CN108072761B - Application of MASP-1 in distinguishing initial outbreak from classical type 1diabetes - Google Patents

Abstract

The present invention provides the application of MASP-1 in distinguishing fulminant and classic type 1 diabetes. Specifically, the present invention relates to the use of mannan-associated serine protease-1 (MASP-1) as a disease molecular classification index in the diagnosis of new onset fulminant type 1 diabetes (initial onset FT1DM), differentiation of onset FT1DM and classic type 1 diabetes (T1DM), and also relates to a method for determining the level of serum MASP-1 protein.

Description

Application of MASP-1 in distinguishing fulminant and classic type 1 diabetes

technical field

The present invention belongs to the fields of biotechnology and medicine. In particular, the present invention relates to the use of MASP-1 in differentiating new onset fulminant type 1 diabetes (FT1DM) from other types of diabetes, especially classic type 1 diabetes.

Background technique

1. Diabetes

Diabetes mellitus is a series of clinical syndromes caused by absolute or relative insufficiency of insulin secretion in the body. The Diabetes Atlas published by the International Diabetes Federation (IDF) is an international authoritative reference book and an important data source on the global prevalence of diabetes and related information. According to the latest data from IDF, in 2013, there were about 387 million adults with diabetes in the world, accounting for 8.3% of the world's population. Among them, 98.4 million Chinese patients with diabetes ranked first in the world, accounting for about a quarter of the total number of people with diabetes in the world. By 2035, if these trends continue, there will be approximately 592 million people worldwide. Compared with developed countries, China still needs a certain amount of time to achieve an ideal diagnosis and treatment environment, and some clinical complications brought about by diabetes are becoming more and more obvious, bringing huge physical, mental and economic consequences to patients and their families. burden.

According to the WHO etiological classification system of diabetes, diabetes can be divided into four categories, namely type 1 diabetes, type 2 diabetes, gestational diabetes and secondary diabetes. Among them, type 1 diabetes mellitus (Type 1 Diabetes Mellitus, T1DM), also known as insulin-dependent diabetes mellitus, is diabetes caused by the absolute deficiency of insulin caused by the damage of autologous pancreatic islet B cells.

In 1999, WHO divided type 1 diabetes into classic type 1A diabetes (Autoimmune type 1 diabetes, type 1A diabetes) and idiopathic type 1 diabetes (Idiopathic type 1 diabetes, type 1B diabetes). In patients with type 1A diabetes, islet-related autoantibodies such as islet cell antibody (ICA), glutamic acid decarboxylase antibody (GADAb), and islet autoantibodies (IA-2/IA-2βAb) can As an immune marker, those who were negative for autoantibodies were classified as type 1B diabetes.

2. Fulminant type 1 diabetes

In 2000, Japanese scholar Imagawa et al proposed Fulminant Type 1 Diabetes Mellitus (FT1DM). This type of diabetes is a new subtype of type 1 diabetes, which is characterized by rapid onset, increased pancreatic enzymes, and severe destruction of beta cells in a short period of time, leading to hyperglycemia and ketoacidosis. The initial onset is often accompanied by fever, cough or Abdominal pain, diarrhea and other respiratory and digestive system symptoms, combined with electrolyte imbalance, liver and kidney insufficiency, rhabdomyolysis, etc., if not treated in time, the mortality rate is very high.

The disease was classified as idiopathic type 1B diabetes mellitus according to the classification of diabetes because the test for islet autoantibodies was negative and the pancreas pathology showed no symptoms of insulitis such as islet exudation or edema. FT1DM patients are more common in adults, and only 8.7% of adolescent patients younger than 20 years old, and the incidence rate has no gender and seasonal differences. In addition, the incidence of FT1DM is high in East Asia, and has been reported in Japan, South Korea, China and the Philippines. According to a report by Imagawa in Japan in 2003, among T1DM (type 1 diabetes) patients with onset of ketosis or ketoacidosis, FT1DM is as high as 15%-20%. Among Korean T1DM patients, FT1DM accounts for approximately 7.1% and 30.4% of adult-onset diabetes patients. There are not many reports about FT1DM in China.

Compared with ordinary T1DM, FT1DM has the following notable characteristics: (1) the course of the disease is very short, and it often develops into ketoacidosis within a week; (2) the onset is accompanied by severe metabolic disorders, such as disturbance of consciousness, ketoacidosis Poisoning and electrolyte imbalance; (3) almost complete and irreversible damage to islet function during the onset; (4) some patients may be combined with multiple organ dysfunction, and in severe cases, rhabdomyolysis, acute renal failure and even cardiac arrest may occur. Without timely treatment, FT1DM has a high mortality rate.

3. Mannose-Associated Serine Protease-1 (MASP-1)

Mannose-Associated Serine Protease-1 (MASP-1), also known as Mannan-binding lectin serineprotease-1 (Mannan-binding lectin serineprotease 1) Encoded serine protease that functions as a lectin pathway component of the complement system involved in innate and adaptive immune responses.

MASP-1 is synthesized as a zymogen that is activated when it complexes with the pathogen recognition molecules of the lectin pathway (mannan-binding lectin and collagen lectin). This protease is not directly involved in complement activation, but may amplify complement activity by cleaving complement C2 or by activating other complement serine proteases such as MASP-2.

MASP-1 also cleaves fibrinogen and factor eight, which may play a role in coagulation. Splice variants of this gene lacking the serine protease domain have been reported to act as inhibitors of the complement pathway.

There are no high-sensitivity, high-specificity identification indicators and effective methods for distinguishing newly onset fulminant type 1 diabetes mellitus from other diabetes mellitus (especially non-fulminant type 1 diabetes mellitus) in the art. For more symptomatic prevention and treatment of diabetes, there is an urgent need in the art to develop biological and clinical indicators that can classify type 1 diabetes more precisely.

SUMMARY OF THE INVENTION

The present invention provides an application of using MASP-1 as a molecular marker to diagnose newly onset fulminant type 1 diabetes mellitus and to distinguish the onset of fulminant type 1 diabetes mellitus from other diabetes mellitus. The present invention also provides a method for detecting the level of mannan-associated serine protease-1 (MASP-1) protein in a sample, as well as a method for testing the therapeutic effect of the drug on fulminant type 1 diabetes mellitus and/or screening candidate therapeutic drugs. method

In some aspects of the invention, there is provided a substance for detecting mannan-associated serine protease-1 (MASP-1) levels in a sample of a subject in the manufacture of a product for diagnosing onset fulminant type 1 diabetes (onset FT1DM) Applications.

In other aspects of the invention, there is also provided a method of diagnosing new onset fulminant type 1 diabetes mellitus (onset FT1DM), the method comprising the step of detecting the level of MASP-1 in a sample from a subject.

In some embodiments of the invention, the method comprises: (a) detecting the level of MASP-1 in a sample of the subject; (b) comparing the level of MASP-1 detected in (a) with a normal control value; If the comparison results show that the MASP-1 level in the subject's sample is higher than that of the normal control, it is suggested that the subject suffers from the onset of fulminant type 1 diabetes mellitus.

In some embodiments of the invention, the diagnosing further comprises combining the MASP-1 level-based diagnosis of the invention with other methods of diagnosing diabetes.

In other aspects of the present invention, there is provided the use of a substance for detecting the level of mannan-associated serine protease-1 (MASP-1) in a sample of a subject in the preparation of a product for diabetes typing, the diabetes typing distinguishing New onset fulminant type 1 diabetes (initial onset FT1DM) and other types of diabetes, wherein the other types of diabetes include: non-initial onset fulminant type 1 diabetes mellitus, non-fulminant type 1 diabetes mellitus, and type 2 diabetes mellitus.

In other aspects of the present invention, there is also provided a diabetes classification method, the diabetes classification distinguishes between the onset of fulminant type 1 diabetes (initial FT1DM) and other types of diabetes, wherein the other types of diabetes Diabetes includes: non-initial fulminant type 1 diabetes mellitus, non-fulminant type 1 diabetes mellitus, type 2 diabetes mellitus, and the method includes the step of detecting the level of MASP-1 in a sample of the subject.

In some embodiments of the invention, the method comprises: (a) detecting the level of MASP-1 in a sample of the subject; (b) comparing the level of MASP-1 detected in (a) with a normal control value; If the comparison results show that the MASP-1 level in the subject's sample is higher than that of the normal control, it is suggested that the subject suffers from the onset of fulminant type 1 diabetes mellitus.

In some embodiments of the present invention, the typing further comprises combining the MASP-1 level-based typing of the present invention with other diabetes typing methods.

In other aspects of the present invention, there is provided a substance for detecting the level of mannan-associated serine protease-1 (MASP-1) in a sample of a subject in preparation for determining the therapeutic effect of a drug on newly fulminant type 1 diabetes mellitus and/or Or the application in the product screening of candidate drugs for the treatment of fulminant type 1 diabetes.

In some embodiments of the present invention, there is provided a method for determining the therapeutic effect of a drug on fulminant type 1 diabetes mellitus and/or for screening candidate drugs for treating fulminant type 1 diabetes mellitus, the method comprising: determining and comparing The subject's MASP-1 levels before and after administration of the drug or drug candidate, if the MASP-1 level is decreased, the drug or drug candidate has a therapeutic effect on fulminant type 1 diabetes.

As described in various aspects herein, wherein the substance is a substance for detecting MASP-1 DNA level, mRNA level and/or protein level, such as an antibody against MASP-1, a specific probe against MASP-1, Gene chip or protein chip for MASP-1.

In some embodiments of the present invention, MASP-1 has the amino acid sequence shown in SEQ ID NO: 1, or its homologous sequence, such as the MASP-1 polypeptide sequence corresponding to the target species; the MASP-1 gene has SEQ ID NO: 1 The nucleotide sequence shown in ID NO: 1, 21, 22 or 23, or its homologous sequence, for example, the MASP-1 gene sequence corresponding to the target species.

As described in various aspects herein, the detection is performed by detecting full-length MASP-1, a MASP-1 signature peptide and/or its coding sequence, preferably the MASP-1 signature peptide is selected from SEQ ID NO: 2- one or more of 20.

As described in various aspects herein, the substance is a substance for detecting MASP-1 by one or more of the following methods: multidimensional liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis, immunohistochemistry method, chemiluminescence method, radioisotope method, fluorescence luminescence method (such as immunofluorescence method), enzyme labeling method, colloidal gold method, real-time quantitative reverse transcription PCR, biochip detection method, Southern blotting method, Northern blotting method in situ hybridization method, and Western blotting.

In some embodiments, the substance bears a detectable label, such as a radioisotope, fluorophore, chemiluminescent moiety, enzyme, enzyme substrate, enzyme cofactor, enzyme inhibitor, dye, metal ion, ligand (eg, , biotin or hapten), or any combination thereof.

As described in various aspects herein, the sample is obtained from the subject, eg, from a human or non-human mammal, such as a primate, rodent, livestock, pet, etc., preferably a human, rat, mouse , dog, horse, cow, rabbit or monkey; and/or, the sample is selected from: fresh tissue or cell samples, formalin-fixed samples, paraffin-embedded samples, blood (such as whole blood, serum, plasma), Body fluids (such as lymph fluid, cerebrospinal fluid, tissue fluid), ascites.

As described in various aspects herein, the presence of MASP-1 in the sample above a control level indicates that the subject is suffering from fulminant type 1 diabetes, eg, the control level is selected from the group consisting of MASP when the subject is healthy -1 level, a statistically determined healthy population standard level, or a normalized level.

As described in various aspects herein, the product is selected from the group consisting of: a test kit, a test strip, a test card, a test pen, a test instrument, or any combination thereof; and/or, the product further comprises a product selected from the group consisting of One or more substances: containers, buffers, auxiliaries, solvents, positive controls, negative controls, instructions for use, other substances used for diabetes diagnosis and/or typing.

In other aspects of this paper, there is also provided a method for detecting the level of mannan-associated serine protease-1 (MASP-1) in a sample, comprising: detecting one selected from the group consisting of SEQ ID NOs: 2-20 in the sample or levels of multiple polypeptides or their coding sequences.

In some embodiments, the detection is by multidimensional liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis, immunohistochemistry, chemiluminescence, radioisotope, fluoroluminescence (eg, immunofluorescence), enzyme labeling method, colloidal gold method, real-time quantitative reverse transcription PCR, biochip detection method, Southern blotting, Northern blotting in situ hybridization, Western blotting and other methods.

Those skilled in the art can arbitrarily combine the foregoing technical solutions and technical features without departing from the inventive concept and protection scope of the present invention. Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

Description of drawings

The present invention will be further described below with reference to the accompanying drawings, wherein these representations are only for illustrating the embodiments of the present invention, rather than for limiting the scope of the present invention.

Figure 1: The quantification of MASP-1 in different groups, where the ordinate is the ratio of the normalized identification intensity of mass spectrometry, and their calculation comes from the expression of each peptide segment in Table 2, which is used to characterize the expression of the protein as a whole quantity information. In the figure, "FC" means fold change, "*" means p≤0.05, and "***" means p≤0.005;

Figure 2: The ROC curve obtained when MASP-1 was used as a diagnostic indicator in the early stage of fulminant type 1 diabetes.

Detailed ways

Through long-term and in-depth research, the inventors found that the level of MASP-1 in patients with initial fulminant type 1 diabetes was significantly higher than that in normal people, fulminant diabetes patients after treatment, patients with type 1 diabetes and patients with type 2 diabetes. This suggests that MASP-1 can be used as a molecular marker for diagnosing the onset of fulminant type 1 diabetes and for the classification of diabetes (especially the subdivision of type 1 diabetes).

In addition, the inventors further found in the methodological study that using MASP-1 as a molecular marker for diagnosing initial onset fulminant type 1 diabetes and diabetes classification (especially type 1 diabetes subdivision) can be used in the diagnosis and/or classification of diabetes. High sensitivity, specificity and accuracy can be obtained in the type, and it has high clinical application value. In addition, based on related research, the MASP-1 of the present invention can be further used as a molecular marker for determining the therapeutic effect of a drug on fulminant type 1 diabetes mellitus and/or for screening candidate drugs for treating fulminant type 1 diabetes mellitus.

In addition, the present inventors have also discovered the characteristic peptides of MASP-1, which provide an advantageous tool for the characterization and quantification of MASP-1 and the methods and applications of the present invention.

As used herein, the term "primitive" refers to a disease that occurs for the first time and has not been treated. As used herein, the term "initial fulminant type 1 diabetes" refers to untreated fulminant type 1 diabetes. As used herein, the term "non-initial fulminant type 1 diabetes" or "other types of diabetes" corresponding to onset fulminant type 1 diabetes includes: non-initial fulminant type 1 diabetes, non-fulminant type 1 diabetes , Type 2 diabetes, etc.

As used herein, the terms "mannan-associated serine protease-1" or "MASP-1" are used interchangeably and have the meanings known in the art. For example, the MASP-1 protein can be the sequence set forth in Gene ID: 5648, or can be a homologous sequence thereof, or an isoform sequence thereof, such as the sequence set forth in SEQ ID NO: 1, 21, 22, or 23, . MASP-1 may be the full-length MASP-1 protein or its coding sequence, or may be a characteristic fragment of MASP-1 (eg, see Table 2) or its coding sequence.

As used herein, the terms "detection substance" or "substance that detects MASP-1" are used interchangeably, and both refer to reagents and/or agents that are specific for MASP-1 and that can be used to directly or indirectly detect levels of MASP-1 equipment.

Since the sequences of MASP-1 molecules are known in the art, those of ordinary skill in the art can prepare reagents specific for MASP-1 molecules based on conventional means or obtain commercially available reagents. For example, the detection reagents that can be used in the present invention include, but are not limited to: antibodies and probes with detection specificity for MASP-1 molecules.

In order to facilitate detection, the detection reagents of the present invention may also carry detectable labels, including but not limited to: radioisotopes, fluorophores, chemiluminescent moieties, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors , dyes, metal ions, ligands (eg, biotin or hapten), etc.

As used herein, the terms "product" or "product of the present invention" are used interchangeably and refer to a product that contains a substance that detects MASP-1 in a biological sample that can be used in the applications of the present invention. The products of the present invention include but are not limited to: test kits, test strips, test cards, test pens, test instruments or any combination thereof. The product of the present invention further comprises one or more substances selected from the group consisting of containers, buffers, auxiliaries, solvents, positive controls, negative controls, instructions for use, and others for the diagnosis and/or typing of diabetes substance.

As used herein, the term "normal control" refers to MASP-1 levels used as a reference, including, but not limited to, MASP-1 levels measured in pre-diseased samples of the same subject, statistically determined population standards level, or normalized level.

As used herein, "comprising", "having" or "including" includes "comprising", "consisting essentially of", "consisting essentially of", and "consisting of"; "consisting essentially of... Consists of", "consisting essentially of" and "consisting of" are subordinate concepts of "contains", "has" or "includes".

All numerical ranges provided herein are intended to expressly include all numerical values falling between the endpoints of the range and numerical ranges therebetween. The features mentioned in the present invention or the features mentioned in the embodiments may be combined. All features disclosed in this specification may be used in combination with any form of composition, and each feature disclosed in the specification may be replaced by any alternative feature serving the same, equivalent or similar purpose. Therefore, unless otherwise stated, the disclosed features are only general examples of equivalent or similar features.

Example

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. Those skilled in the art can make appropriate modifications and changes to the present invention, and these modifications and changes are all within the scope of the present invention.

The experimental method of unreceipted specific conditions in the following examples, can adopt the conventional method in this area, for example with reference to " molecular cloning experiment guide " (Third Edition, New York, Cold Spring Harbor Laboratory Press, New York: Cold Spring Harbor Laboratory Press, 1989) or as suggested by the supplier. DNA sequencing methods are routine methods in the art, and tests can also be provided by commercial companies.

Percentages and parts are by weight unless otherwise indicated. Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be used in the methods of the present invention. Methods and materials for preferred embodiments described herein are provided for illustrative purposes only.

Reagents and experimental procedures

(1) Chemical reagents

All buffers were prepared in Milli-Q water (Millipore, Bedford, MA, USA). Dithiothreitol (DTT), Iodoacetamide (IAA), Urea (Urea), Sodium Dodecyl Sulfonate (SDS), Tris(hydroxymethyl)aminoethane-hydrochloric acid (Tris-HCl), etc. Purchased from Bio-Rad Company (Hercules, CA, USA); Formic acid (Formic acid, FA), ammonium bicarbonate (NH4HCO3), etc. were purchased from Sigma Company (St.Lous, MO, USA); HPLC grade acetonitrile (ACN) and Methanol (Methanol) was purchased from Fisher Company (Fair Lawn, NJ, USA); Trypsin (Trypsin) was purchased from Promega Company (Madison, WI, USA); TMTMass Tagging Kits (90066) were purchased from Thermo Scientific Company (Waltham, MA, USA).

(2) Collection and preparation of human serum samples

The present invention involves a total of 60 human serum samples: fulminant type 1 diabetes patients (5 cases, clinically diagnosed as FT1DM and untreated patients) and 1 year of treatment (5 cases, conventional treatment), normal blood sugar (15 cases), type 1 diabetes (T1DM, 20 cases, untreated), type 2 diabetes (T2DM, 15 cases, untreated).

All 60 samples were from the Sixth People's Hospital Affiliated to Shanghai Jiaotong University. The acquisition of human blood samples was carried out strictly in accordance with the guidelines of Chinese law and ethics committee, and each patient signed an informed consent.

(3) Removal of high-abundance proteins

Agilent's MARS (multiple affinity removal system 5188-6411) was used to remove 7 high-abundance components of human serum proteins (ie, albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin, and fibrinogen). .

The specific operation is as follows: take 28 μL of serum and add 400 μL of A solution, use a 0.22 μm ultrafiltration tube, 4° C., 1000 g, and centrifuge for 15 min. After equilibrating the MARS column with 4 mL A solution, take 200 μL of the filtered filtrate onto the column, centrifuge at 100 g at 4°C for 1 min. Add 300 μL of solution A, centrifuge for 1 min at 4°C, 100 g, and transfer all the filtrate to a new centrifuge tube. The high-abundance protein fraction bound to the column was slowly eluted with 2 mL of B solution. After equilibrating the column with 4 mL of A solution, repeat the above operation once for the remaining 200 μL of serum samples, combine the filtrate into the centrifuge tube in the previous step, freeze-dry, and store at -80°C.

(4) Fluorescence quantification of protein concentration

60 serum samples after removal of high-abundance proteins and samples formed by mixing multiple samples (called mix, obtained by mixing all 60 serum samples after removal of high-abundance proteins) were lyophilized and lysed with 300 μL of 2D solution (8 Murea, 40 mM Tris, 65 mM DTT), and then quantified by fluorescence. The specific operations are: tryptophan fluorescence method is used for protein quantification. 20 μL of protein solution was added to 1 mL of urea solution (8M urea, 20 mM Tris-HCl, pH 7.6). The excitation fluorescence wavelength was set to 295 nm, and the absorption measurement fluorescence wavelength was set to 350 nm. The standard tryptophan fold dilution was used to draw a standard curve and calculate the protein concentration in the sample. The average tryptophan content in the samples was calculated as 1.3%.

(5) Enzymatic hydrolysis in solution

The in-solution enzymatic hydrolysis step is based on FASP enzymatic hydrolysis. According to the results of fluorescence quantification, 14 μg of the sample obtained in step (4) was added to 50 μL of 8M UA buffer (8M urea, 0.1M Tris-HCl, pH 8.5), respectively placed on 10k ultrafiltration tubes (OMEGA filter, OD010C35), and then 2 μL of 1M DTT was added and placed in a 37°C incubator for 1.5h. After that, 10 μL of 1M IAA was added to the system, and the reaction was carried out in the dark at room temperature for 30 min.

After the above operations, the protein is completely denatured, the disulfide bonds are opened, and the sulfhydryl groups are blocked. After that, 300 μL of 0.2M TEAB was added to the system, and centrifuged at 20° C. and 13000 g for 15 min. After that, the filtrate was discarded, and the membrane was washed three times with 200 μL of 0.2M TEAB buffer, and the ultrafiltration conditions were 4° C. and centrifugation at 13,000 g for 15 min. Finally, 100 μL of 0.2M TEAB containing trypsin was added to the ultrafiltration tube according to the mass ratio (trypsin:protein=1:25), and placed in a shaker at 37°C for enzymatic hydrolysis for 16 hours. Then add trypsin to the ultrafiltration tube according to enzyme:protein=1:25 and continue enzymolysis for 4h. The filtrate was collected by centrifugation at 13,000 g of the peptide mixture after enzymatic hydrolysis, and freeze-dried at -80°C for use in subsequent experiments.

(6) TMT isotope labeling in vitro

TMT Mass Tagging Kits (Thermo Scientific, 90066) has a total of six channels (respectively 126, 127, 128, 129, 130, 131), and each channel marks 1 sample, of which 126 channels are used to label mix internal reference samples, and a total of 12 groups, marked in the following order in Table 1.

TMT labeling step: firstly dissolve the enzymatically hydrolyzed peptide with 14 μL 0.2M TEAB buffer, and 43 μL ACN to dissolve the labeling reagent; then add the labeling reagent to the peptide, mix well and place at room temperature for 1.5 h for labeling; finally add 8 μL 5 The reaction was terminated by % hydroxylamine for 15 min, the 6 channel peptides in each group were mixed, and finally the combined 14-tube samples were added to ddH ₂ O and lyophilized three times, and stored at -80°C.

Table 1. Sample Labeling Information

Group No 126 127 128 129 130 131 G1 mix FT1DMa1 FT1DMa2 FT1DMa3 FT1DMa4 FT1DMa5 G2 mix FT1DMc1 FT1DMc2 FT1DMc3 FT1DMc4 FT1DMc5 G3 mix N4 N5 N17 N23 N22 G4 mix N2 N6 N7 N8 N9 G5 mix N10 N11 N12 N13 N15 G7 mix T1-7 T1-10 T1-18 T1-20 T1-23 G8 mix T1-9 T1-11 T1-21 T1-26 T1-27 G9 mix T1-1 T1-2 T1-4 T1-5 T1-6 G10 mix T1-8 T1-13 T1-16 T1-24 T1-25 G11 mix T2-1 T2-4 T2-13 T2-24 T2-30 G13 mix T2-6 T2-8 T2-14 T2-15 T2-17 G14 mix T2-19 T2-21 T2-23 T2-25 T2-27

Among them, FT1DMa and FT1DMc represent the initial onset of fulminant type 1 diabetes and the corresponding treatment for 1 year, respectively, N represents normal blood sugar, T1 represents type 1 diabetes, T2 represents type 2 diabetes, and the numbers after the above letters indicate different The serial number of the sample, that is, different individuals of the same disease type; mix is a mixed sample, which is used as an internal reference

(7) Peptide segment desalting

The labeled and lyophilized samples obtained in step (6) were fully dissolved in 900 μL of 0.2% TFA aqueous solution, and 200 μL of each sample was desalted with a stage-tip filled with C18, and the operations were performed in parallel.

First, the stage-tip filled with two layers of C18 was activated with 100 μL methanol, centrifuged at 1500 g for 2 min at room temperature, and the filtrate was removed. Afterwards, the packing was washed with 100 μL of 80% acetonitrile containing 0.2% TFA to maximize the removal of contaminants, and the centrifugation conditions were the same as above. Then, the filler was equilibrated three times with 100 μL of an aqueous solution containing 0.2% TFA, centrifuged at 1500 g for 2 min at room temperature, and the filtrate was removed. Take 200 μL of each sample and bind it to the stage-tip, and centrifuge at low speed 800g for 5 min at room temperature to ensure that the sample is fully bound to the C18 filler. Afterwards, the filler was washed three times with 100 μL of an aqueous solution containing 0.2% TFA, and centrifuged at 2000 g for 2 min at room temperature to remove salts and other hydrophilic components that could not be combined with the C18 filler. Finally, it was eluted with 100 μL of 90% acetonitrile containing 0.2% TFA, and the centrifugation conditions were the same as above, and the filtrate was collected and lyophilized for mass spectrometry analysis.

(8) Multidimensional liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis

LC-MS/MS mass detection was performed on a nanoHPLC-Orbitrap Fusion (Thermo Fisher Scientific, San Jose, CA) system.

The liquid chromatography was an Easy-nLC-1000 system (ThermoFisher Scientific); the reversed-phase column was a needle reversed-phase column (tip column, 75 μm×150 mm, C18, 3 μm); the reversed-phase eluent was a 0.1% formic acid aqueous solution ( A) and 0.1% formic acid in acetonitrile (B). The sample was loaded onto the reversed-phase column and eluted through a reversed-phase elution system with a flow rate of 0.3 μL/min. The effective elution gradient was: 5min, 4%B; 150min, 26%B, 155min, 40%B; 160min , 90%B, mass spectrometry analysis time was 180min.

The peptides eluted from the reversed-phase column enter the quadrupole-electrostatic field orbitrap-linear ion trap triple mass spectrometer Orbitrap Fusion for analysis, using the data-dependent data acquisition mode to maximize the acquisition rate ( top speed) for secondary mass spectrum acquisition. The dynamic exclusion setting is: if a precursor ion is identified once, the precursor ion of this mass will not be detected in the next 2 minutes. The precursor ion fragmentation mode adopts the HCD mode, and the resolutions of the primary and secondary spectra are set to 60,000 and 15,000 (at 200 m/z), respectively.

(9) Database search

The original files collected by mass spectrometry were analyzed with Maxquant1.5.2.8 software, and the database used was Uniprot homo sapiens database. The library search parameters are set as follows: the mass deviation of the fragment ion is within 20ppm, the mass deviation of the precursor ion is within 6ppm, the protease is set to trypsin, the maximum allowable deletion site is 2, and the fixed modification is set to cysteine carbamidomethylation of amino acids (+57Da), variable modification set to oxidation of methionine (oxidation, +16Da) and TMT-6plex labeling at the free amino terminus of lysine residues were all set to For variable modifications, the false discovery rate (FDR) for proteins and peptides was set to 0.01.

(10) Statistical and bioinformatics analysis

All sample data were median-corrected and then divided by the internal reference sample for logarithmic processing. Pairwise comparisons between groups were performed using the rank sum test, corrected by FDR. All data analysis and statistical testing were done using the R package or Excel.

Example 1. Differential protein analysis and marker finding

Differential protein analysis is based on the statistical numerical P value (P value), which is the probability of a sample observation or more extreme results obtained when the null hypothesis is true. If the P value is small, it means that the probability of the occurrence of the null hypothesis is very small, and if it occurs, according to the principle of small probability, we have a reason to reject the null hypothesis. The smaller the P value, the stronger the reason for us to reject the null hypothesis. In conclusion, the smaller the P value, the more significant the result.

The diagnostic value of markers can be judged by the area under the curve of the ROC curve (receiver operating characteristic curve). The relationship between sensitivity and specificity, it calculates a series of sensitivity and specificity by setting multiple different critical values for continuous variables, and then takes the sensitivity as the ordinate and (1-specificity) as the The abscissa is drawn as a curve, and the larger the area under the curve, the higher the diagnostic accuracy. It is generally believed that the diagnostic value is higher when the area under the curve is higher than 0.9.

Pairwise comparison of patients with fulminant diabetes mellitus (before treatment), after treatment of fulminant diabetes mellitus, normoglycemia group, type 2 diabetes mellitus group, and type 1 diabetes mellitus group showed that MASP-1 was significantly higher in serum of fulminant diabetes mellitus patients. The expression level in the medium was significantly higher than that after treatment, normal control group, type 1 diabetes group and type 2 diabetes group.

Compared with the initial onset of fulminant diabetes (FT1DMa), the abundance of MASP-1 decreased by 2.14 times after treatment (FT1DMc), and the paired t-test showed that its P value was 0.0015. Compared with the normal control (N), the abundance of MASP-1 increased by 2.66 times in the initial onset of fulminant diabetes mellitus (FT1DMa), and the BH-corrected rank sum test p value was less than 0.05. MASP-1 was used as the test Receiver operating characteristic curve (ROC curve), the area under the curve is 1.

Compared with the normal control group, the fold change of the abundance of fulminant diabetes patients after treatment (FT1DMc), type 1 diabetes mellitus (T1DM), and type 2 diabetes mellitus (T2DM) were all below 1.3. The test p-values were all greater than 0.05. In addition, patients with fulminant diabetes mellitus after treatment (FT1DMc), type 1 diabetes mellitus (T1DM), type 2 diabetes mellitus (T2DM) were compared with the normal control group using MASP-1 to make receiver operating characteristic curves (ROC curve), under the curve The areas are 0.747, 0.523 and 0.662, respectively (see Figure 2).

It can be seen from the above results that at the early stage of the onset of fulminant type 1 diabetes, the abundance of serum MASP-1 was significantly increased compared with the normal controls, while the abundance of serum MASP-1 in patients with type 1 diabetes and type 2 diabetes was higher than that in the control group. There was no significant change in the normal control,

Therefore, serum MASP-1 can be used as a molecular diagnostic marker for distinguishing the initial stage of fulminant type 1 diabetes from other type 1 diabetes.

Example 2. Identification of MASP-1 characteristic peptides

Using mass spectrometry, we identified the following characteristic peptides belonging to MASP-1 in serum for the first time. They can be used to characterize serum expression levels of the protein MASP-1:

Table 2. List of MASP-1 signature peptides identified in serum

MASP-1 signature peptide SEQ ID NO. AAGNECPELQPPVHGK 2 APGELEHGLITFSTR 3 DMICAGEK 4 DNVEMDTFQIECLK 5 DQVLVSCDTGYK 6 DSDLLSPDFK 7 DTCLGDSGGAFVIFDDLSQR 8 ETTDTEQTPGQEVVLSPGSFMSITFR 9 FPETLMEIEIPIVDHSTCQK 10 IEPSQAK 11 LPVVPHAECK 12 SDENEQHLGVK 13 SLPTCLPVCGLPK 14 TGVITSPDFPNPYPK 15 TLSDVLQYVK 16 VECSDNLFTQR 17 VLGPFCGEK 18 VLKDNVEMDTFQIECLK 19 YSCQEPYYK 20

Example 3. Quantitative display of MASP-1 within different groups

As shown in Figure 1, the quantification of MASP-1 in different groups is shown respectively, where the ordinate is the ratio of the normalized identification intensity of mass spectrometry, and their calculation comes from the expression of each peptide in Table 2 (using Maxquant1.5.2.8 software automatically calculates), which is used to characterize the expression level information of the protein as a whole.

Specifically, the expression level of MASP-1 in the serum of patients with fulminant type 1 diabetes at the early stage of onset was significantly higher than that of the normal group, while the expression level of the serum of type 1 diabetes patients was not significantly different from that of the normal group.

Example 4. ROC of MASP-1 used to differentiate the initial onset of fulminant type 1 diabetes from the diagnosis of type 1 diabetes curve

The diagnostic value of markers can be judged by the area under the curve of the ROC curve (receiver operating characteristic curve). The relationship between sensitivity and specificity, it calculates a series of sensitivity and specificity by setting multiple different critical values for continuous variables, and then takes the sensitivity as the ordinate and (1-specificity) as the The abscissa is drawn as a curve, and the larger the area under the curve, the higher the diagnostic accuracy. It is generally believed that the diagnostic value is higher when the area under the curve is higher than 0.9.

As shown in Fig. 2, the ROC curves obtained when MASP-1 was used as the initial diagnosis index of fulminant type 1 diabetes patients were respectively shown. As shown in the figure, using MASP-1 as an indicator to diagnose the onset of fulminant type 1 diabetes mellitus, the area under the ROC curve is 1, which has a high value for judging fulminant type 1 diabetes mellitus patients. However, using MASP-1 as an indicator to judge the occurrence of type 1 diabetes, the area under the ROC curve is 0.523, so there is no diagnostic effect.

To sum up, the present invention revealed that serum MASP-1 has value as a diagnostic marker for distinguishing initial fulminant type 1 diabetes mellitus and classic type 1 diabetes mellitus, and in specific experiments in different human groups (normal blood sugar group, 1 The characteristic peptides belonging to MSAP-1 (Table 2) were identified in the serum of people with type 2 diabetes and type 2 diabetes, and these characteristic peptides can be used to characterize the protein expression information.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

sequence listing

<110> Shanghai Institute of Biological Sciences, Chinese Academy of Sciences

Shanghai Sixth People's Hospital

<120> The application of MASP-1 in distinguishing fulminant and classic type 1 diabetes

<130> 167469 1CNCN

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<170> PatentIn version 3.3

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Gln Pro Pro Val His Gly Lys Ile Glu Pro Ser Gln Ala Lys Tyr Phe

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Trp Arg Leu Ser Tyr Arg Ala Ala Gly Asn Glu Cys Pro Glu Leu Gln

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Asp Asn Val Glu Met Asp Thr Phe Gln Ile Glu Cys Leu Lys Asp Gly

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Thr Trp Ser Asn Lys Ile Pro Thr Cys Lys Ile Val Asp Cys Arg Ala

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Pro Gly Glu Leu Glu His Gly Leu Ile Thr Phe Ser Thr Arg Asn Asn

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Leu Thr Thr Tyr Lys Ser Glu Ile Lys Tyr Ser Cys Gln Glu Pro Tyr

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Tyr Lys Met Leu Asn Asn Asn Thr Gly Ile Tyr Thr Cys Ser Ala Gln

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Gly Val Trp Met Asn Lys Val Leu Gly Arg Ser Leu Pro Thr Cys Leu

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Pro Glu Cys Gly Gln Pro Ser Arg Ser Leu Pro Ser Leu Val Lys Arg

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Ile Ile Gly Gly Arg Asn Ala Glu Pro Gly Leu Phe Pro Trp Gln Ala

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Leu Ile Val Val Glu Asp Thr Ser Arg Val Pro Asn Asp Lys Trp Phe

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Gly Ser Gly Ala Leu Leu Ser Ala Ser Trp Ile Leu Thr Ala Ala His

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Val Leu Arg Ser Gln Arg Arg Asp Thr Thr Val Ile Pro Val Ser Lys

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Glu His Val Thr Val Tyr Leu Gly Leu His Asp Val Arg Asp Lys Ser

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Gly Ala Val Asn Ser Ser Ala Ala Arg Val Val Leu His Pro Asp Phe

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Asn Ile Gln Asn Tyr Asn His Asp Ile Ala Leu Val Gln Leu Gln Glu

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Pro Val Pro Leu Gly Pro His Val Met Pro Val Cys Leu Pro Arg Leu

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Glu Pro Glu Gly Pro Ala Pro His Met Leu Gly Leu Val Ala Gly Trp

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Gly Ile Ser Asn Pro Asn Val Thr Val Asp Glu Ile Ile Ser Ser Gly

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Thr Arg Thr Leu Ser Asp Val Leu Gln Tyr Val Lys Leu Pro Val Val

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Pro His Ala Glu Cys Lys Thr Ser Tyr Glu Ser Arg Ser Gly Asn Tyr

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Leu Ser Gln Arg Trp Val Val Gln Gly Leu Val Ser Trp Gly Gly Pro

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Glu Glu Cys Gly Ser Lys Gln Val Tyr Gly Val Tyr Thr Lys Val Ser

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Claims (13)

1. Use of a substance for the detection of the level of mannan-related serine protease-1 (MASP-1) protein in a human blood sample for the preparation of a product for the diagnosis of first-onset and untreated fulminant type1 diabetes.

2. Use of a substance for detecting the level of mannan-related serine protease-1 (MASP-1) protein in a human blood sample for the preparation of a product for use in diabetes typing, said diabetes typing differentiating first-onset and untreated fulminant type 1diabetes from other types of diabetes, wherein said other types of diabetes include: non-first-onset or treated fulminant type 1diabetes, non-fulminant type 1diabetes, type 2 diabetes.

3. Use of a substance for determining the level of a mannan-related serine protease-1 (MASP-1) protein in a human blood sample for the manufacture of a product for determining the therapeutic effect of a drug on first-occurring and untreated fulminant type 1diabetes mellitus and/or for screening a candidate drug for the treatment of fulminant type 1diabetes mellitus.

4. Use according to claim 1 or 2 or 3, wherein the substance is selected from: antibodies against MASP-1, specific probes against MASP-1, protein chips against MASP-1.

5. Use according to claim 1 or 2 or 3, wherein the detection is performed by detecting full-length MASP-1 and/or MASP-1 signature peptides.

6. The use according to claim 5, wherein the sequence of MASP-1 is selected from the group consisting of: 1, 21, 22 and 23, wherein the MASP-1 characteristic peptide fragment is selected from: 2-20 of SEQ ID NO.

7. Use according to claim 1 or 2 or 3, wherein the substance is a substance for detecting MASP-1 by one or more of the following methods: multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, immunohistochemistry, chemiluminescence, radioisotope method, fluorescence, enzyme labeling, colloidal gold, biochip detection, and western blotting.

8. Use according to claim 7, wherein the fluorescence method is immunofluorescence.

9. Use according to claim 1 or 2 or 3, wherein the sample is selected from: whole blood, serum, plasma.

10. The use of claim 1 or 2, wherein the presence of MASP-1 in the sample above a control level indicates that the subject suffers from first-onset, untreated, fulminant type1 diabetes.

11. The use of claim 10, wherein the control level is selected from the group consisting of: a level of MASP-1 when the subject is healthy, a standard level of a healthy population determined by statistics, or a normalized level.

12. Use according to claim 1 or 2 or 3, wherein the product is selected from: a detection kit, a detection strip, a detection card, a detection pen, a detection instrument or any combination thereof; and/or, the product further comprises one or more substances selected from the group consisting of: containers, buffers, adjuvants, solvents, positive controls, negative controls, instructions for use, and other materials useful in the diagnosis and/or typing of diabetes.

13. A method of detecting mannan-associated serine protease-1 (MASP-1) protein levels in a human sample comprising: detecting the level of one or more polypeptides selected from SEQ ID NO 2-20 in a human sample.

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