CN108072761B - Application of MASP-1 in distinguishing initial outbreak from classical type 1diabetes - Google Patents
Application of MASP-1 in distinguishing initial outbreak from classical type 1diabetes Download PDFInfo
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Abstract
The invention provides the use of MASP-1 to distinguish between incipient outbreak and classical type1 diabetes. In particular, the invention relates to the use of mannan-related serine protease-1 (MASP-1) as an indicator for disease molecular typing for the diagnosis of incipient fulminant type 1diabetes (incipient FT1DM), for the differentiation of incipient FT1DM from classical type 1diabetes (T1DM), and to methods for determining serum MASP-1 protein levels.
Description
Technical Field
The present invention belongs to the field of biotechnology and medicine. In particular, the invention relates to the use of MASP-1 to distinguish between incipient fulminant type 1diabetes (FT1DM) and other types of diabetes, particularly classical type1 diabetes.
Background
1. Diabetes mellitus
Diabetes mellitus is a series of clinical syndromes caused by absolute or relative insufficiency of insulin secretion in the body. International Diabetes mellitus Federation, IDF) is an authoritative reference and important data source for International information about global conditions and related information about Diabetes mellitus. According to the latest data of IDF, about 3.87 hundred million adult diabetics account for 8.3 percent of the population all over the world in 2013, wherein 9840 million Chinese diabetics account for the first part of the world and account for about one fourth of the total number of the worldwide diabetics. By 2035, there are approximately 5.92 billion people worldwide if these trends continue. Compared with developed countries, China still needs a certain time to reach an ideal diagnosis and treatment environment, some clinical complications caused by diabetes become more obvious, and huge physical, mental and economic burdens are brought to patients and families of the patients.
According to the WHO diabetes etiological classification system, diabetes can be classified into 4 major categories, namely type 1diabetes, type 2 diabetes, gestational diabetes, and secondary diabetes. Among them, Type 1Diabetes Mellitus (T1DM), also called insulin-dependent Diabetes Mellitus, is Diabetes caused by absolute deficiency of insulin due to the damage of B cells of the own islets.
WHO classified type 1diabetes into classical type 1A diabetes (Autoimmune type 1diabetes, type 1A diabetes) and Idiopathic type 1B diabetes (idiophatic type 1diabetes, type 1B diabetes) in 1999 in type 1A diabetic patients, islet-associated autoantibodies such as Islet Cell Antibodies (ICA), glutamate decarboxylase antibodies (GADAb), islet autoantibodies (IA-2/IA-2 β Ab) may be used as immune markers, while those WHO are negative for autoantibodies are classified as type 1B diabetes.
2. Fulminant type 1diabetes mellitus
Japanese scholars Imagawa et al proposed, in 2000, Fulminant Type 1Diabetes mellitis, FT1DM, which is a new subtype of Type 1Diabetes and is characterized by an acute onset of disease, elevated pancreatic enzymes, β cells being largely and severely destroyed in a short period of time and causing hyperglycemia and ketoacidosis, the onset of disease often accompanied by respiratory and digestive system symptoms such as fever, cough or abdominal pain, diarrhea, and the like, with accompanying electrolyte disturbance, hepatic and renal insufficiency, rhabdomyolysis, and the like, and the mortality is high if the treatment is not timely obtained.
The disease is classified as idiopathic type 1B diabetes according to the classification of diabetes because the islet autoantibody detection is negative, and the pancreas pathology does not have insulitis symptoms such as islet exudation or edema. FT1DM patients are common in adults, with only 8.7% of adolescent patients younger than 20 years of age, and the incidence is gender-free and seasonally different. In addition, the incidence of FT1DM is high in east asia, and has been reported in japan, korea, china and the philippines. According to the report of Imagawa in japan 2003, FT1DM is as high as 15% -20% in T1DM (type 1 diabetes) patients with ketosis or ketoacidosis. Among korean T1DM patients, FT1DM accounts for about 7.1%, and 30.4% of adult type diabetic patients. There are few reports on FT1DM in China.
Compared with the common T1DM, FT1DM has the following remarkable characteristics: (1) the disease course is very short, and ketoacidosis is often developed within one week; (2) the onset is accompanied by serious metabolic disturbances, such as: disturbance of consciousness, ketoacidosis and electrolyte disturbance; (3) almost complete and irreversible damage to islet function during disease onset; (4) in some patients, the function of multiple organs can be damaged when the disease occurs, striated muscle lysis, acute renal failure and even sudden cardiac arrest can occur in severe cases, and if the disease cannot be treated in time, the death rate of FT1DM is high.
3. Mannan-related Serine Protease-1 (Mannose-Associated spring Protease-1, MASP-1)
Mannan-Associated Serine Protease-1 (MASP-1), also known as Mannan-binding lectin-Associated Serine Protease-1 (Mannan-binding lectin serineprotease 1), is a Serine Protease encoded by the MASP1 gene that functions as a lectin pathway component of the complement system involved in both innate and acquired immune responses.
MASP-1 is synthesized as a zymogen which is activated when complexed with pathogen recognition molecules of the lectin pathway (mannan-binding lectin and collectin). This protease is not directly involved in complement activation, but it may amplify complement activity by cleaving complement C2 or by activating other complement serine proteases (e.g., MASP-2).
MASP-1 also cleaves fibrinogen and factor VIII, and may play a role in coagulation. Splice variants of this gene lacking the serine protease domain are reported to be useful as inhibitors of the complement pathway.
There is no high sensitivity, high specificity identification index and effective method in the art for distinguishing incipient fulminant type 1diabetes from other diabetes, especially non-fulminant type1 diabetes. In order to prevent and treat diabetes more symptomatically, there is an urgent need in the art to develop biological and clinical indicators that can more finely classify type1 diabetes.
Disclosure of Invention
The invention provides the application of MASP-1 as a molecular marker for diagnosing the initial fulminant type 1diabetes and distinguishing the initial fulminant type 1diabetes from other diabetes. The invention also provides a method for detecting the protein level of mannan-related serine protease-1 (MASP-1) in a sample, and a method for testing the treatment effect of a medicament on fulminant type 1diabetes and/or screening candidate treatment medicaments in the invention
In some aspects of the invention, there is provided the use of a substance that detects the level of mannan-associated serine protease-1 (MASP-1) in a sample from a subject in the manufacture of a product for use in the diagnosis of incipient, fulminant type 1diabetes (incipient FT1 DM).
In other aspects of the invention, there is also provided a method of diagnosing incipient fulminant type 1diabetes (incipient FT1DM) 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 from the subject; (b) comparing the level of MASP-1 detected in (a) to a normal control value; if the comparison shows that the level of MASP-1 in the subject's sample is higher than that of the normal control, the subject is indicated to have incipient fulminant type1 diabetes.
In some embodiments of the invention, the diagnosis further comprises combining the MASP-1 level-based diagnosis of the invention with other methods of diabetes diagnosis.
In further aspects of the invention, there is provided the use of a substance that detects the level of mannan-associated serine protease-1 (MASP-1) in a sample from a subject for the preparation of a product for diabetes typing that distinguishes incipient, fulminant type 1diabetes (incipient FT1DM) from other types of diabetes, wherein the other types of diabetes include: non-incipient fulminant type 1diabetes, non-fulminant type 1diabetes, and non-fulminant type 2 diabetes.
In other aspects of the present invention, there is also provided a diabetes typing method that distinguishes incipient fulminant type 1diabetes (incipient FT1DM) from other types of diabetes, wherein the other types of diabetes include: non-incipient, non-fulminant type 1diabetes, type 2 diabetes, said method comprising the step of detecting the level of MASP-1 in a sample from the subject.
In some embodiments of the invention, the method comprises: (a) detecting the level of MASP-1 in a sample from the subject; (b) comparing the level of MASP-1 detected in (a) to a normal control value; if the comparison shows that the level of MASP-1 in the subject's sample is higher than that of the normal control, the subject is indicated to have incipient fulminant type1 diabetes.
In some embodiments of the invention, the typing further comprises combining the MASP-1 level-based typing of the invention with other diabetes typing methods.
In other aspects of the invention, there is provided use of a substance that detects the level of mannan-associated serine protease-1 (MASP-1) in a sample from a subject in the manufacture of a product for determining the therapeutic effect of a drug on incipient type 1diabetes and/or screening for a candidate drug for the treatment of fulminant type1 diabetes.
In some embodiments of the present invention, there is provided a method of determining the therapeutic effect of a drug on incipient fulminant type 1diabetes and/or screening for a candidate drug for the treatment of fulminant type 1diabetes, the method comprising: determining and comparing the level of MASP-1 in said subject before and after administration of said drug or drug candidate, wherein if the level of MASP-1 is reduced the drug or drug candidate has a therapeutic effect on fulminant type1 diabetes.
As described in various aspects herein, wherein the substance is a substance for detecting the DNA level, mRNA level and/or protein level of MASP-1, e.g., an antibody against MASP-1, a specific probe against MASP-1, a gene chip or a protein chip against MASP-1.
In some embodiments of the invention, MASP-1 has the amino acid sequence shown in SEQ ID NO. 1, or is a homologous sequence thereof, such as a MASP-1 polypeptide sequence corresponding to the subject species; the MASP-1 gene has the nucleotide sequence shown in SEQ ID NO. 1, 21, 22 or 23, or is a homologous sequence thereof, such as the MASP-1 gene sequence corresponding to the subject species.
As described in various aspects herein, the assay is performed by assaying for full-length MASP-1, MASP-1 characteristic peptides and/or coding sequences thereof, preferably the MASP-1 characteristic peptide is selected from one or more of SEQ ID NOs 2-20.
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, chemiluminescence, radioisotope methods, fluorescence methods (such as immunofluorescence), enzyme labeling, colloidal gold methods, real-time quantitative reverse transcription PCR, biochip detection methods, DNA blotting, RNA blotting in situ hybridization methods, and Western blotting methods.
In some embodiments, the substance carries a detectable label, such as a radioisotope, a fluorophore, a chemiluminescent moiety, an enzyme substrate, an enzyme cofactor, an enzyme inhibitor, a dye, a metal ion, a ligand (e.g., biotin or hapten), or any combination thereof.
As described in various aspects herein, the sample is obtained from the subject, e.g., 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 (e.g., whole blood, serum, plasma), bodily fluids (e.g., lymph fluid, cerebrospinal fluid, interstitial fluid), ascites fluid.
As described in various aspects herein, the presence of MASP-1 in the sample above a control level indicates that the subject suffers from an incipient fulminant type 1diabetes, e.g., the control level is selected from: a level of MASP-1 when the subject is healthy, a standard level of a healthy population determined by statistics, or a normalized level.
As described in aspects herein, 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.
In other aspects herein, there is also provided a method of detecting mannan-associated serine protease-1 (MASP-1) levels in a sample, comprising: detecting the level of one or more polypeptides selected from SEQ ID NO 2-20 or coding sequences thereof in the sample.
In some embodiments, the detection is performed by multidimensional liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis, immunohistochemistry, chemiluminescence, radioisotopes, fluorescence (e.g., immunofluorescence), enzyme labeling, colloidal gold, real-time quantitative reverse transcription PCR, biochip detection, southern blotting, northern blotting in situ hybridization, western blotting, and the like.
Any combination of the above-described solutions and features may be made by those skilled in the art without departing from the spirit and scope of the present invention. Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.
Drawings
The present invention will now be further described with reference to the accompanying drawings, wherein the showings are for the purpose of illustrating embodiments of the invention only and not for the purpose of limiting the scope of the invention.
FIG. 1: quantitative conditions of MASP-1 in different groups are determined, wherein the ordinate is the ratio of mass spectrum identification intensity after normalization, and the calculation is derived from the expression quantity of each peptide segment in the table 2 and is used for overall characterization of the expression quantity information of the protein. In the figure, "FC" represents the fold change (fold change), "+" represents p ≦ 0.05, and "+" represents p ≦ 0.005;
FIG. 2: and (3) adopting MASP-1 as a diagnosis index of the onset initial stage of the fulminant type 1diabetes patients to obtain an ROC curve.
Detailed Description
The inventor finds that the level of MASP-1 in the initial patient with fulminant type 1diabetes is obviously higher than that of normal people, the treated fulminant diabetes patient, the type 1diabetes patient and the type 2 diabetes patient through long-term and intensive research, thereby proposing that the MASP-1 can be used as a molecular marker for diagnosing the initial fulminant type 1diabetes and the diabetes classification (particularly the type 1diabetes subdivision).
In addition, the inventor further discovers that MASP-1 is adopted as a molecular marker for diagnosing the initial fulminant type 1diabetes and the diabetes typing (particularly the type 1diabetes subdivision) in the methodology research, so that high sensitivity, specificity and accuracy can be obtained in the diagnosis and/or the typing, and the method has high clinical application value. In addition, based on relevant research, the MASP-1 of the invention can be further used as a molecular marker for determining the treatment effect of the medicine on the initial fulminant type 1diabetes mellitus and/or screening candidate medicines for treating the fulminant type 1diabetes mellitus.
Furthermore, the present inventors have found that the characteristic peptides of MASP-1 provide an advantageous tool for the characterization and quantification of MASP-1 and the methods and uses of the present invention.
As used herein, the term "initial onset" refers to a disease that occurs first and is untreated. As used herein, the term "incipient fulminant type1 diabetes" refers to untreated fulminant type1 diabetes. As used herein, the term "non-incipient type1 diabetes" or "other types of diabetes" corresponding to incipient type 1diabetes include: non-incipient fulminant type 1diabetes, non-fulminant type 1diabetes, type 2 diabetes, and the like.
As used herein, the terms "mannan-related serine protease-1" or "MASP-1" are used interchangeably and have the meaning known in the art. For example, the MASP-1 protein may be the sequence shown in Gene ID:5648, or may be a homologous sequence thereof, or an isoform thereof, e.g.as shown in SEQ ID NO:1, 21, 22 or 23. MASP-1 may be a full-length MASP-1 protein or its coding sequence, or may be a characteristic fragment of MASP-1 (see, e.g., Table 2) or its coding sequence.
As used herein, the terms "detection substance" or "substance which detects MASP-1" are used interchangeably and refer to reagents and/or devices which are specific for MASP-1 and which can be used to directly or indirectly detect the level of MASP-1.
Since the sequence of MASP-1 molecules is known in the art, one of ordinary skill in the art can prepare reagents specific for MASP-1 molecules based on routine means or by commercially available methods. For example, detection reagents useful in the present invention include, but are not limited to: antibodies and probes having detection specificity for MASP-1 molecules.
To facilitate detection, the detection reagents of the 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 (e.g., biotin or haptens), and the like.
As used herein, the terms "product" or "product of the invention" are used interchangeably and refer to a product comprising a substance for detecting MASP-1 in a biological sample that can be used in the application of the invention. Products of the invention include, but are not limited to: a test kit, a test strip, a test card, a test pen, a test instrument, or any combination thereof. The product of the invention 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.
As used herein, the term "normal control" refers to the level of MASP-1 used as a reference, including but not limited to: the level of MASP-1 measured in a pre-disease sample from the same subject, a population standard level determined by statistics, or a normalized level.
As used herein, "comprising," having, "or" including "includes" comprising, "" consisting essentially of … …, "" consisting essentially of … …, "and" consisting of … …; "consisting essentially of … …", "consisting essentially of … …", and "consisting of … …" are subordinate concepts of "comprising", "having", or "including".
All numerical ranges provided herein are intended to expressly include all numbers between the end points of the ranges and numerical ranges there between. The features mentioned with reference to the invention or the features mentioned with reference to the embodiments can be combined. All the features disclosed in this specification may be combined in any combination, and each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the features disclosed are merely generic examples of equivalent or similar features.
Examples
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art can make appropriate modifications and alterations to the present invention, which fall within the scope of the invention.
The experimental procedures for the conditions not specified in the examples below can be carried out by methods conventional in the art, for example, by referring to the molecular cloning, A Laboratory Manual, New York, Cold spring harbor Laboratory Press, 1989 or according to the conditions recommended by the supplier. Methods for sequencing DNA are conventional in the art and tests are also available from commercial companies.
Unless otherwise indicated, percentages and parts are by weight. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
Reagent and experimental process
(1) Chemical reagent
All buffers were made in Milli-Q water (Millipore, Bedford, MA, USA). Dithiothreitol (DTT), Iodoacetamide (IAA), Urea (Urea), Sodium Dodecylsulfate (SDS), Tris (hydroxymethyl) aminoethane-hydrochloric acid (Tris-HCl), etc. were purchased from Bio-Rad corporation (Hercules, Calif., USA); formic Acid (FA), ammonium bicarbonate (NH4HCO3), etc. are available from Sigma, Inc. (St. Lous, MO, USA); HPLC grade Acetonitrile (ACN) and Methanol (Methanol) were purchased from Fisher corporation (Fair law, NJ, USA); pancreatin (Trypsin) was purchased from Promega corporation (Madison, WI, USA); TMTMass Tagging Kits (90066) were purchased from Thermo Scientific Inc. (Waltham, MA, USA).
(2) Human serum sample collection and preparation
The present invention relates to 60 human serum samples in total: patients with incipient outbreaks of fulminant type 1diabetes (5, untreated patients who had been clinically diagnosed as FT1DM) and treated for 1 year (5, conventional treatment), normoglycemia (15), type 1diabetes (T1DM, 20, untreated), type 2 diabetes (T2DM, 15, untreated).
All 60 samples were sourced from the sixth national hospital affiliated with Shanghai university of transportation. The acquisition of human blood samples was carried out in strict compliance with the guidelines of the Chinese law and ethical committee, each patient signed an informed consent.
(3) Removal of high abundance proteins
The 7 high abundance components of human serum proteins (i.e., albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin and fibrinogen) were removed using the MARS (multiple affinity removal system 5188-6411) from Agilent.
The specific operation is as follows: 28 μ L of serum was added to 400 μ L A solution, and centrifuged at 4 deg.C and 1000g for 15min using a 0.22 μm ultrafiltration tube. After equilibration of the MARS column with 4mL of solution A, 200. mu.L of the filtered filtrate was applied to the column, centrifuged at 100g for 1min at 4 ℃. Add 300. mu. L A solution, centrifuge at 4 ℃ and 100g for 1min and transfer all the filtrate to a new centrifuge tube. The high abundance protein fraction bound to the column was eluted slowly with 2mL of B solution. After the column was equilibrated with 4mL of solution A, the above procedure was repeated once for the remaining 200. mu.L or so of serum sample, and the filtrates were combined in the centrifuge tube of the above step, lyophilized and stored at-80 ℃.
(4) Fluorescence quantification of protein concentration
The 60 serum samples after removal of the abundant proteins and the samples formed by mixing the multiple samples (called mix, obtained by mixing equal amounts of all 60 serum samples after removal of the abundant proteins) were lyophilized and then reconstituted with 300. mu.L of 2D lysate (8Murea, 40mM Tris, 65mM DTT) and then quantified by fluorescence. The specific operation is as follows: the protein quantification was performed by tryptophan fluorescence. mu.L of the protein solution was added to 1mL of urea solution (8M urea, 20mM Tris-HCl, pH 7.6). The excitation fluorescence wavelength was set at 295nm and the absorption measurement fluorescence wavelength was set at 350 nm. The standard tryptophan was diluted in multiple ratios and used to plot a standard curve and calculate the protein concentration in the sample. The average tryptophan content of the samples was calculated as 1.3%.
(5) Enzymolysis in solution
The in-solution enzymatic step is based on FASP enzymatic hydrolysis. According to the result of the fluorescence quantification, 14. mu.g of the sample obtained in step (4) was added to 50. mu.L of 8M UA buffer (8M urea, 0.1M Tris-HCl, pH8.5), placed on 10k ultrafiltration tubes (OMEGA filter, OD010C35), added with 2. mu.L of 1M DTT, and placed in an incubator at 37 ℃ for 1.5 hours. Thereafter, 10. mu.L of 1M IAA was added to the system, and the reaction was carried out in the dark at room temperature for 30 min.
Through the above operation, the protein is completely denatured, the disulfide bond is opened, and the thiol group is blocked. Thereafter, 300. mu.L of 0.2M TEAB was added to the system, and centrifuged at 13000g for 15min at 20 ℃. Thereafter, the filtrate was discarded, and the membrane was washed three times with 200. mu.L of 0.2M TEAB buffer under ultrafiltration conditions of 4 ℃ and 13000g for 15 min. Finally, 100 μ L of 0.2M TEAB containing trypsin was added to the ultrafiltration tube in mass ratio (pancreatin: protein ═ 1: 25), and the mixture was placed in a shaker at 37 ℃ for enzymatic hydrolysis for 16 h. Then according to enzyme: protein 1: 25 adding trypsin into the ultrafiltration tube to continue enzymolysis for 4 h. 13000g of the peptide fragment mixture after enzymolysis is centrifuged to collect filtrate, and the filtrate is freeze-dried and stored at-80 ℃ for subsequent experiments.
(6) In vitro isotopic labeling of TMT
The TMT Mass Tagging Kits (Thermo Scientific, 90066) had six channels (126, 127, 128, 129, 130, 131, respectively) in total, each channel labeled with 1 sample, wherein 126 channels were used to label mix internal reference samples, and 12 groups were labeled in the order shown in table 1 below.
TMT marking step: dissolving the peptide fragments after enzymolysis by 14 mu L of 0.2M TEAB buffer solution respectively, and dissolving the labeling reagent by 43 mu L of ACN; then adding a marking reagent into the peptide segment, uniformly mixing, standing at room temperature for 1.5h, and marking; finally adding 8mu L of 5% hydroxylamine to stop the reaction for 15min, mixing the 6 channel peptide fragments in each group, and finally adding ddH into the combined 14-tube sample2Freeze-drying for three times, and storing at-80 deg.C.
TABLE 1 sample labelling information
| Group number | 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 |
Wherein, FT1DMa and FT1DMc respectively represent patients with the onset of fulminant type 1diabetes and corresponding 1 year of treatment, N represents normal blood sugar, T1 represents type 1diabetes, T2 represents type 2 diabetes, wherein the numbers following the above letters represent the serial numbers of different samples, i.e. different individuals of the same disease type; mix is a mixed sample for use as an internal reference
(7) Peptide de-salting
The labeled and lyophilized samples obtained in step (6) were taken and dissolved well in 900. mu.L of 0.2% TFA in water, 200. mu.L of each sample was desalted with stage-tip filled with C18, and two samples were run in parallel.
First, the stage-tip filled with two layers of C18 was activated with 100. mu.L of methanol, centrifuged at 1500g for 2min at room temperature, and the filtrate was removed. Thereafter, the packing was washed with 100 μ L of 80% acetonitrile containing 0.2% TFA to maximize contaminant removal and the centrifugation conditions were the same as above. The packing was then equilibrated with 100. mu.L of 0.2% TFA in water 3 times, centrifuged at 1500g for 2min at room temperature, and the filtrate was removed. 200 μ L of each sample was bound to stage-tip and centrifuged at low speed 800g for 5min at room temperature to ensure adequate binding of the sample to the C18 filler. The filler was then washed 3 times with 100 μ L of 0.2% TFA in water and centrifuged at 2000g for 2min at room temperature to remove salts and other hydrophilic components that were not bound to the C18 filler. Finally, the column was eluted with 100. mu.L of 90% acetonitrile containing 0.2% TFA, centrifuged as above, and the filtrate was collected, lyophilized and subjected to mass spectrometry.
(8) Multidimensional liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis
LC-MS/MS mass spectrometric detection was performed on a nano HPLC-Orbitrap Fusion (Thermo Fisher Scientific, San Jose, Calif.) system.
The liquid chromatography is Easy-nLC-1000 system (ThermoFisher Scientific); the reverse phase column is a spray needle reverse phase column (tip column, 75 μm × 150mm, C18, 3 μm); the eluents in reverse phase were 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). After the sample is loaded on the reversed-phase column, the sample is eluted by a reversed-phase elution system with the flow rate of 0.3 mu L/min, and the effective elution gradient is as follows: 5min, 4% B; 150min, 26% B, 155min, 40% B; 160min, 90% B, and 180min for mass spectrometry.
The peptide fragments eluted from the reversed phase column enter a quadrupole rod-electrostatic field orbit trap-linear ion trap three-in-one combined mass spectrum Orbitrap Fusion for analysis, and a data-dependent data acquisition mode is adopted to acquire a secondary mass spectrum at a maximum acquisition rate (top speed). The dynamic exclusion settings are: if a parent ion is identified 1 time, the mass parent ion is not detected within the next 2 min. The parent ion fragmentation mode assumed the HCD mode, with primary and secondary spectral resolutions set at 60000 and 15000 (at 200 m/z), respectively.
(9) Database search
The raw file collected by mass spectrum is analyzed by Maxquant1.5.2.8 software, and the database is Uniprot homo sapiens database. The library searching parameters are set as follows: the mass deviation of the ion (fragment) is within 20ppm, the mass deviation of the parent ion (precursor ion) is within 6ppm, the protease is set as trypsin, the maximum allowable deletion enzyme cutting site is 2, the fixed modification is set as carboxymethylation of cysteine (ca mida methylation, +57Da), the variable modification is set as oxidation of methionine, +16Da) and the TMT-6plex mark positioned at the free amino terminal of the lysine residue are set as variable modification, and the false positive rate (FDR) of the protein and peptide fragment is set as 0.01.
(10) Statistical and bioinformatics analysis
And (4) carrying out median correction on all sample data, and then carrying out logarithmic processing on the data except for the internal reference sample. Pairwise comparisons between groups were checked with a rank sum and corrected by FDR. All data analysis and statistical tests were done using the R-install package or Excel.
Example 1 differential protein analysis and marker search
Differential protein analysis is based on a statistical value P value (P value), which is the probability that a sample observation or more extreme result occurs when the original assumption is true. If the P value is small, the probability of the situation of the original hypothesis is small, and if the P value is small, the reason why the original hypothesis is rejected is reasonable according to the small probability principle. In summary, a smaller P value indicates a more pronounced result.
The diagnostic value of the marker can be judged by the area under the curve of the ROC curve (subject working characteristic curve), wherein the ROC curve refers to the subject working characteristic curve which is a comprehensive index reflecting continuous variables of sensitivity and specificity, the correlation of the sensitivity and the specificity is revealed by a composition method, a series of sensitivity and specificity are calculated by setting the continuous variables to be different critical values, then the sensitivity is used as an ordinate, and the (1-specificity) is used as an abscissa to draw a curve, and the larger the area under the curve is, the higher the diagnostic accuracy is. It is generally considered that the diagnostic value is higher when the area under the curve is higher than 0.9.
The comparison of the initial onset (before treatment) of the fulminant diabetes patients, the treatment of the fulminant diabetes patients, the normal blood sugar group, the type 2 diabetes group and the type 1diabetes group shows that the expression level of MASP-1 in the serum of the fulminant diabetes patients is obviously higher than that of the treatment of the patients, the normal group, the type 1diabetes group and the type 2 diabetes group.
Post-treatment (FT1DMc) MASP-1 abundance was reduced by a factor of 2.14 compared to the initial disease (FT1DMa) in patients with fulminant diabetes, where paired t-tests showed a P value of 0.0015. Compared with normal control (N), the abundance of MASP-1 is increased by 2.66 times in the early onset stage (FT1DMa) of a fulminant diabetes patient, the rank sum test p value after BH correction is less than 0.05, and MASP-1 is used as a receiver working characteristic curve (ROC curve), and the area under the curve is 1.
After the treatment of the patients with the initial outbreak of the fulminant diabetes (FT1DMc), the patients with the 1-type diabetes (T1DM) and the patients with the 2-type diabetes (T2DM) are respectively compared with the normal control group, the abundance change multiples are below 1.3, and the BH-corrected rank sum test p value is more than 0.05. In addition, after the treatment of patients with initial onset of fulminant diabetes (FT1DMc), type 1diabetes (T1DM) and type 2 diabetes (T2DM), the MASP-1 was used as the test subject working characteristic curve (ROC curve) for the normal control group, and the areas under the curve were 0.747, 0.523 and 0.662 (see FIG. 2).
From the above results, it can be seen that in the early stage of onset of fulminant type 1diabetes, the abundance of MASP-1 in serum is significantly increased compared with that in the normal control, while in the patients with type 1diabetes and the patients with type 2 diabetes, the abundance of MASP-1 in serum is not significantly changed compared with that in the normal control,
therefore, serum MASP-1 can be used as a molecular typing diagnostic marker for distinguishing the initial stage of the fulminant type 1diabetes mellitus from other type 1diabetes mellitus.
Example 2 identification of MASP-1 characteristic peptide fragments
Using mass spectrometry, we identified for the first time the following characteristic peptide fragments belonging to MASP-1 in serum. They can be used to characterize the serum expression level of the protein MASP-1:
TABLE 2 list of MASP-1 characteristic peptides identified in serum
| MASP-1 characteristic peptide fragment | SEQ ID NO. |
| |
2 |
| |
3 |
| DMICAGEK | 4 |
| DNVEMDTFQIECLK | 5 |
| DQVLVSCDTGYK | 6 |
| DSDLLSPSDFK | 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 in different groups
As shown in FIG. 1, the quantitative status of MASP-1 in different groups is shown, wherein the ordinate is the ratio after the mass spectrometric identification intensity is normalized, and the calculation is derived from the expression level of each peptide segment in Table 2 (automatically calculated by Maxquant1.5.2.8 software) and is used for overall characterization of the protein expression level information.
Specifically, the expression level of MASP-1 in the serum of patients with fulminant type 1diabetes at the initial stage of onset is obviously higher than that of normal people, and the expression level in the serum of patients with type 1diabetes is not obviously different from that of normal people.
Example 4 use of MASP-1 to differentiate between the early stages of onset of fulminant type 1diabetes and the ROC for diagnosis of type 1diabetes
Curve line
The diagnostic value of the marker can be judged by the area under the curve of the ROC curve (subject working characteristic curve), wherein the ROC curve refers to the subject working characteristic curve which is a comprehensive index reflecting continuous variables of sensitivity and specificity, the correlation of the sensitivity and the specificity is revealed by a composition method, a series of sensitivity and specificity are calculated by setting the continuous variables to be different critical values, then the sensitivity is used as an ordinate, and the (1-specificity) is used as an abscissa to draw a curve, and the larger the area under the curve is, the higher the diagnostic accuracy is. It is generally considered that the diagnostic value is higher when the area under the curve is higher than 0.9.
As shown in FIG. 2, ROC curves obtained when MASP-1 was used as an index for the initial diagnosis of patients with fulminant type 1diabetes are shown, respectively. As shown in the figure, MASP-1 is used as an index to diagnose the outbreak of the fulminant type 1diabetes mellitus patient, the area under the ROC curve is 1, and the value of judging the fulminant type 1diabetes mellitus patient is higher. And MASP-1 is used as an index to judge the occurrence of type 1diabetes, and the area under the ROC curve is 0.523, so that no diagnosis effect exists.
In conclusion, the serum MASP-1 disclosed by the invention has the value as a diagnostic marker for distinguishing the initial fulminant type 1diabetes from the classical type 1diabetes, and characteristic peptide fragments (table 2) belonging to the MSAP-1 are identified in the serum of different groups (a normal blood sugar group, a type 1diabetes group and a type 2 diabetes group) in specific experiments, and can be used for representing the expression quantity information of the protein.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Shanghai Life science research institute of Chinese academy of sciences
The sixth people hospital in Shanghai City
<120> use of MASP-1 for differentiating between incipient outbreak and classical type 1diabetes
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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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Ser Val Thr Glu Asn Met Phe Cys Ala Gly Tyr Tyr Glu Gly Gly Lys
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Asp Thr Cys Leu Gly Asp Ser Gly Gly Ala Phe Val Ile Phe Asp Asp
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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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Inventor after: Zeng Rong Inventor after: Jia Weiping Inventor after: Wu Jiarui Inventor after: Li Rongxia Inventor after: Zhou Jian Inventor before: Zeng Rong Inventor before: Jia Weiping Inventor before: Wu Jiarui Inventor before: Li Rongxia Inventor before: Zhou Jian |