression of type 1 diabetes have been impeded due to a shortage of biomarkers of the immune processes that ultimately are the origin of the disease (4). Hence, there is an unmet medical need to identify novel biomarkers of type 1 diabetes.

[0007] Known biomarkers such as Prostate specific antigen (PSA) in prostate cancer (6) and beta-amyloid in Alzheimer's disease (AD) (2) serve as readouts that clearly evaluate the risk of developing the disease and disease status. In the case of AD, the beta- amyloid biomarker shows specificity of above 80%, in distinguishing AD from dementia. Biomarkers are assessors of disease risk that can be developed into clinical diagnostic, prognostic, and treatment tools. Early detection biomarkers are essential in disease management and the treatment decision making process. These early biomarkers have the overall potential of improving favorable outcomes, with reduced health and economic burden to both the patient and society (7).

[0008] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

BRIEF SUMMARY OF THE INVENTION

[0009] In one embodiment of the present invention, the purified biomarker for type I diabetes comprises the syncollin protein. [0010] In another embodiment of the present invention, the purified biomarker for type I diabetes comprises the pancreatic triacylglycerol lipase protein. [0011] In another embodiment of the present invention, the purified biomarker for type 1 diabetes comprises the amino acid sequence GILGDWSNAISALYCR. [0012] In another embodiment of the present invention, the purified biomarker for type 1 diabetes comprises the amino acid sequence TNDVGQKFYLDTGDASNFAR. [0013] In still another embodiment of the present invention, the purified biomarker for type 1 diabetes comprises the amino acid sequence FIWYNNVF PTLPR. [0014] In yet another embodiment of the present invention, the purified biomarker for type 1 diabetes comprises the amino acid sequence NILSQIVDIDGIWEGTR. [0015] In further embodiments of the present invention, the purified biomarker for type 1 diabetes is selected from the group consisting of syncollin, pancreatic

triacylglycerol lipase, pancreatic amylase, bile salt-activated lipase, fatty acid binding protein, glutathione reductase, pancreatic secretory trypsin inhibitor, phosphoglycerate kinase, profilin-l, quinine oxidoreductase, and superoxide dismutase, or fragments or variants thereof. [0016] Another embodiment of the invention provides a method for screening for type 1 diabetes in a subject comprising the steps of: (a) providing a biological sample from a subject; (b) detecting at least one biomarker in said sample, said biomarker selected from the group consisting of peptides with the amino acid sequence comprising the syncollin protein, the pancreatic triacylglycerol lipase protein,

GILGDWSNAISALYCR, TNDVGQKFYLDTGDASNFAR, FIWYNNVINPTLPR, and NILSQIVDIDGIWEGTR; and (c) correlating said detection with a status of type 1 diabetes or no type 1 diabetes. [0017] In a further embodiment, the detecting at least one biomarker is performed by mass spectrometry. [0018] In a further embodiment, the mass spectrometry is MALDI-IMS. [0019] In another embodiment, the detecting at least one biomarker is performed by immunoassay. [0020] In a further embodiment, the immunoassay is an enzyme immunoassay. [0021] In still another embodiment, the biological sample is selected from the group consisting of biological fluid and tissue. [0022] In yet another embodiment, the biological fluid is whole blood, serum, plasma, or urine. [0023] In a further embodiment, the tissue is a pancreatic tissue sample. [0024] In yet another embodiment, the invention provides a method of diagnosing type 1 diabetes in a subject, comprising the steps of: (a) obtaining one or more test samples from a subject; (b) detecting the differential expression of at least one biomarker in the one or more test samples, wherein the biomarker is selected from: syncollin, pancreatic triacylglycerol lipase, pancreatic amylase, bile salt-activated lipase, fatty acid binding protein, glutathione reductase, pancreatic secretory trypsin inhibitor,

phosphoglycerate kinase, profilin-l, quinine oxidoreductase, and superoxide dismutase; and (c) correlating the detection of differential expression of at least one biomarker with a diagnosis of type 1 diabetes, wherein the correlation takes into account the amount of the at least one biomarker in the one or more test samples compared to a control amount of the at least one biomarker. [0025] In a further embodiment, one test sample is selected from the group consisting of urine, whole blood, serum, plasma, and pancreatic tissue. [0026] Kits for diagnosing type 1 diabetes are also contemplated in the present invention. [0027] In one embodiment, the invention includes a kit for diagnosing type 1 diabetes, the kit comprising (a) a solid support comprising at least one biospecific capture reagent attached thereto, wherein the biospecific capture reagent is capable of capturing a type 1 diabetes biomarker, the type 1 diabetes biomarker comprising the amino acid sequence comprising syncollin, pancreatic triacylglycerol lipase,

GILGDWSNAISALYCR, TNDVGQKFYLDTGDASNFAR, FIWYNNVINPTLPR, or NILSQIVDIDGIWEGTR, or a combination thereof; and (b) instructions for using the solid support to detect the type 1 diabetes biomarker. [0028] In a further embodiment, the invention provides a method of treating type 1 diabetes by administering an agent that reduces the expression of one or more biomarkers for type 1 diabetes selected from the group consisting of the amino acid sequence comprising the syncollin protein, the amino acid sequence comprising the pancreatic triacylglycerol lipase protein, the amino acid sequence comprising

GILGDWSNAISALYCR, the amino acid sequence comprising

TNDVGQKFYLDTGDASNFAR, the amino acid sequence comprising

FIWYNNVINPTLPR, and the amino acid sequence comprising

NILSQIVDIDGIWEGTR. [0029] In a further embodiment, the invention provides a method of treating type 1 diabetes by administering an agent that reduces the expression of one or more biomarkers for type 1 diabetes selected from the group consisting of: syncollin, pancreatic triacylglycerol lipase pancreatic amylase, bile salt-activated lipase, fatty acid binding protein, glutathione reductase, pancreatic secretory trypsin inhibitor, phosphoglycerate kinase, profilin-1, quinine oxidoreductase, and superoxide dismutase. [0030] In one embodiment, the invention includes a kit comprising: (a) a solid support comprising at least one biospecific capture reagent attached thereto, wherein the biospecific capture reagent is capable of capturing at least one type 1 diabetes biomarker selected from the group consisting of (i) the amino acid sequence comprising the syncollin protein, (ii) the amino acid sequence comprising the pancreatic triacylglycerol lipase protein, (iii) the amino acid sequence comprising GILGDWSNAISALYCR, (iv) the amino acid sequence comprising TNDVGQKFYLDTGDASNFAR, (v) the amino acid sequence comprising FIWYNNVINPTLPR, and (vi) the amino acid sequence comprising NILSQIVDIDGIWEGTR; and (b) instructions for using the solid support to detect the at least one type 1 diabetes biomarker. [0031] In a further embodiment, the kit includes instructions for using the solid support to detect a plurality of said biomarkers. [0032] In one embodiment, the invention includes a method for detecting type 1 diabetes in a subject comprising the steps of: (a) providing a biological sample from the subject; (b) contacting the biological sample with a biospecific capture reagent capable of capturing at least one type 1 diabetes biomarker comprising: (i) the amino acid sequence comprising pancreatic triacylglycerol lipase (ii) the amino acid sequence comprising TNDVGQKFYLDTGDASNFAR, (iii) the amino acid sequence comprising FIWYNNVINPTLPR, and (iv) the amino acid sequence comprising

NILSQIVDIDGIWEGTR; (c) determining the amount of the bound pancreatic triacylglycerol lipase biomarker having a molecular weight of about 25 kDa +/- 2.5 kDa; (d) correlating the amount of the bound pancreatic triacylglycerol lipase biomarker having a molecular weight of about 25 kDa +/- 2.5 kDa to a status of Type 1 diabetes. [0033] In a further embodiment, the screening differentiates between type 1 diabetes versus normal. [0034] In a further embodiment, the detecting is part of a diagnosis or prognosis of type 1 diabetes in the subject. [0035] In a further embodiment, the biological sample is selected from the group consisting of biological fluid and tissue.

[0036] a further embodiment, the biological fluid is whole blood, serum, plasma, or urine. [0037] In a further embodiment, the tissue is a pancreatic tissue sample. [0038] In a further embodiment, the biospecific capture reagent is attached to a solid support. [0039] In a further embodiment, the solid support is a mass spectrometry probe and the biospecific capture reagent comprises an antibody attached to the probe and wherein step (c) comprises detecting the bound biomarker by mass spectrometry. [0040] In a further embodiment, the antibody is selected from the group consisting of polyclonal antibodies, monoclonal antibodies, bispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, and Fv, FAb, and Fab2 fragments thereof. [0041] In a further embodiment, the method further comprises the step of: (e) comparing the amount of the pancreatic triacylglycerol lipase biomarker having a molecular weight of about 25 kDa +/- 2.5 kDa in the biological sample with the amount of pancreatic triacylglycerol lipase biomarker having a molecular weight of about 25 kDa +/- 2.5 kDa in a biological sample from one or more subjects free from type 1 diabetes or with a previously determined reference range for a pancreatic triacylglycerol lipase biomarker having a molecular weight of about 25 kDa +/- 2.5 kDa in subjects free from type 1 diabetes. [0042] In a further embodiment, step (c) comprises high-performance liquid chromatography. [0043] In a further embodiment, step (c) comprises polyacrylamide gel

electrophoresis (PAGE) and Western blotting. [0044] In a further embodiment, PAGE is 2-dimensional PAGE. [0045] In another embodiment, the invention includes a method for detecting type 1 diabetes in a subject comprising the steps of: (a) providing a biological sample from the subject; (b) contacting the biological sample with a biospecific capture reagent capable of capturing at least one type I diabetes biomarker comprising: (i) the amino acid sequence of syncollin, (ii) the amino acid sequence GILGDWSNAISALYCR; and (c) determining the amount of the bound syncollin biomarker having a molecular weight of about 15 kDa +/- 1.5 kDa; and (d) correlating the amount of the bound syncollin biomarker having a molecular weight of about 15 kDa +/- 1.5 kDa to a status of Type 1 diabetes. [0046] In a further embodiment, the screening differentiates between type 1 diabetes versus normal. [0047] In a further embodiment, the detecting is part of a diagnosis or prognosis of type 1 diabetes in the subject. [0048] In a further embodiment, the biological sample is selected from the group consisting of biological fluid and tissue. [0049] In a further embodiment, the biological fluid is whole blood, serum, plasma, or urine. [0050] In a further embodiment, the tissue is a pancreatic tissue sample. [0051] In a further embodiment, the biospecific capture reagent is attached to a solid support. [0052] In a further embodiment, the solid support is a mass spectrometry probe and the biospecific capture reagent comprises an antibody attached to the probe and wherein step (c) comprises detecting the bound biomarker by mass spectrometry.

[0053] In a further embodiment, the antibody is selected from the group consisting of polyclonal antibodies, monoclonal antibodies, bispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, and Fv, FAb, and Fab2 fragments thereof. [0054] In a further embodiment, the method further comprises the step of: (e) comparing the amount of the syncollin biomarker having a molecular weight of about 15 kDa +/- 1.5 kDa in the biological sample with the amount of syncollin biomarker having a molecular weight of about 15 kDa +/- 1.5 kDa in a biological sample from one or more subjects free from typel diabetes or with a previously determined reference range for a syncollin biomarker having a molecular weight of about 15 kDa +/- 1.5 kDa in subjects free from type 1 diabetes. [0055] In a further embodiment, step (c) comprises high-performance liquid chromatography. [0056] In a further embodiment, step (c) comprises polyacrylamide gel

electrophoresis (PAGE) and Western blotting. [0057] In a further embodiment, PAGE is 2-dimensional PAGE. [0058] In another embodiment, the invention includes a method of imaging beta cell tissue comprising the steps of: (a) administering to a subject a type 1 diabetes biomarker selected from the group consisting of syncollin, pancreatic triacylglycerol lipase pancreatic amylase, bile salt-activated lipase, fatty acid binding protein, glutathione reductase, pancreatic secretory trypsin inhibitor, phosphoglycerate kinase, profilin-1, quinine oxidoreductase, superoxide dismutase, the amino acid sequence comprising GILGDWSNAISALYCPv, the amino acid sequence comprising

TNDVGQKFYLDTGDASNFAR, the amino acid sequence comprising

FIWYNNVINPTLPR, and the amino acid sequence comprising

NILSQIVDIDGIWEGTR; and (b) using an imaging method to determine the amount of beta cell tissue. [0059] In a further embodiment, the imaging method is selected from the group consisting of (MRI); proton emission tomography (PET), ultrasonography, bio- luminescence, fluorescence, and nuclear medicine techniques. [0060] In another embodiment, the invention includes a method of monitoring the effect of an anti-diabetes drug or therapy on a subject comprising: (a) providing a biological sample from the subject; (b) contacting the biological sample with a biospecific capture reagent capable of capturing at least one type I diabetes biomarker selected from the group consisting of syncollin, pancreatic triacylglycerol lipase pancreatic amylase, bile salt-activated lipase, fatty acid binding protein, glutathione reductase, pancreatic secretory trypsin inhibitor, phosphoglycerate kinase, profilin-1, quinine oxidoreductase, superoxide dismutase, the amino acid sequence comprising GILGDWSNAISALYCR, the amino acid sequence comprising

TNDVGQKFYLDTGDASNFAR, the amino acid sequence comprising

FIWYNNVINPTLPR, and the amino acid sequence comprising

NILSQIVDIDGIWEGTR; (c) measuring the amount of the at least one type-I biomarker; (d) providing the subject with an anti-diabetes drug or therapy; (e) measuring the amount of the at least type-I biomarker using steps (a) and (b); and (f) correlating the two measurements with a diagnosis that the diabetes is regressing or progressing. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] The following figures are provided for the purpose of illustration only and are not intended to be limiting.

[0062] FIGURE 1: Figure 1A is averaged protein spectra, extracted from MALDI- IMS region of interest, within pancreatic tissue section. Figure 1 Ai is the full spectrum of the differentially expressed peak at 15 kiloDaltons ("kDa") , in type one diabetic

("TID") versus non-type one diabetic (Non-TID") tissue samples. Figure ΙΑζϊ is a graph that shows extracted imaging mass spectrometry spectra representing differential expression of 15 kDa in Type I diabetes ("TID"), non-diabetes ("non-TID"), and insulin antibody positive ("Ab+") samples viewed at full spectrum (2-20 kDa) showing the differential expression between type one diabetic, auto-antibody positive and non-type one diabetic tissue samples. Figure IB is a SDS PAGE analysis of pancreatic tissue extracts, showing differentially expressed protein bands between type-one diabetic ("TID"), auto-antibody positive ("Ab+") and non-type one diabetic ("Non-T ID") tissue lysates. Figure 1C is a Venn diagram illustrating all protein hits identified between type one diabetic ("TID") (53 protein hits), auto-antibody positive ("AB") (28 protein hits) and non-type one diabetic ("Non-TID") (23 protein hits) tissue lysates. The two biomarkers; syncollin and pancreatic triacylglycerol lipase were identified in the sector emphasized by the black oval along with 29 other biomarkers for type 1 diabetes.

[0063] FIGURE 2: Figure 2A is a MS-MS identification of human syncollin (top panel) and human pancreatic triacylglycerol lipase (bottom panel). The identified peptides are shown in underlined italics. Figure 2B is MS-MS Fragmentation and annotation data for human syncollin. The masses of the identified fragments are shown in underlined italics. Figure 2C is MS-MS Fragmentation and annotation data for pancreatic triacylglycerol lipase.

[0064] FIGURE 3:. Western blot analysis of syncollin in human pancreatic tissue. Figure 3A is the original western blot. Figure 3B is a repeat of western blot with 8 kD ubiquitin as loading control. Figure 3C is a densitometric quantitation of syncollin. All samples are normalized to ubiquitin control.

[0065] FIGURE 4: Figure 4 A is a quantitative multiple reaction monitoring assay for the pancreatic triacylglycerol lipase peptide with the sequence of

FIWYNNVTNPTLPPv. Figure 4B are the bar graphs with standard errors corresponding to the area under the curves for the extracted ion chromatogram from triplicate MRM experiments using the peptide parent ion of (Ql) MH³⁺ 873.969 and the Q3 ylO MH⁺ fragment mass of 1137.637. The ylO refers to a fragment of the described peptide that is numbered from the c-terminal residue of the described peptide (i.e. NNVINPTLPR).

[0066] FIGURE 5: Quantitative multiple reaction monitoring assay for the pancreatic triacylglycerol lipase peptide with the sequence NILSQIVDIDGIWEGTR. The MRM transition pairs for this peptide are the parent ion (Ql) MH³⁺ 643.674 and (Ql) MH²⁺ 965.007 (not shown) and the Q3 fragment masses are y6 MH⁺, 761.394 and ylO MH⁺, 1161.553. The y6 refers to a fragment of the peptide that is numbered from the c-terminal residue of the described peptide (i.e. IWEGTR).

[0067] FIGURE 6: Quantitative multiple reaction monitoring assay for the the pancreatic triacylglycerol lipase peptide with the sequence FIWYNNVINPTLPR. The MRM transition pairs for this peptide are the parent ion (Ql) MH³⁺ 582.982 (not shown) and MH²⁺ 873.969 and the Q3 fragment masses are y7 MH⁺810.483 and ylO MH⁺ 1137.637.

[0068] FIGURE 7: Figure 7 is a series of charts, which show the results of gene expression in pancreatic tissue analysis of syncollin in non-obese diabetic mice ("NOD") and non-obese diabetic mice that are deficient in 12/15 lipoxygenase ("NOD-Aloxl5") null mice at 4, 8, and 12 weeks of age (FIGS. 7A, B, and C, respectively) and syncollin gene expression in cytokine treated and untreated pancreatic duct tissue (FIG. 7D).

[0069] FIGURE 8: Figure 8 is a summary table of 9 additional biomarkers for type 1 diabetes provided by this invention. Included in the table are the Swiss Prot Accession numbers, human gene that codes for the protein and a description of the protein.

[0070] FIGURE 9: The amino acid sequence of pancreatic alpha-amylase.

[0071] FIGURE 10: The amino acid sequence of bile salt-activated lipase.

[0072] FIGURE 11: The amino acid sequence of fatty acid-binding protein, epidermal.

[0073] FIGURE 12: The amino acid sequence of glutathione reductase,

mitochrondrial.

[0074] FIGURE 13: The amino acid sequence of pancreatic secretory trypsin inhibitor.

[0075] FIGURE 14: The amino acid sequence of phosphoglycerate kinase 1.

[0076] FIGURE 15: The amino acid sequence of profilin-1.

[0077] FIGURE 16: The amino acid sequence of quinone oxidoreductase.

[0078] FIGURE 17: The amino acid sequence of superoxide dismutase. DETAILED DESCRIPTION OF THE INVENTION

[0079] The invention is directed to biomarkers for type I diabetes. The invention is also directed to methods of detecting the presence of one or more biomarkers in order to make a diagnosis of type I diabetes. The measurement of these markers, alone or in combination, in patient samples provides information that the diagnostician can correlate with a diagnosis of type I diabetes or risk of developing type I diabetes. In some embodiments, the biomarkers are syncollin, pancreatic triacylglycerol lipase, or fragments or variants thereof. [0080] Syncollin, pancreatic triacylglycerol lipase, and other biomarkers for type I diabetes, as well as methods and uses thereof, are disclosed. These biomarkers (including syncollin and pancreatic triacylglycerol lipase) are overexpressed in patients with Type I (insulin-dependent) diabetes and in insulin antibody positive patients compared to non- diabetic patients (see FIG. 1). These novel biomarkers can be utilized in diagnosis, and as potentially therapeutic targets for treatment of type 1 diabetes. In some embodiments, the invention provides a method of diagnosing type I diabetes in a subject, comprising detecting the differential expression of at least one biomarker in the one or more test samples obtained from the subject, wherein the protein marker is syncollin, pancreatic triacylglycerol lipase, or fragments and variants thereof.

[0081] Syncollin, pancreatic triacylglycerol lipase, and fragments and variants thereof contained in biological fluids (e.g. blood or urine) and tissues can be used as biomarkers to diagnose patients with Type I diabetes, or to diagnose patients at risk for developing Type I diabetes.

[0082] In one embodiment of the present invention, a method of diagnosing type I diabetes or risk for developing type I diabetes in a subject is provided. This method comprises the steps of (a) providing a biological sample from the subject; (b) contacting the biological sample with a biospecific capture reagent capable of capturing a type I diabetes biomarker comprising (i) the amino acid sequence of syncollin, (ii) the amino avid sequence of pancreatic triacylglycerol lipase, (iii) the amino acid sequence

GILGDWSNAISALYCR, (iv) the amino acid sequence

TNDVGQKFYLDTGDASNFAR, (v) the amino acid sequence FIWYNNVINPTLPR, and/or (vi) the amino acid sequence NILSQIVDIDGIWEGTR; (c) determining the amount of the bound type I diabetes biomarker or biomarkers; and (d) correlating the amount of the bound biomarker or biomarkers to a type I diabetes diagnosis. [0083] The amount of type I diabetes biomarkers (i.e., syncollin or pancreatic triacylglycerol lipase) in normal (i.e., non-diabetic) biological samples can be assessed in a variety of ways as described herein. In one embodiment, the " normal" or control amount of type I diabetes biomarkers expression may be determined by assessing the amount of syncollin or pancreatic triacylglycerol lipase in one or more samples obtained from one or more non-diabetic individuals. [0084] Using the methods of the invention, levels of type I diabetes biomarkers (i.e., syncollin or pancreatic triacylglycerol lipase or fragments or variants thereof) are determined in a biological sample from a subject suspected of having type I diabetes and in one or more comparable biological samples from normal or healthy subjects (i.e., control samples). A level of type I diabetes biomarker (i.e, syncollin or pancreatic triacylglycerol lipase or fragments or variants thereof ) detected in a biological sample from a subject suspected of having type I diabetes that is higher than the type I diabetes biomarker level detected in a comparable biological sample from a normal or healthy subject, indicates that the subject suspected of having type I diabetes has or is likely to have type I diabetes. [0085] Methods for detecting and/or quantifying type I diabetes biomarkers (i.e., syncollin and pancreatic triacylglycerol lipase and fragments and variants thereof) in biological fluids such as serum and urine include enzyme linked immunosorbent assays (ELISAs; including sandwich ELISA and competitive ELISA),, gene expression arrays,multiple reaction monitoring mass spectrometry (MRM-MS), proteomic analysis, western blot, and Coomassie Stain (see FIGS. 1-4). Useful assays also include immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmune assay (RIA), and/or immunoradiometric assay (IRMA). These assays are well known in the art.

[0086] A biomarker is an organic biomolecule, the presence of which in a sample is used to determine the phenotypic status of the subject (e.g., diabetes patient v. normal patient). In a preferred embodiment, the biomarker is differentially present in a sample taken from a subject of one phenotypic status (e.g., having a disease) as compared with another phenotypic status (e.g., not having the disease). Biomarkers, alone or in combination, provide measures of relative risk that a subject belongs to one phenotypic status or another. Therefore, they are useful as markers for disease (diagnostics), therapeutic effectiveness of a drug (theranostics), drug toxicity, and predicting and identifying the immune response. [0087] In accordance with the invention, at least one biomarker may be detected. It is to be understood, and is described herein, that one or more biomarkers may be detected and subsequently analyzed, including several or all of the biomarkers identified.

[0088] Biomarkers according to the invention include proteins, protein fragments, and peptides. Peptides, polypeptides and proteins of the present invention include amino acid polymers having D- and L-isoforms of individual amino acid residues, as well as other amino acid variants, as described herein.

[0089] Biomarkers of the present invention may be detected from a biological sample from a subject. The biological sample may be a biological fluid such as whole blood or serum. The biological sample may also be from tissue such as pancreatic tissue. Other examples of tissue specimen useful to practice the methods of the present invention include samples taken from the prostate, central nervous system, bone, breast tissue, renal tissue, endometrium, head/neck, gall bladder, parotid tissue, brain, pituitary gland, kidney tissue, muscle, esophagus, stomach, small intestine, colon, urethra, liver, spleen, pancreas, thyroid tissue, heart, lung, bladder, adipose tissue, lymph node tissue, adrenal tissue, testis tissue, tonsils, and thymus.

[0090] Biomarkers of the present invention may also be detected from biological fluid such as whole blood, serum, plasma, urine, tears, mucus ascites fluid, oral fluid, salivia, semen, seminal fluid, mucus, stool, sputum, cerebrospinal fluid, bone marrow, lymph, and fetal fluid. The biological fluid samples may include cells, proteins, or membrane extracts of cells.

[0091] "Subject" includes living and dead organisms. Examples of subjects include mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic nonhuman animals. Most preferably the subject is a human.

[0092] The biomarkers of this invention can be isolated and purified from biological fluids, such as urine or serum. They can be isolated by any method known in the art, based on their mass, their binding characteristics and their identity as a syncollin, pancreatic triacylglycerol lipase, pancreatic alpha-amylase, bile salt-activated lipase, fatty acid binding protein (epidermal), glutathione reductase (mitochondrial), pancreatic secretory trypsin inhibitor, phosphoglycerate kinase 1, profilin-1, quinine oxidoreductase , or superoxide dismutase polypeptide . For example, a biological sample comprising the biomarkers can be subject to chromatographic fractionation and subject to further separation by, e.g., acrylamide gel electrophoresis. Knowledge of the identity of the biomarker also allows their isolation by immunoaffmity chromatography. [0093] In one aspect, this invention provides these biomarkers in isolated form. In one embodiment of the present invention, a purified biomarker is provided, the biomarker comprising (i) an amino acid sequence comprising syncollin, (ii) an amino acid sequence comprising pancreatic triacylglycerol lipase, (iii) an amino acid sequence comprising GILGDWSNAISALYCR, (iv) an amino acid sequence comprising

TNDVGQKFYLDTGDASNFAR, (v) an amino acid sequence comprising

FIWYNNVINPTLPR, or (vi) an amino acid sequence comprising

NILSQIVDIDGIWEGTR. In other aspects of the invention the purified biomarker comprises the amino acid sequence comprising the pancreatic alpha-amylase, bile salt- activated lipase, fatty acid binding protein (epidermal), glutathione reductase

(mitochondrial), pancreatic secretory trypsin inhibitor, phosphoglycerate kinase 1, profilin-1, quinine oxidoreductase , or superoxide dismutase polypeptide.

[0094] Some purified biomarkers of this invention comprise amino acid sequences that are found in the amino acid sequences of syncollin and pancreatic triacylglycerol lipase. Specifically, the amino acid sequence GILGDWSNAISALYCR of the purified biomarker of the present invention is found at position 117 to 132 of the human syncollin protein. This biomarker has a m/z of MH⁺¹ 1738.858, MH⁺² 869.933 and MH⁺³ 580.291. MH⁺¹ refers to the first ion created by the addition of a proton and MH⁺² refers to an ion created by adding two protons, etc. The human syncollin protein has a m/z of ~15 kDa +/- 1.5 kDa (theoretically calculated to be 14,405 Da). The error of the molecular weight is 100 ppm of the total observed weight. This is based on the current accuracy of the mass spectrometry equipment used to characterize the present invention. The amino acid sequence of TNDVGQKFYLDTGDASNFAR of the purified biomarker of the present invention is found at position 336 to 353 of the human pancreatic triacylglycerol lipase protein and has a m/z of MH⁺¹ 2219.036, MH⁺² 1110.022 and MH⁺³ 740.350. The amino acid sequence of FIWYNNVINPTLPR of the purified biomarker of the present invention is found at position 417 to 430 of the human pancreatic triacylglycerol lipase protein and has a m/z of MH⁺¹ 1746.9326 , MH⁺² 873.969 and MH⁺³ 582.982. The amino acid sequence of NILSQIVDIDGIWEGTR of the purified biomarker of the present invention is found at position 257 to 273 of the human pancreatic triacylglycerol lipase protein and has a m/z of MH⁺¹ 1929.007, MH⁺² 965.007 and MH⁺³ 643.674. The human pancreatic triacylglycerol lipase protein has an molecular mass of ~51 kDa and it undergoes processing to produce a -25 kDa +/- 2.5 kDa version of the protein. [0095] "Purified" means substantially pure and refers to biomarkers that are substantially free of other proteins, lipids, carbohydrates or other materials with which they are naturally associated. Purified syncollin and pancreatic triacylglycerol lipase, for example, may retain, however, covalent posttranslational modifications. Individually, the purified syncollin and pancreatic triacylglycerol lipase of the present invention will yield a single major peak by, for example, SELDI analysis. The purity of the type-I diabetes biomarkers can also be determined by PAGE, amino-terminal amino acid sequence analysis and/or tryptic peptide analysis.

[0096] The biomarkers may be characterized by mass-to-charge ratio as determined by mass spectrometry, by the shape of their spectral peak in time-of-flight mass spectrometry and by their binding characteristics to adsorbent surfaces. These characteristics provide one method to determine whether a particular detected

biomolecule is a biomarker of this invention. These characteristics represent inherent characteristics of the biomolecules and not process limitations in the manner in which the biomolecules are discriminated.

[0097] Biomarkers of this invention may be initially characterized by mass-to-charge ratio, binding properties and spectral shape. Thus, they can be detected by mass spectrometry without knowing their specific identity. However, biomarkers can be identified by, for example, determining the amino acid sequence of the polypeptides. For example, a biomarker can be peptide -mapped with a number of enzymes, such as trypsin or V8 protease, and the molecular weights of the digestion fragments can be used to search databases for sequences that match the molecular weights of the digestion fragments generated by the various enzymes. Alternatively, protein biomarkers can be sequenced using tandem MS technology. In this method, the protein is isolated by, for example, gel electrophoresis. A band containing the biomarker is cut out and the protein is subject to protease digestion. Individual protein fragments are separated by a first mass spectrometer. The fragment is then subjected to collision-induced cooling, which fragments the peptide and produces a polypeptide ladder. A polypeptide ladder is then analyzed by the second mass spectrometer of the tandem MS. The difference in masses of the members of the polypeptide ladder identifies the amino acids in the sequence. An entire protein can be sequenced this way, or a sequence fragment can be subjected to database mining to find identity candidates.

[0098] In prior work, insulin within β-cells of islets of Langerhans was identified by MALDI-TOF-MS/MS after in-situ reduction of disulphide bonds (8). Endocrine Reviews, December 2012, 33(6):892-919, which is incorporated by reference herein in its entirety.

[0099] In certain embodiments, the invention provides biomarkers that are differentially present in samples of diabetic subjects and non-diabetic subjects identified by the use of MALDI-IMS, or Matrix- Assisted Laser Desorption/Ionization Mass Spectrometric Tissue Imaging. MALDI-IMS is described in, for example U.S. Pat. No. 5,808,300, which is incorporated by reference herein in its entirety.

[0100] MALDI-IMS is a technique that allows for imaging of biological samples and has been shown to be quite versatile in its many applications to the analysis of biological samples, such as peptides and proteins. Typically, samples are mixed with an organic compound which acts as a matrix to facilitate ablation and ionization of compounds in the sample. The presence of this matrix is necessary to provide the required sensitivity and specificity to use laser desorption techniques in the analysis of biological material. The application of thin layers of matrix has special advantages, particularly when very high sensitivity is needed.

[0101] MALDI-IMS may be used to generate images of samples in one or more m/z pictures, providing the capability for mapping the concentrations of specific molecules in X, Y coordinates of the original biological sample. A MALDI-IMS "image" is achieved by desorption and measurement of tissue proteins/peptides from focused regions, which is subsequently summed across the entire tissue field. Each "spot" is a piece of the composite picture resulting from the grid arrangement of the spots. In this way, a protein/peptide that is overexpressed or underexpressed can have the related expression associated with the tissue region. In effect a region of the tissue that selectively expresses a discrete peptide will display an area of high expression that can be seen from the MS data. In certain embodiments, such images may be matched to a mirror tissue that is reviewed by the pathologist and provides supplemental information to the pathologist to aid in diagnosis and staging/grading of disease.

[0102] In the present invention, a comparative analysis of MALDI-TOF-MS spectra from type 1 diabetes tissues to those of Non-type 1 diabetes revealed several uniquely expressed proteins between the two groups. In these analyses, insulin, with an m/z 5812 was over expressed in spectra in the Non-type 1 diabetes compared to the type 1 diabetes tissue sections. On the other hand, a polypeptide with an m/z of ~15 kDa was over expressed in the type 1 diabetes samples compared to the Non-type 1 diabetes tissues. Aspects of the present invention were designed as an experimental strategy to identify potential biomarkers. MALDI-IMS, LC-ESI-MS/MS, and multiple reaction monitoring (MRM) was used to identify and verify syncollin, pancreatic triacylglycerol lipase, and 9 other proteins as potential biomarkers of type 1 diabetes.

[0103] Biomarker discovery has paved the way for diagnosis and the development of treatment of many diseases including cancer (6) however, there are no clinically useful biomarkers for human type 1 diabetes other than antibody and metabolic testing. We have identified syncollin and pancreatic triacylglycerol lipase as potential biomarkers for human type 1 diabetes. Syncollin is a ~15 kDa secretory granule protein important for insulin granule exocytosis within the β-cells of the pancreatic islets of Langerhans. In the exocrine pancreas, a subset of the syncollin chain is linked to the inner surface of the granule membrane (13). In the acinar cells of the pancreas, secretory proteins are produced in the rough endoplasmic reticulum and transported to the Golgi apparatus while mature granules are stored in the apical pole. The condensing vacuoles of the trans-Golgi network serve as chambers that separate the proteins into two subsets: those destined to become granular content proteins and those fated to become constitutive secretion proteins. After segregation, they are further matured and condensed into zymogen granules. Upon hormonal or nervous stimulation, local Ca²⁺ concentration increases at the apical pole, which causes the granule content to be discharged into the pancreatic duct by exocytosis (14).

[0104] Triglyceride lipases are lipolytic enzymes involved in the hydrolysis of ester linkages of triglycerides (19). The main known function of lipoprotein lipase is to hydro lyze triglycerides of chylomicrons and very low density lipoproteins (VLDL) (20). Pancreatic triacylglycerol lipase hydrolyzes dietary long chain triacylglycerol to free fatty acids and monoacylglycerols in the intestinal lumen (21) thus, aiding in fat absorption. Here, we identified pancreatic triacylglycerol lipase as a potential novel biomarker in type 1 diabetes.

[0105] This invention has illustrated the utility of mass spectrometry as a tool to identify potential biomarkers for type 1 diabetes.

[0106] The present invention also contemplates additional proteins that may be important in type 1 diabetes pathogenesis and could form a panel of potential biomarkers to discriminate between disease and non-disease states. These proteins are: Pancreatic alpha-amylase (SwissProt Accession # P04746; gene name AMYP_HUMAN), bile salt- activated lipase (SwissProt Accession # P19835; gene name CEL_HUMAN), fatty acid binding protein, epidermal (SwissProt Accession # Q01469; gene name FABP5 HUMAN), glutathione reductase, mitochondrial (SwissProt Accession # P00390; gene name GSHR HUMAN), pancreatic secretory trypsin inhibitor (SwissProt Accession #P00995; gene name ISK1 HUMAN), phosphoglycerate kinase 1 (SwissProt Accession # P00558; gene name PGK1_HUMAN), profilin-1 (SwissProt Accession # P07737; gene name PROF 1 HUMAN), quinine oxidoreductase (SwissProt Accession # Q08257; gene name QOR_HUMAN), superoxide dismutase (SwissProt Accession # P00441; gene name SODC HUMAN) (See Figure 8). The sequences for these proteins are shown in FIGS. 9-17. Some of these proteins indicate the presence of acute inflammatory response and excessive cellular oxidative stress, while others are mitochondrial proteins that could indicate mitochondrial dysfunction. These proteins, along with syncollin and pancreatic triacylglycerol lipase, and fragments and variants thereof, could provide a panel of biomarkers to identify risk onset for type 1 diabetes.

[0107] The biomarkers of the invention include amino acid sequence variants of the various biomarkers identified. These variants may, for instance, be minor sequence variants of the polypeptide which arise due to natural variation within the population or they may be homologues found in other species. They also may be sequences which do not occur naturally but which are sufficiently similar that they function similarly and/or elicit an immune response that cross-reacts with natural forms of the polypeptide.

Sequence variants may be prepared by standard methods of site-directed mutagenesis that are well-known in the art.

[0108] Amino acid sequence variants of the polypeptide may be substitutional, insertional or deletion variants. Deletion variants lack one or more residues of the native protein which are not essential for function or immunogenic activity, such as variants lacking a transmembrane sequence. Another common type of deletion variant is one lacking secretory signal sequences or signal sequences directing a protein to bind to a particular part of a cell. An example of the latter sequence is the SH2 domain, which induces protein binding to phosphotyrosine residues.

[0109] Substitutional variants typically contain an alternative amino acid at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide such as stability against proteolytic cleavage. Substitutions may be are conservative, that is, one amino acid is replaced with one of similar size and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamate; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.

[0110] Insertional variants include fusion proteins such as those used to allow rapid purification of the polypeptide and also may include hybrid proteins containing sequences from other proteins and polypeptides which are homologues of the polypeptide. For example, an insertional variant may include portions of the amino acid sequence of the polypeptide from one species, together with portions of the homologous polypeptide from another species. Other insertional variants may include those in which additional amino acids are introduced within the coding sequence of the polypeptide. These typically are smaller insertions than the fusion proteins described above and are introduced, for example, to disrupt a protease cleavage site.

[0111] The markers of the invention identified by MALDI- TOF-MS/MS and MALDI-IMS can be detected by other methods, also within the scope of the invention. Such methods may include chromatographic methods, such as liquid chromatography or gel chromatography, or immunoassays. Using the purified markers or their nucleic acid sequences, antibodies that specifically bind to a marker can be prepared using any suitable methods known in the art. See, e.g., Current Protocols in Immunology (2007); Harlow & Lane, Antibodies: A Laboratory Manual (1988); Goding, Monoclonal Antibodies: Principles and Practice (3d ed. 1996); and Kohler & Milstein, Nature 256:495-497 (1975). Such techniques include, but are not limited to, antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors, as well as preparation of polyclonal and monoclonal antibodies by immunizing rabbits or mice (see, e.g., Huse et al, Science 246: t275-1281 (1989); Ward et al, Nature 341 :544-546 (1989)).

[0112] After the antibody is provided, a marker can be detected and/or quantified using any of a number of well recognized immunological binding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). Useful assays include, for example, an enzyme immune assay (EIA) such as enzyme-linked immunosorbent assay (ELISA), a radioimmune assay (RIA), a Western blot assay, or a slot blot assay. For a review of the general immunoassays, see also, Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology (Stites & Teff, eds., 7th ed. 1991).

Kits for Diagnosing Type 1 Diabetes

[0113] In another aspect, the present invention provides kits for diagnosing type 1 diabetes, wherein the kits are used to detect type 1 diabetes biomarkers according to the invention.

[0114] In one embodiment of the present invention a kit for diagnosing type 1 diabetes is provided, the kit comprising (a) a solid support comprising at least one biospecific capture reagent attached thereto, wherein the biospecific capture reagent is capable of capturing at least one type 1 diabetes biomarker selected from the group consisting of (i) the protein sequence of syncollin, (ii) the protein sequence of pancreatic triacylglycerol lipase (iii) the amino acid sequence comprising GILGDWSNAISALYCR, (iv) the amino acid sequence comprising TNDVGQKFYLDTGDASNFAR, (v) the amino acid sequence comprising FIWYNNVINPTLPR, and (vi) the amino acid sequence comprising NILSQIVDIDGIWEGTR; and (b) instructions for using the solid support to detect the at least one type 1 diabetes biomarker. The solid support comprising the biospecific capture reagent may be, for example, a SELDI probe, a chip, a microtiter plate, a bead or a resin.

[0115] "Biospecific capture reagent" refers to any material capable of capturing a biomarker. The term includes reagents comprising a biomolecule, e.g., a nucleic acid molecule (e.g., an aptamer), a polypeptide, a polysaccharide, a lipid, a steroid or a conjugate of these (e.g., a glycoprotein, a lipoprotein, a glycolipid, a nucleic acid (e.g., DNA)-protein conjugate). In certain instances, the biospecific adsorbent can be a macromolecular structure such as a multiprotein complex, a biological membrane or a virus. Examples of biospecific capture reagents are antibodies, receptor proteins and nucleic acids. A biospecific capture reagent of this invention includes a biospecific capture reagent that captures syncollin or pancreatic triacylglycerol lipase or fragments or variants thereof. In other embodiments, the biospecific capture reagent of this invention includes a biospecific capture reagent that captures pancreatic alpha-amylase, bile salt-activated lipase, fatty acid binding protein (epidermal), glutathione reductase (mitochondrial), pancreatic secretory trypsin inhibitor, phosphoglycerate kinase 1 , profilin-1, quinine oxidoreductase , or superoxide dismutase polypeptide, or fragments or variants thereof. The terms "biospecific capture reagent" and "biospecific adsorbent" are used interchangeably. [0116] "Capture," "capturing" or grammatical equivalents thereof refer to the capability of a biospecific capture reagent to recognize and bind to a target molecule. Target molecule includes syncollin or pancreatic triacylglycerol lipase or fragments or variants thereof. Target molecule also includes pancreatic alpha-amylase, bile salt- activated lipase, fatty acid binding protein (epidermal), glutathione reductase

(mitochondrial), pancreatic secretory trypsin inhibitor, phosphoglycerate kinase 1, profilin-1, quinine oxidoreductase , or superoxide dismutase polypeptide, or fragments or variants thereof. Synonyms of the term "capture" are contemplated within the scope of the present invention and include, but are not limited to, adsorbing, preserving, keeping, holding, retaining. Generally it refers to a detectable binding between a biospecific capture reagent and a marker, such as syncollin or pancreatic triacylglycerol lipase (or the other presently disclosed biomarkers of this invention).

[0117] In one embodiment of the present invention, the biospecific capture reagent is attached to a solid support. The solid support can be a mass spectrometry probe. The biospecific capture reagent may comprise an antibody attached to the probe. The step of determining the amount of the bound biomarker may comprise detecting the bound biomarker by laser desorption-ionization mass spectrometry.

[0118] The antibody may be selected from the group consisting of polyclonal antibodies, monoclonal antibodies, bispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, and active fragments thereof (including Fv, FAb, and Fab2 fragments).

[0119] In one embodiment, the kit comprises a solid support, such as a chip, a microtiter plate or a bead or resin having a capture reagent attached thereon, wherein the capture reagent binds a biomarker of the invention. Thus, for example, the kits of the present invention can comprise mass spectrometry probes for SELDI, such as

ProteinChip.RTM. arrays. In the case of biospecific capture reagents, the kit can comprise a solid support with a reactive surface, and a container comprising the biospecific capture reagent.

[0120] Thus, for example, the kits of this invention could include a solid support having a hydrophobic function, such as a protein biochip (e.g., a Ciphergen H50

ProteinChip array, e.g., ProteinChip array) and a sodium acetate buffer for washing the solid support as well as instructions providing a protocol to measure the biomarkers of this invention on the chip and to use these measurements to diagnose type I diabetes status. [0121] The kit can also comprise a washing solution or instructions for making a washing solution, in which the combination of the biospecific capture reagent and the washing solution allows capture of the biomarker or biomarkers on the solid support for subsequent detection by, e.g., mass spectrometry. The kit may include more than one type of adsorbent, each present on a different solid support.

[0122] In a further embodiment, such a kit can comprise instructions for suitable operational parameters in the form of a label or separate insert. For example, the instructions may inform a consumer about how to collect a biological sample, how to wash the probe or the particular biomarker to be detected.

[0123] In yet another embodiment, the kit can comprise one or more containers with samples of, e.g., syncollin or pancreatic triacylglycerol lipase biomarkers (or the other biomarkers disclosed herein), or fragments or variants thereof, to be used as standard(s) for calibration. Monitoring the Effect of an Anti-Diabetic Drug or Therapy Administered to a Subject with Type I Diabetes

[0124] In another embodiment of the present invention, the type I diabetes status is determined as part of monitoring the effect of an anti-diabetes drug or a therapy administered to the subject diagnosed with diabetes. The effect of an anti-diabetes drug or a therapy administered to a subject with diabetes may include the worsening or improvement of diabetes processes such as inflammation. This also could be utilized to evaluate the health of beta cells and whether therapy is effective in helping to prevent loss of beta cell mass or help in regeneration or restoration of beta cell mass or function.

[0125] Using the methods of the invention, levels of syncollin or pancreatic triacylglycerol lipase (or the other type-I diabetes biomarkers disclosed herein) are determined in a biological sample from a subject at various times of having been given an anti-diabetes drug or a therapy. A type I diabetes biomarker level detected in a biological sample from a subject at a first time (tl; e.g., before giving an anti-diabetes drug or a therapy) that is higher than the type I diabetes biomarker level detected in a comparable biological sample from the same subject taken at a second time (t2; e.g., after giving an anti-diabetes or a therapy), indicates that the diabetes in the subject is regressing. Likewise, a higher type I diabetes biomarker level at a second time compared to a type I diabetes biomarker level at a first time, indicates that the diabetes in the subject is progressing. [0042] In another embodiment, this method involves measuring one or more type-I diabetes biomarkers, one of which may be syncollin or pancreatic triacylglycerol lipase, in a subject at least at two different time points, e.g., a first time and a second time, and comparing the change in amounts, if any. The effect of the anti-diabetes drug or therapy on the progression or regression of diabetes is determined based on these comparisons. Thus, this method is useful for determining the response to treatment. If a treatment is effective, then the type-I diabetes biomarker will trend toward normal, while if treatment is ineffective, the type-I diabetes biomarker will trend toward disease indications. Use of Biomarkers for Diabetes in Screening Assays and Methods of Treating Diabetes

[0126] The methods of the present invention have other applications as well. For example, the type-I diabetes biomarkers can be used to screen for compounds that modulate the expression of the syncollin or pancreatic triacylglycerol lipase or other type-I diabetes biomarkers in vitro or in vivo, which compounds in turn may be useful in treating or preventing type-I diabetes in patients. Moreover, the expression of the genes corresponding to syncollin and pancreatic triacylglycerol lipase in the acinar of the pancreas can be reduced or inhibited to decrease or eliminate the deleterious effects of Type I diabetes. Syncollin specific targets could also be used to regulate exocytotic events of insulin, one of the main causes of malfunction in Type I diabetes. Further, targets aimed at reducing pancreatic triacylglycerol lipase would decrease lipotoxicity, thus reducing inflammation typically seen in Type I diabetes. Also, small molecule inhibitors that target the syncollin and pancreatic triacylglycerol lipase or other type-I diabetes biomarker pathways can be used to modulate the expression of these proteins in vivo.

[0127] Modulating the expression of the genes that encode for syncollin and pancreatic

triacylglycerol lipase (or other type-I diabetes biomarkers) could reduce or eliminate the need for invasive, lifelong daily insulin injections. Treatments that target syncollin and pancreatic triacylglycerol lipase (or other type-I diabetes biomarkers) for treating type I diabetes would be more efficacious than exogenous insulin replacement therapies.

Moreover, in addition to being highly efficacious, inhibitors that target syncollin and pancreatic triacylglycerol lipase (or other type-I diabetes biomarkers) could be developed for less than the cost of daily insulin therapy. Use of Biomarkers To Develop Imaging Probes to Diagnose and Treat Diabetes

[0128] Various types of imaging techniques can be used to monitor β-cell mass in vivo. These techniques include but are not limited to magnetic resonance imaging (MRI); proton emission tomography (PET), ultrasonography, bio- luminescence, fluorescence, and nuclear medicine techniques. See Gialleonardo et al, Imaging of β- Cell Mass and Insulitis in Insulin-Dependent (Type 1) Diabetes Mellitus, Endocrine Reviews, December 2012, 33(6):892-919. Endocrine Reviews, December 2012, 33(6):892-919. However, there are difficulties in obtaining probes that target pancreatic β-cell mass. Therefore, in one aspect of the present invention, the biomarkers are used as probes for imaging techniques. In another aspect, the biomarkers are used to develop probes for imaging techniques. EXAMPLES

[0129] The following examples are presented for the purpose of illustration only and are not intended to be limiting.

METHODS OF IDENTIFICATION OF BIOMARKERS OF THE PRESENT INVENTION

Materials

[0130] Trifluoroacetic acid (TFA) was purchased from Pierce Biotechnology (Rockford, IL, USA), high purity grade reagents, acetonitrile (ACN), HPLC water, 3, 5- dimethoxy-4-hydroxycinnamic acid, iodoacetamide, 2-methylbutane, methanol, acetone and ethanol from Sigma (St. Louis, MO, USA), a-cyano-4-hydroxycinnamic acid (HCCA) from Bruker Daltonics (Billerica, MA, USA)., 16% Novex^® Tricine Gels from Invitrogen (Carlsbad, CA, USA). Syncollin antibody (ab72208) and pancreatic triacylglycerol lipase antibody (ab49288) from Abeam (Cambridge MA, USA), and colloidal Coomassie blue from Bio-Rad (Hercules, CA, USA).

Biospecimens

[0131] Healthy human pancreatic tissues were obtained from the nPOD

Biorepository located in Gainesville, Florida, USA. nPOD defines patients as being type 1 diabetes if they had a clinical history of diabetes and whose medical history, donor questionnaire, and additional laboratory data (Aab, C-peptide) confirmed the

designation. Autoantibody positive (Aab+) donors, in the nPOD bio repository, are those that have no history or next of kin reported diabetes, but whose serum or plasma tested positive for at least one diabetes-related autoantibody, including islet cell antigen 512 (ICA512), glutamic acid decarboxylase 65 (GADA65), insulin auto antibodies (mlAA), anti-Protein Tyrosine Phosphatase IAZ (anti-IAZ) and lately, Zinc transporter 8 (ZnT8) antibodies. Finally, non-type 1 diabetic patients are defined as donors that do not have any type of autoantibodies, diabetes (type one or two), and no rare disease or other dramatic co-morbidity. (9).

Tissue Processing for Imaging Mass Spectrometry

[0132] The nPOD pathology Core Facility provided 7 μΜ thick tissue slices mounted on glass slides for hematoxylin and eosin staining. Sequential slices were cut and mounted onto an indium-tin oxide-coated mass spectrometry slide for MALDI-IMS. The tissue sections were washed and fixed to slides. The dehydrated slides were air dried and stored in a desiccator for 1 h before matrix deposition. A fine homogenous layer of sinapinic acid matrix was automatically sprayed and deposited on the dried tissue using the ImagePrep workstation (Bruker Daltonics) and the tissue was subjected to MALDI- IMS to profile the proteins in the type 1 diabetes, Aab+, and Non-type 1 diabetes pancreatic tissue sections, as we have discussed previously (8)

Sample preparation for MALDI-TOF mass spectrometry

[0133] Approximately 5-10 mg of the frozen pancreatic tissue was mixed with 3000 μΐ of 0.1% TFA and incubated for 30 minutes at 4° C. The mixture was transferred to a glass homogenizer and ground to a liquid consistency. A two stage fractionation strategy was used to enrich for proteins with molecular mass range between 3-30 kDa. In a first step, the suspension was filtered through a 30 kDa Amicon ultrafiltration device

(Millipore) by centrifugation at 4,000 x g for 30 minutes at 23° C. In the second step, the filtrate was concentrated by centrifugation using a 3 kDa Amicon ultrafiltration device at 4,000 x g for 30 min. The concentration of the retentate consisting of

polypeptides/proteins with a molecular mass between 3-30 kDa was determined by the Lowry Protein assay and stored at -80° C until ready for use.

[0134] For protein profiling by linear mode MALDI-TOF, 2.7 μg of the samples were mixed in a ratio of 1 :2 with freshly prepared a-cyano-4-hydroxycinnamic acid (50 mg/mL HCCA with 25% TFA in 66% ethanol and 33% acetone) and spotted on a MALDI steel target plate (Bruker MTP 384 ground steel). The mixture was allowed to dry and crystallize before MS analysis on an Ultraflex III MALDI TOF/TOF (Bruker Daltonics, Billerica MA, USA) equipped with a Smart Beam laser operating at 200 Hz. An optimized high mass acquisition method was used and the instrument was calibrated externally using peptide and protein calibration standards II (Bruker Daltonics, Billerica MA, USA) for accuracy. The spectra were acquired in linear mode over a mass range of 2000 to 20,000 m/z in the positive ion mode summing a total of 2000 laser shots for each spectrum with a raster width of 200 μΜ and a laser spot diameter of 100 μΜ. Acquisition was executed automatically as defined in the Auto Execute module in FlexControl™ (Bruker Daltonics, Billerica MA, USA). The resulting spectra were processed and analyzed, as defined in the FlexAnalysis™ (Bruker Daltonics, Billerica MA, USA) method.

SDS-PAGE separation and protein identification by mass spectrometry

[0135] Identical protein concentrations of the fractionated lysates from different disease states were separated using 16% SDS-Tricine gel under reducing conditions. The separated proteins were stained with colloidal Coomassie overnight and destained with deionized water to visualize the polypeptide bands. The gel bands were excised and digested with trypsin, and the extracted peptides were analyzed by ESI-LC MS/MS in a LTQ linear ion trap (Thermo Fisher) mass spectrometer using data dependent acquisition as previously described (8). Briefly, normalized peptide extracts were automatically loaded on a CapTrap column (TR1/25109/32 CI 8; Michrom Bioresources Inc) via an Accela autosampler (Thermo Fisher), followed by chromatographic separation under the following conditions: Solvent A (0.1 % formic acid, 0.005% HFBA) and Solvent B (95% acetonitrile in 0.1% formic acid, 0.005% HFBA). The tryptic digests were eluted at 500 nl/min with PicoFrit columns (75 μΜ inner diameter, 2 μΜ tip opening, New Objective, Woburn, MA) slurry-packed in house with 10 cm of reverse phase 5 μΜ, 100 Angstrom Magic C18 resin (Michrom Bioresources, Auburn, CA). The acquisition cycle consisted of a survey MS scan with a set mass range from 350 m/z to 1800 m/z at the highest resolving power, followed by 5 data-dependent MS/MS scans using collision

dissociation fragmentation (CID) assisted with helium gas. Dynamic exclusion was used with the following parameters: exclusion size 50, repeat count 3, repeat duration 120 s, exclusion time 180 s, exclusion window ± 0.8 Da. Target values were set at 5 x 10⁵ and 10⁴ for the survey and tandem MS scans, respectively, and the maximum ion

accumulation times were set at 200 ms in both cases. Regular scans were used both for the precursor and tandem MS with no averaging. Multiple MS runs were performed with extensive blanks between each sample to avoid carry-over of peptides that could affect protein identification. Peak lists were generated using XCalibur (version 2.1) prior to database searching. [0136] Protein database sequence analysis was performed with MASCOT (version 2.2.03) [www.matrixscience.com] using SwissProt 2010x (SwissProt 57.1) database with a human taxonomy filter enabled that contained 516,603 sequences entries. Database searches were performed with fixed modification as carbamidomethyl (C) and variable modifications as oxidation (M), deamidation (N, D) and phosphorylation (STY). Enzyme specificity was selected to trypsin with 2 missed cleavage sites. The mass tolerance was 0.8 Da for both precursor ions and fragment ions. Threshold score for acceptance of individual spectra was set at 0.05. All the MS/MS spectra were manually inspected to verify the validity of the database search results. To determine the false discovery rates (FDRs), we utilized the decoy version of SwissProt protein database for searches. FDRs on the protein identification were calculated to be 0.25%.

Western blot detection of potential biomarkers

[0137] To verify the identity of proteins identified by mass spectrometry, western blots were performed using the tissue lysate fractions described above. 65 μg protein lysates from each sample category were loaded per lane and separated using a 16% SDS- Tricine gel under reducing conditions. Proteins that showed qualitative and quantitative differences in expression, in MS analyses, between the three groups and those that were expressed in the type 1 diabetes and Aab+ groups, but not in the non-type one diabetes groups, were specifically targeted for orthogonal verification via western blot. Western blot transfer of separated proteins was done using standard procedures and the intensity of the detected bands was quantified using the LI-COR Odyssey 3133 system (Lincoln Nebraska, USA) per the manufacturer's instructions. Three samples from each condition group were analyzed, the mean band intensities were calculated, and the significance of the differences determined using the student's t-test.

Multiple reaction monitoring (MRM) detection of potential biomarkers in pancreatic tissues

[0138] MRM on a triple quadrupole mass spectrometer provides superior rapid, sensitive, and specific identification and quantitation of targeted compounds in highly complex samples. MRM has been utilized as a quantitative and qualitative method for analysis of low molecular weight compounds over the last 3 decades and has now being optimized for protein and peptide analysis (10, 11). The necessary requirements for the technology are the capability to identify the ion of interest (Ql m/z), followed by the ability to segregate and fragment that precursor ion in the collision-induced chamber (in Q2) and lastly, identification of the ion of interest from the fragmented precursor ion (Q3 m/z). Only ions with this exact transition are detected. Nano-LC-MRM/MS analyses using tryptic peptides from lysates from the three disease conditions was performed in a 4000 QTRAP® mass spectrometer (Applied Biosystems, Foster City, CA) attached to a Tempo NanoLC system (Eksigent Technologies, Dublin, CA) as we have previously described (12). Acquisition parameters were optimized for maximum transmission and sensitivity of the MRM transitions for the syncollin and pancreatic triacylglycerol lipase peptides. Two MRM transitions pairs for each peptide were monitored in quadrupoles Ql and Q3, in order the validate biomarker identity. The following MRM transition pairs were used for detection: Pancreatic triacylglycerol lipase peptide sequence

FIWYNNVINPTLPR with optimized transition pairs 873.9699/810.4832 and

873.9699/1137.6375 and for the peptide with sequence NILSQIVDIDGIWEGTR, the optimized transition pairs of 643.67/761.394 and 643.67/1161.553 were used. The following MRM transition peptides pairs were used for the syncollin peptide

GILGDWSNAISALYCR: 869.933/1398.647 and 869.933/825.4287. These peptide fragments were further characterized as described below, but as seen in Figure 2A, many additional fragments of both the syncollin protein and the pancreatic triacylglycerol lipase protein have been identified that may also be useful as biomarkers of the present invention.

RESULTS:

Imaging data revealed differential protein expression between type 1 diabetes, Aab+ and non-type 1 diabetes tissue samples

[0139] To verify that the presence of the 15 kDa peak was unique to type 1 diabetes and Aab+ patient samples, several type 1 diabetes, Aab+ and Non-type 1 diabetes tissues were collected from nPOD. The samples were processed as previously described, and the expression and spatial distribution of proteins were evaluated via MALDI-IMS [8]. Regions of interest were delineated around the islets as previously described [8]. The spectra from each group were averaged, overlaid and compared. The compared spectra revealed that the 15 kDa peak previously observed in the type 1 diabetes samples was present in all three samples; however, it showed a 3 fold increase in expression in the type 1 diabetes compared to the Non-type 1 diabetes (Fig. 1 A). The protein was also highly expressed in the Aab+ tissue samples, but at slightly lower levels than the type 1 diabetes tissue samples. Two proteins with approximate masses of 15 kDa and 25 kDa showed differential expression in type 1 diabetes and Aab+ samples compared to Non-type 1 diabetes samples

[0140] To verify the expression profiles observed in MALDI-IMS experiments in the previous section, protein lysates were made from type 1 diabetes, Aab+ and Non-type 1 diabetes tissue samples and the 3-30 kDa fraction were separated on a 16% Tricine gel. Colloidal Coomassie staining showed that the band at 15 kDa was present at significantly higher concentrations in the type 1 diabetes and Aab+ compared to the Non-type 1 diabetes lysates. Although there was an increase in the concentration of the 15 kDa protein in the Aab+ lysates compared to the Non-type 1 diabetes lysates, the Aab+ samples still had a significantly lower expression of the protein compared to the type 1 diabetes as seen by the reduced intensity of the band (Fig. IB). Further analysis of the separated protein bands showed a differentially expressed protein at 25 kDa. This protein was highly abundant in the type 1 diabetes tissue lysates but with a progressive decrease in expression from the Aab+ to the Non-type 1 diabetes tissue samples. This mass is beyond the optimal range of resolution and detection for the linear mode of the MALDI- TOF instrument (Fig. IB).

Tandem MS/MS identification of pancreatic biomarkers of type 1 diabetes

[0141] To characterize the differentially expressed proteins, the stained bands were excised from the gel in three major sectors: type 1 diabetes bands 1-9 (lane 1), Aab+ bands 10-18 (lane two) and Non-type 1 diabetes bands 19-26 (lane 3) (Fig. IB). Tryptic digestion was performed on all sectors from each lane as previously described. The protein extracts from these sectors were subjected to LC-ESI-MS/MS analysis. The MS data was processed and subjected to Mascot database searches using the parameters described above. A total of 414 proteins with significant mascot scores that fulfilled the criteria were identified.

[0142] The Venn diagram illustrates the similarities and differences between all protein hits among the three sectors (Fig. 1C). The diagram showed that there were 163 proteins in the type 1 diabetes group, 133 in the Aab+ and 118 in the non-type 1 diabetes group. Of these, 53 proteins were unique to type 1 diabetes, 28 were unique to Aab+ and 23 were unique to the non-type 1 diabetes group. There were 31 proteins shared between the type 1 diabetes and Aab+ groups, 16 between the Aab+ and non-type 1 diabetes groups and 21 common to the type 1 diabetes and non-type 1 diabetes groups. A total of 58 proteins are common to all three groups. Since the objective of the study was to identify potential biomarkers of the disease, we focused on characterizing the 31 proteins that are exclusively shared between the type 1 diabetes and Aab+ individuals. These individuals are either established type 1 diabetes or at risk for progression to the disease. Of the 31 shared proteins, two proteins, syncollin and pancreatic triacylglycerol lipase corresponded to the bands of interest at -15 and -25 kDa respectively, (Fig. IB).

[0143] Band numbers 8, 16 and 24 corresponded to the 15 kDa band of interest in the type 1 diabetes, Aab+ and non-TID samples respectively. The Mascot search results identified the 15 kDa protein band as Syncollin (SwissProt Accession # Q0VAF6) with 61% peptide sequence coverage. MS/MS fragmentation of several peptides confirmed the identity of the protein. The MS/MS fragmentation and annotation data for the syncollin peptide GILGDWSNAISALYCR are shown in Figures 2Az and 2B, which illustrate the quality and significance of the mass spectrometry and identification data used. The peptide is identified with a significant ion score of 74 and an expect score of 2.5e-05. The protein was only identified in the type 1 diabetes and Aab+ and not in the Non-type 1 diabetes sample.

[0144] Bands numbered 4, 12 and 20 corresponded to the 25 kDa band of interest in the type 1 diabetes, Aab+ and Non-TID samples respectively. LC-ESI-MS/MS analysis identified the band as Pancreatic Triacylglycerol Lipase (SwissProt Accession # P16233) with 50% peptide sequence coverage. MS/MS fragmentation of several peptides confirmed the identity of the protein. The MS/MS fragmentation and annotation data for pancreatic triacylglycerol lipase peptide TNDVGQKFYLDTGDASNFAR is shown in Figures 2Αζϊ and 2C. The peptide is identified with a significant ion score of 86 and an expect score of 1.9e-06. The protein was only identified in the type 1 diabetes and Aab+ and not in the Non-type 1 diabetes sample.

Validation of 15 kDa protein by western blot

[0145] To further validate the results of the mass spectrometry data and protein identification results, western blotting was performed using an antibody against syncollin. The results confirmed the presence of the -15 kDa band as syncollin (Fig. 3).

MRM analysis and relative quantitation of pancreatic triacylglycerol lipase

[0146] The relative concentrations of syncollin and pancreatic triacylglycerol lipase peptides in different categories of pancreatic tissue sections were quantified in LC- MRM-MS experiments. Each sample was analyzed in 3 technical replicate experiments and 3 samples were used for each medium category. Quantitation was achieved by comparing the area under the curve for peptides in the different extracted ion chromatograms (XIC). Figure 4a shows the LC-MRM-MS/MS results for the pancreatic triacylglycerol lipase peptide. It also shows the total ion current (TIC), the detection of the MRM transition pairs, the XIC and the enhanced product ion scan (EPI) scans for the FIWYNNVTNPTLPR peptide. The concentrations of both proteins are significantly higher in the type 1 diabetes compared to the non-type 1 diabetes samples as shown for pancreatic triacylglycerol lipase in Figure 4b.

[0147] Referring to FIG. 7, the fold change in gene expression of syncollin was consistently greater in NOD (non-obese diabetic mice) versus NOD-Aloxl5 (non-obese diabetic deficient in 12/15 lipoxygenase ) null mice when analyzed at 4, 8, and 12 weeks of age (FIGS. 7 A, C, and B, respectively). Further, fold change in syncollin gene expression was reduced in cytokine-treated pancreatic duct tissue compared to untreated pancreatic duct tissue (control) (FIG. 7D).

[0148] As will be apparent to one of ordinary skill in the art from a reading of this disclosure, aspects of the present disclosure can be embodied in forms other than those specifically disclosed above. The particular embodiments described above are, therefore, to be considered as illustrative and not restrictive. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described herein.

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Claims

CLAIMS:

1. A purified biomarker for type 1 diabetes comprising the amino acid sequence GILGDWSNAISALYCR.

2. A purified biomarker for type 1 diabetes comprising the amino acid sequence TNDVGQKFYLDTGDASNFAR.

3. A purified biomarker for type 1 diabetes comprising the amino acid sequence FIWYNNVINPTLPR.

4. A purified biomarker for type 1 diabetes comprising the amino acid sequence NILSQIVDIDGIWEGTR.

5. A purified biomarker for type 1 diabetes selected from the group consisting of syncollin, pancreatic triacylglycerol lipase, pancreatic amylase, bile salt-activated lipase, fatty acid binding protein, glutathione reductase, pancreatic secretory trypsin inhibitor, phosphoglycerate kinase, profilin-l, quinine oxidoreductase, and superoxide dismutase, or fragments or variants thereof.

6. A method for screening for type 1 diabetes in a subject comprising the steps of:

(a) providing a biological sample from a subject;

(b) detecting at least one biomarker in said sample, said biomarker selected from the group consisting of peptides with the amino acid sequence comprising the syncollin protein, the pancreatic triacylglycerol lipase protein,

GILGDWSNAISALYCR, TNDVGQKFYLDTGDASNFAR,

FIWYNNVINPTLPR, and NILSQIVDIDGIWEGTR;

(c) correlating said detection with a status of type 1 diabetes or no type 1 diabetes.

7. The method of claim 6 wherein said detecting at least one biomarker is performed by mass spectrometry.

8. The method of claim 7 wherein the mass spectrometry is MALDI-IMS.

9. The method of claim 6 wherein said detecting at least one biomarker is performed by immunoassay.

10. The method of claim 9, wherein said immunoassay is an enzyme immunoassay.

11. The method of claim 6, wherein the biological sample is selected from the group consisting of biological fluid and tissue.

12. The method according to claim 11, wherein the biological fluid is whole blood, serum, plasma, or urine.

13. The method according to claim 11, wherein the tissue is a pancreatic tissue

sample.

14. A method of diagnosing type 1 diabetes in a subject, comprising the steps of:

(a) obtaining one or more test samples from a subject;

(b) detecting the differential expression of at least one biomarker in the one or more test samples, wherein the biomarker is selected from: syncollin, pancreatic triacylglycerol lipase, pancreatic amylase, bile salt-activated lipase, fatty acid binding protein, glutathione reductase, pancreatic secretory trypsin inhibitor, phosphoglycerate kinase, profilin-1, quinine oxidoreductase, and superoxide dismutase; and

(c) correlating the detection of differential expression of at least one biomarker with a diagnosis of type 1 diabetes, wherein the correlation takes into account the amount of the at least one biomarker in the one or more test samples compared to a control amount of the at least one biomarker.

15. The method of claim 14 wherein one test sample is selected from the group

consisting of urine, whole blood, serum, plasma, and pancreatic tissue.

16. A kit for diagnosing type 1 diabetes, the kit comprising

(a) a solid support comprising at least one biospecific capture reagent attached thereto, wherein the biospecific capture reagent is capable of capturing a type 1 diabetes biomarker, the type 1 diabetes biomarker comprising the amino acid sequence comprising syncollin, pancreatic triacylglycerol lipase,

GILGDWSNAISALYCR, TNDVGQKFYLDTGDASNFAR,

FIWYNNVINPTLPR, or NILSQIVDIDGIWEGTR, or a combination thereof; and (b) instructions for using the solid support to detect the type 1 diabetes biomarker.

17. A method of treating type 1 diabetes by administering an agent that reduces the expression of one or more biomarkers for type 1 diabetes selected from the group consisting of the amino acid sequence comprising the syncollin protein, the amino acid sequence comprising the pancreatic triacylglycerol lipase protein, the amino acid sequence comprising GILGDWSNAISALYCR, the amino acid sequence comprising TNDVGQKFYLDTGDASNFAR, the amino acid sequence comprising FIWYNNVINPTLPR, and the amino acid sequence comprising NILSQIVDIDGIWEGTR.

18. A method of treating type 1 diabetes by administering an agent that reduces the expression of one or more biomarkers for type 1 diabetes selected from the group consisting of: syncollin, pancreatic triacylglycerol lipase pancreatic amylase, bile salt-activated lipase, fatty acid binding protein, glutathione reductase, pancreatic secretory trypsin inhibitor, phosphoglycerate kinase, profilin-1, quinine oxidoreductase, and superoxide dismutase.

19. A kit comprising :

(a) a solid support comprising at least one biospecific capture reagent attached thereto, wherein the biospecific capture reagent is capable of capturing at least one type 1 diabetes biomarker selected from the group consisting of (i) the amino acid sequence comprising the syncollin protein, (ii) the amino acid sequence comprising the pancreatic triacylglycerol lipase protein, (iii) the amino acid sequence comprising GILGDWSNAISALYCR, (iv) the amino acid sequence comprising TNDVGQKFYLDTGDASNFAR, (v) the amino acid sequence comprising FIWYNNVINPTLPR, and (vi) the amino acid sequence comprising NILSQIVDIDGIWEGTR; and

(b) instructions for using the solid support to detect the at least one type 1 diabetes biomarker.

20. The kit of claim 19, comprising instructions for using the solid support to detect a plurality of said biomarkers.

21. A method for detecting type 1 diabetes in a subject comprising the steps of: (a) providing a biological sample from the subject;

(b) contacting the biological sample with a biospecific capture reagent capable of capturing at least one type 1 diabetes biomarker comprising:

(i) the amino acid sequence comprising pancreatic triacylglycerol lipase

(ii) the amino acid sequence comprising

TNDVGQKFYLDTGDASNFAR, (iii) the amino acid sequence comprising FIWYNNVINPTLPR, and (iv) the amino acid sequence comprising NILSQIVDIDGIWEGTR;

(c) determining the amount of the bound pancreatic triacylglycerol lipase biomarker having a molecular weight of about 25 kDa +/- 2.5 kDa;

(d) correlating the amount of the bound pancreatic triacylglycerol lipase biomarker having a molecular weight of about 25 kDa +/- 2.5 kDa to a status of Type 1 diabetes.

22. The method of claim 21, wherein the screening differentiates between type 1 diabetes versus normal.

23. The method of claim 21, wherein the detecting is part of a diagnosis or prognosis of type 1 diabetes in the subject.

24. The method of claim 21, wherein the biological sample is selected from the group consisting of biological fluid and tissue.

25. The method according to claim 24, wherein the biological fluid is whole blood, serum, plasma, or urine.

26. The method according to claim 24, wherein the tissue is a pancreatic tissue

sample.

27. The method according to claim 21, wherein the biospecific capture reagent is attached to a solid support.

28. The method according to claim 27, wherein the solid support is a mass

spectrometry probe and the biospecific capture reagent comprises an antibody attached to the probe and wherein step (c) comprises detecting the bound biomarker by mass spectrometry.

29. The method according to claim 28, wherein the antibody is selected from the group consisting of polyclonal antibodies, monoclonal antibodies, bispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, and Fv, FAb, and Fab2 fragments thereof.

30. The method according to claim 21, further comprising the step of:

(e) comparing the amount of the pancreatic triacylglycerol lipase biomarker having a molecular weight of about 25 kDa +/- 2.5 kDa in the biological sample with the amount of pancreatic triacylglycerol lipase biomarker having a molecular weight of about 25 kDa +/- 2.5 kDa in a biological sample from one or more subjects free from type 1 diabetes or with a previously determined reference range for a pancreatic triacylglycerol lipase biomarker having a molecular weight of about 25 kDa +/- 2.5 kDa in subjects free from type 1 diabetes.

31. The method according to claim 21 , wherein step (c) comprises high-performance liquid chromatography.

32. The method according to claim 21, wherein step (c) comprises polyacrylamide gel electrophoresis (PAGE) and Western blotting.

33. The method according to claim 32, wherein PAGE is 2-dimensional PAGE.

34. A method for detecting type 1 diabetes in a subject comprising the steps of:

(a) providing a biological sample from the subject;

(b) contacting the biological sample with a biospecific capture reagent capable of capturing at least one type I diabetes biomarker comprising:

(i) the amino acid sequence of syncollin, (ii) the amino acid sequence GILGDWSNAISALYCR; and

(c) determining the amount of the bound syncollin biomarker having a molecular weight of about 15 kDa +/- 1.5 kDa; and

(d) correlating the amount of the bound syncollin biomarker having a molecular weight of about 15 kDa +/- 1.5 kDa to a status of Type 1 diabetes.

35. The method of claim 34, wherein the screening differentiates between type 1 diabetes versus normal.

36. The method of claim 34, wherein the detecting is part of a diagnosis or prognosis of type 1 diabetes in the subject.

37. The method of claim 34, wherein the biological sample is selected from the group consisting of biological fluid and tissue.

38. The method according to claim 37, wherein the biological fluid is whole blood, serum, plasma, or urine.

39. The method according to claim 37, wherein the tissue is a pancreatic tissue

sample.

40. The method according to claim 34, wherein the biospecific capture reagent is attached to a solid support.

41. The method according to claim 40, wherein the solid support is a mass

spectrometry probe and the biospecific capture reagent comprises an antibody attached to the probe and wherein step (c) comprises detecting the bound biomarker by mass spectrometry.

42. The method according to claim 40, wherein the antibody is selected from the group consisting of polyclonal antibodies, monoclonal antibodies, bispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, and Fv, FAb, and Fab2 fragments thereof.

43. The method according to claim 34, further comprising the step of:

(e) comparing the amount of the syncollin biomarker having a molecular weight of about 15 kDa +/- 1.5 kDa in the biological sample with the amount of syncollin biomarker having a molecular weight of about 15 kDa +/- 1.5 kDa in a biological sample from one or more subjects free from typel diabetes or with a previously determined reference range for a syncollin biomarker having a molecular weight of about 15 kDa +/- 1.5 kDa in subjects free from type 1 diabetes.

44. The method according to claim 34, wherein step (c) comprises high-performance liquid chromatography.

45. The method according to claim 34, wherein step (c) comprises polyacrylamide gel electrophoresis (PAGE) and Western blotting.

46. The method according to claim 45, wherein PAGE is 2-dimensional PAGE.

47. A method of imaging beta cell tissue comprising the steps of:

(a) administering to a subject a type 1 diabetes biomarker selected from the group consisting of syncollin, pancreatic triacylglycerol lipase pancreatic amylase, bile salt-activated lipase, fatty acid binding protein, glutathione reductase, pancreatic secretory trypsin inhibitor, phosphoglycerate kinase, profilin-1, quinine oxidoreductase, superoxide dismutase, the amino acid sequence comprising GILGDWSNAISALYCR, the amino acid sequence comprising

TNDVGQKFYLDTGDASNFAR, the amino acid sequence comprising

FIWYNNVINPTLPR, and the amino acid sequence comprising

NILSQIVDIDGIWEGTR; and

(b) using an imaging method to determine the amount of beta cell tissue.

48. The method of claim 47, wherein the imaging method is selected from the group consisting of (MRI); proton emission tomography (PET), ultrasonography, bio- luminescence, fluorescence, and nuclear medicine techniques.

49. A method of monitoring the effect of an anti-diabetes drug or therapy on a

subject comprising:

(a) providing a biological sample from the subject;

(b) contacting the biological sample with a biospecific capture reagent capable of capturing at least one type I diabetes biomarker selected from the group consisting of syncollin, pancreatic triacylglycerol lipase pancreatic amylase, bile salt-activated lipase, fatty acid binding protein, glutathione reductase, pancreatic secretory trypsin inhibitor, phosphoglycerate kinase, profilin-1, quinine oxidoreductase, superoxide dismutase, the amino acid sequence comprising GILGDWSNAISALYCR, the amino acid sequence comprising TNDVGQKFYLDTGDASNFAR, the amino acid sequence comprising

FIWYNNVINPTLPR, and the amino acid sequence comprising

NILSQIVDIDGIWEGTR;

(c) measuring the amount of the at least one type-I biomarker;

(d) providing the subject with an anti-diabetes drug or therapy;

(e) measuring the amount of the at least type-I biomarker using steps (a) and (b); and

(f) corrlating the two measurements with a diagnosis that the diabetes is regressing or progressing.