Application of Amino Acid Site Modification in Collagen in Pancreatic Cancer Detection

Abstract

The present invention relates to the application of amino acid site modification in collagen in the detection of pancreatic cancer. The present invention belongs to the field of cancer diagnosis, and particularly relates to a protein biomarker related to the detection of pancreatic cancer and its application. The present invention for the first time reports that the proline mono-oxidation modification in the α2 chain of type V collagen (COL5A2) and the α2 chain of type I collagen (COL1A2) can be used as a diagnostic biomarker for pancreatic cancer. Compared with the traditional abnormal protein quantification, it has earlier and more effective diagnostic value, as well as higher therapeutic reference value, and thus has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of pancreatic cancer diagnosis, and particularly relates to the application of mono-hydroxylated proline modification in type V collagen α2 and type I collagen α2 in the detection of pancreatic cancer. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the overall understanding of the present invention, and is not necessarily to be regarded as an admission or any form of implication that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Pancreatic cancer is a highly malignant tumor in the digestive system. The main reason is that there are no typical symptoms in the early stage of the disease, making it impossible to detect and diagnose it early. At present, the clinical diagnosis of pancreatic cancer mostly relies on clinical manifestations, color Doppler ultrasound, CT, and magnetic resonance. Most patients are diagnosed at the middle and late stages. Surgical treatment is difficult, and even if surgical treatment is performed, it is easy to relapse, and the drug treatment effect is poor, thus seriously affecting the prognosis of pancreatic cancer patients.

[0004] The integrity of the arterial wall structure and function is closely related to the extracellular matrix (ECM) in the arterial wall. ECM is a stable macromolecular complex secreted by fibroblasts, including collagen fibers, elastic fibers, glycoproteins, proteoglycans, etc. The most important component is collagen. Collagen is the main component of the extracellular matrix, a rope-like molecule formed by three peptide chains (α1, α2, α3) coiled in a helix. Collagen is classified into two groups according to its function. The first group is fibrillar collagen, including types I, II, III, XI, etc.; the rest is the second group, non-fibrillar collagen. According to the different structures of the three peptide chains (α1, α2, α3), different types of collagen can be divided. Among them, types I, III, IV, V, etc. are related to the arterial wall. Type I is mainly distributed in the adventitia, types III and VI are mainly distributed in the media, type IV is mainly distributed in the basement membrane, type V is mainly distributed in the media and basement membrane, and types I and III collagen account for 80 - 90% of all collagen.

[0005] Type I collagen is a heterotrimer formed by two α1 chains and one α2 chain in a right-handed helix. Each α2 polypeptide chain includes three regions: an amino-terminal region (N-terminal domain), a collagen region, and a carboxyl-terminal region (C-terminal domain). The collagen region domain has 1040 amino acids, including an 11-amino acid N-terminal peptide, a 15-amino acid C-terminal tail peptide, and a 1014-amino acid triple helix structure, that is, 338 Gly-X-Y repeat structures. The collagen region domain of the α1 chain has 1057 amino acids, including a 17-amino acid N-terminal peptide, a 26-amino acid C-terminal tail peptide, and a 1014-amino acid triple helix structure, that is, 338 Gly-X-Y repeat structures. Glycine residues are buried in the center of the triple helix structure and cannot accommodate other residues. The strict conservation of glycine is necessary to maintain the triple helix structure. If one glycine in the Gly-X-Y repeat structure is replaced by a missense mutation or other amino acid residues, osteogenesis imperfecta will ultimately result from the deficiency of type I collagen.

[0006] Type V collagen is a member of the collagen fibril subclass, which commonly has a triple helix domain composed of a continuous series of Gly-X-Y triplets. Type V collagen is a quantitatively minor component of the major type I collagen fibrils in most non-cartilaginous tissues. There are several type V collagen isotypes with different types and ratios of constituent chains. The most abundant and widely distributed isoform is α1(V)2α2(V), which forms heterotypic fibrils with type I collagen. The role of type V collagen in the organization and biological properties of the collagen extracellular matrix is poorly understood. A negative correlation between the ratio of type V collagen to type I collagen and the diameter of collagen fibrils was observed in in vitro fibril assembly experiments, and this function may be mediated by the retention of the non-collagenous amino-terminal propeptide after the incorporation of type V collagen molecules into the fibrils. This non-collagenous domain protrudes outward through the gaps between adjacent type I collagen molecules, mostly remaining on the surface of the fibrils, and may limit the lateral growth of the fibrils through steric hindrance and charge interactions.

[0007] The inventors' research found that the current molecular diagnosis of pancreatic cancer is only limited to the expression levels of genes or proteins, and the detection methods are limited. Developing modifications at a single site for the detection of pancreatic cancer helps to achieve a detection effect with strong specificity, high accuracy, and simple and easy operation. Summary of the Invention

[0008] In view of the above problems of the prior art, the present invention provides a protein biomarker related to pancreatic cancer detection and its application. The present invention takes collagen as the research object and finds that the proline mono-oxidation modification in the α2 chain of type I collagen and the α2 chain of type V collagen is significantly down-regulated compared with the control group, and there are significant differences. It can be speculated from this that the oxygen metabolism-related mechanism in the internal environment of pancreatic cancer has changed, resulting in the oxidation modification of proline in the collagen located outside the membrane being different from that of the control group.

[0009] Based on the above findings, the present invention relates to the following technical solutions:

[0010] In the first aspect of the present invention, there is provided the application of amino acid site modification in collagen in the detection of pancreatic cancer, wherein the amino acid modification is the proline mono-oxidation modification in the α2 chain of type V collagen (COL5A2) or the proline mono-oxidation modification in the α2 chain of type I collagen (COL1A2).

[0011] The present invention uses high-precision mass spectrometry (Orbitrap) to rapidly detect the amino acid differences of collagen in pancreatic cancer and perform correlation data analysis.

[0012] Through research, it has been found that the mono-oxidized proline modification at the three sites of positions 578, 593, and 659 in the COL5A2 protein molecule and / or the mono-oxidized proline modification at the three sites of positions 147, 471, and 789 in the COL1A2 protein molecule can be used to judge the occurrence of pancreatic cancer.

[0013] Preferably, the application of the above sites in the detection of pancreatic cancer includes the preparation of a pancreatic cancer diagnostic product, and the pancreatic cancer diagnostic product is used to detect whether the proline mono-oxidation modification occurs at any one or more of the amino acids at positions 578, 593, and 659 of type V collagen α2 (COL5A2) and positions 147, 471, and 789 of type I collagen α2 (COL1A2) to judge the occurrence or progression of pancreatic cancer.

[0014] More preferably, examples of the diagnostic product include a pancreatic cancer diagnostic kit, a pancreatic cancer diagnostic kit product based on the proline mono-oxidation modification at any one or more of the above sites, namely positions 578, 593, and 659 in the COL5A2 protein molecule and positions 147, 471, and 789 in the COL1A2 protein molecule, as markers.

[0015] In the above embodiments, the pancreatic cancer diagnostic kit includes cell / tissue sample processing reagents, protein extraction reagents, peptide digestion reagents, mass spectrometry detection auxiliary reagents, etc.

[0016] Secondly, based on the findings of the present invention, the present invention also claims the application of the proline mono-oxidation sites at positions 578, 593, and 659 in the COL5A2 protein molecule and at positions 147, 471, and 789 in the COL1A2 protein molecule as targets in the preparation of drugs for treating pancreatic cancer.

[0017] The kit also contains a buffer solution.

[0018] Advantages of the above one or more technical solutions:

[0019] The present invention reports for the first time that the proline mono-oxidation modification in the α2 (COL1A2) chain of type I collagen and the proline mono-oxidation modification in the α2 (COL5A2) chain of type V collagen can be used as diagnostic markers for pancreatic cancer. It has been proven by experiments that, compared with the traditional abnormal protein quantification, it has earlier and more effective diagnostic value, and at the same time, it also has higher therapeutic reference value. Therefore, it has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments and descriptions thereof of the present invention are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0021] Figure 1 . Comparison of the total sum of oxidized prolines at three positions 578, 593, and 659 in the COL5A2 protein in pancreatic cancer patients and control group samples;

[0022] Figure 2 . ROC curve of the combined analysis of three positions 578, 593, and 659 in the COL5A2 protein in pancreatic cancer patients and control group samples;

[0023] Figure 3 . Differences in the individual mono-oxidized prolines at three different positions in the COL5A2 protein between the pancreatic cancer group and the control group;

[0024] Figure 4 . ROC curves of the individual mono-oxidized prolines at three positions of COL5A2 in the pancreatic cancer group and the control group;

[0025] Figure 5 . GLTGN P . Secondary spectrum of GVQGPEGK (proline at position 578 in the protein);

[0026] Figure 6 . LGPLGA P . Secondary spectrum of GEDGR (proline at position 593 in the protein);

[0027] Figure 7.DGEVGPSGPVGP P Secondary spectrum of proline at position 659 in GLAGER (protein);

[0028] Figure 8 .Comparison of the total amount of oxidized proline at three positions, 147, 471, and 789, in COL1A2 protein between pancreatic cancer patients and control group samples;

[0029] Figure 9 .ROC curve for the combined analysis of oxidized proline at three positions, 147, 471, and 789, in COL1A2 protein between pancreatic cancer patients and control group samples; Note: AUC of the ROC curve = 0.987;

[0030] Figure 10 .Differences in single oxidized proline at three different positions in COL1A2 protein between the pancreatic cancer group and the control group;

[0031] Figure 11 .ROC curves for single oxidized proline at three positions in COL1A2 for the pancreatic cancer group and the control group respectively;

[0032] Figure 12 .AGEDGH P Secondary spectrum of proline at position 147 in GKPGR (protein);

[0033] Figure 13 .EGPVGL P Secondary spectrum of proline at position 471 in GIDGR (protein);

[0034] Figure 14 .GDGGPPGMTGF P Secondary spectrum of proline at position 789 in GAAGR (protein). Detailed implementation mode

[0035] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0036] It should be noted that the terms used herein are only for describing the specific implementation mode and are not intended to limit the exemplary implementation mode according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0037] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0038] Example 1 Screening for Collagen Amino Acid Modifications in Pancreatic Cancer Specimens

[0039] I. Tissue Sample Processing

[0040] A total of 9 pairs of samples were used in this experimental process, including 9 cases of pancreatic cancer tissue and 9 cases of normal pancreatic tissue.

[0041] The sample tissue was cut into tissue blocks smaller than 1 mm 2 . RIPA lysis buffer (tissue: lysis buffer = 1:100 (1 mg / 100 μl)) was added and shaken well, and incubated on ice for 30 min. The ground beads after ice bath were added to the sample tube, and the tissue was ground by a grinder. The ground beads were taken out and sonicated on ice. After centrifugation at 4°C, 10000 g for 10 min, the supernatant was taken. Acetone was added to precipitate the protein, and 100 mM ammonium bicarbonate solution was added to the precipitate to dissolve the precipitate, and low-temperature sonication could assist in dissolution.

[0042] The protein concentration of the sample was determined by the BCA method. Trypsin[KR|P] (abbreviated as Trypsin) was added to the sample tube at a ratio of total sample protein: Trypsin = 50:1 (μg: μg). A desalting column (Waters Sep-Pak Vac 1cc (50 mg) C18 Cartridges) was used to desalt the digested peptide segments.

[0043] The concentration of the desalted peptide segments was determined using a BCA peptide concentration assay kit.

[0044] Take 100 μg of the peptide sample and perform fractionation using a Waters e2695 (Separations Module) liquid phase. Each sample is fractionated into three levels. Follow the following fractionation process (conventional high-pH reversed-phase fractionation of peptides): ① Buffer A: 10 ml of 100× stock solution, 970 ml of water, 20 ml of CAN (acetonitrile); Buffer B: 10 ml of 100× stock solution, 10 ml of water, 980 ml of ACN (acetonitrile); 100× stock solution (100 ml): 13.4 ml of concentrated ammonia water, 86.6 ml of water. ② Run the chromatograph and flush the chromatograph lines. ③ Take 100 μl of the sample into a residue-free sample vial, start loading the sample, set the liquid phase flow rate to 0.1 ml / min, the chromatographic column oven temperature to 50 °C, Wavelength: 214 nm; bandwidth: 12 nm. ④ Prepare 2 ml flat-bottomed EP tubes, label them in sequence and place them in the sample collector to collect the sample components. ⑤ Divide the collected components into three groups according to the time sequence, combine the sample components of each group, and vacuum centrifuge to dry the sample.

[0045] Dissolve the sample with 0.1% formic acid (water) and centrifuge at 20000 g for 10 min at 4 °C.

[0046] Separate using an EASY-nLC 1200 liquid phase system and perform DDA mode detection using an online Thermo QE-HFX mass spectrometer. Liquid phase separation system parameters, pre-column: 150 μm * 3 cm C18 1.9 μm Reprosil-Pur 120; chromatographic column: 150 μm * 25 cm C18 1.9 μm Reprosil-Pur 120; flow rate: 600 nl / min; mobile phase A: 0.1% formic acid (water); mobile phase B: 0.1% formic acid 80% acetonitrile (water); elution gradient: 4% - 7% B for 1 min, 7% - 25% B for 94 min, 25% - 40% B for 16 min, 40% - 100% B for 5 min, 100% for 4 min; sample loading volume: 5 μl. Mass spectrometry detection parameters, scanning range: 150 - 2000 m / z; data acquisition mode: DDA acquisition mode, top 20 fragment spectra; MS1 resolution (200 m / z): 60,000; MS1 AGC target: 3e 6 , maxIT: 50 ms; MS2 resolution (200 m / z): 15,000; MS2 AGC target: 5e 4 , maxIT: 45 ms; normalized collision energy (NCE): 28%; isolation window: 1.6 m / z.

[0047] II. Data analysis method for the mono-hydroxylation modification of proline at position α2 of type V collagen

[0048] Analysis of the mass spectrometry detection results showed that the mono-hydroxylation modification of proline at positions 578, 593, and 659 in the type V collagen α2 (COL5A2) molecule accounted for 64.1% of the total oxidation modification of this protein. Moreover, the sample data showed that, compared with the control group, the total mono-hydroxylation of these three positions in the COL5A2 protein in pancreatic cancer tissues was down-regulated by 4.1-fold, and there was a significant statistical difference, p = 0.003 < 0.01( Figure 1 ).

[0049] According to the combined analysis of the mono-hydroxylated proline at positions 578, 593, and 659 in the COL5A2 protein molecule of pancreatic cancer and the control group, an ROC curve was plotted( Figure 2 ). From the above information, it can be seen that the sum of the mono-hydroxylated proline at positions 578, 593, and 659 in the COL5A2 protein molecule in pancreatic cancer and the control group has significant statistical differences and diagnostic value. Moreover, the mono-hydroxylated proline at positions 578, 593, and 659 in the COL5A2 protein molecule has the same diagnostic significance when used alone( Figure 3 , 4).

[0050] The ROC curves of the mono-hydroxylated proline at these 3 positions in COL5A2 for pancreatic cancer and the control group are as follows ( Figure 4 ). From the above information with modified secondary spectra( Figures 5 - 7 ), it can be seen that these modifications are all highly reliable.

[0051] The specific Deltamass-related information of the 3 mono-hydroxylated prolines in COL5A2 is as follows: the protein to which it belongs: P05997 (type V collagen α2, COL5A2); the positions in the COL5A2 protein and the peptide segments to which they belong are: proline at position 578 and its peptide segment GLTGN P GVQGPEGK; proline at position 593 and its peptide segment LGPLGA P GEDGR; proline at position 659 and its peptide segment DGEVGPSGPVGP PGLAGER. Deltamass molecular weight: 15.99; average Deltamass score: 0.919 (The Deltamass score means that in mass spectrometry detection, due to various extremely minute effects, the results of individual Deltamass measured by mass spectrometry are in a normal distribution form within the allowable range of precision and error, and the score is the goodness of fit for the normal curve, with a score range of 0 to 1. The higher the score, the more reliable the detection result. A secondary spectrum with a score reaching 0.2 will be recognized).

[0052] In the above content of this example, the subsequent experiments were carried out after trypsinizing the sample protein with Trypsin[KR|P] for the target peptide segment. In mass spectrometry detection, various sample trypsinization schemes can also be adopted, such as Trypsin(semi)[KR|P]; Trypsin / p[KR|-]; TrypsinK[K|P]; TrypsinR[R|P]; ChymoTrypsin[FWYL|P]; ArgC[R|P]; AspN[-|D]n-term; Clostripain[R|-]; CNBr[M|P]; Elastase[GVLIA|P]; Formic Acid[D|P]; GluC[DE|P]; GluC bicarb[E|P]; Iodosobenzoate[W|-]; LysC[K|P]; LysC / P[K|-]; LysN[-|K]n-term; LysN promisc[-|KASR]n-term; PepsinA[FL|P]; Protein endopeptidase[P|-]; Staph protease[E|-]; Trypsin-CNBr[KRM|P]; Trypsin-GluC[DEKR|P], etc. Monohydroxyproline of the COL5A2 protein species can also be detected in the samples treated by these trypsinizations or physical schemes such as ultrasonic treatment. In addition, through schemes such as immunology and protein interaction experiments, the modifications described in this example can also be enriched and detected.

[0053] III. Data analysis method for the monohydroxyproline modification of type I collagen α2.

[0054] Analysis of the mass spectrometry detection results shows that the total monohydroxyproline at the 3 sites described in this example is 14% of the monohydroxyproline modification of the COL1A2 molecule in this study. And the sample data shows that compared with the control group, the sum of monohydroxyproline at the three sites (the 147th, 471st, and 789th positions) in pancreatic cancer is down-regulated by 3.8 times, and there is a significant statistical difference, p = 5e -4 <0.001( Figure 8)。

[0055] Based on the combined analysis of the data of the number of single hydroxylation modifications of three prolines in the COL1A2 protein molecule in the pancreatic cancer group and the control group, an ROC curve was plotted ( Figure 9 )。

[0056] From the above information, it can be seen that there are significant statistical differences and diagnostic significance in the proline mono-hydroxylation in the COL1A2 protein molecule between the pancreatic cancer group and the control group.

[0057] In the proline mono-hydroxylation of the COL1A2 protein molecule, the single hydroxylated prolines at 3 separate positions also have significant statistical differences: the specific information of positions 147, 471, and 789 is shown in the following figure (Figure 10).

[0058] The ROC curves of the single hydroxylated prolines at these 3 positions in COL1A2 for pancreatic cancer and the control group are as follows ( Figure 11 )。

[0059] From the above information, it can be seen that the single hydroxylated proline at each site in the COL1A2 protein molecule can be used for individual diagnosis, or combined with the remaining sites of this protein as a detection and diagnostic indicator, and both have corresponding accuracy.

[0060] The positions of the protein where the single hydroxylated prolines at 3 positions in COL1A2 are located and the secondary spectra of the peptide segments are as follows ( Figures 12 - 14 )。From the above information with modified secondary spectrum information, it can be seen that these modifications have extremely high credibility.

[0061] The specific Deltamass-related information of the 3 single hydroxylated prolines in COL1A2 is as follows: the protein to which they belong: P08123 (type I collagen alpha 2, COL1A2); the positions in the COL1A2 protein and the peptide segments to which they belong are: proline at position 147 and its peptide segment AGEDGH P GKPGR; proline at position 471 and its peptide segment EGPVGL P GIDGR; proline at position 789 and its peptide segment GDGGPPGMTGF P GAAGR. Deltamass molecular weight: 15.99; Deltamass scores are all: 0.919.

[0062] Example 2 Application of single hydroxylated proline in the COL5A2 protein in the diagnosis of pancreatic cancer

[0063] A rapid mass spectrometry detection method for the single hydroxylation modification of COL5A2.

[0064] Using the mass spectrometry data measured by Skyline analysis for "Technical Solution 2", the chromatographic retention times of the target peptide segments GLTGNPGVQGPEGK, LGPLGAPGEDGR, and DGEVGPSGPVGPPGLAGER were obtained. Combining this retention time with the divalent and trivalent parent ions of the target peptide segments and their corresponding daughter ions to construct an ion list for PRM collected during mass spectrometry detection. (See Table 2. Ion list. Each parent ion can correspond to multiple monovalent, divalent, and trivalent daughter ions. For the convenience of detection, only the daughter ion fragments with relatively high abundance are listed in this example):

[0065] Table 1. GLTGN P Divalent and trivalent parent ions of the GVQGPEGK peptide segment and their corresponding daughter ions

[0066]

[0067] Table 2. LGPLGA P Divalent and trivalent parent ions of the GEDGR peptide segment and their corresponding daughter ions

[0068]

[0069] Table 3. DGEVGPSGPVGP P Divalent and trivalent parent ions of the GLAGER peptide segment and their corresponding daughter ions

[0070]

[0071] Process the collected samples according to "1. Tissue Sample Processing" in Example 1. Use the EASY-nLC 1200 liquid phase system for separation and the online Thermo QE-HFX mass spectrometer for detection. The parameters of the liquid phase separation system are as follows: pre-column: 75μm * 2cm PepMap C18 3μm Thermo; chromatographic column: 75μm * 15cm PepMap C18 2μm Thermo; flow rate: 500 nl / min; mobile phase A: 0.1% formic acid (water); mobile phase B: 0.1% formic acid 80% acetonitrile (water); elution gradient: 6% - 25% B for 21 min, 25% - 40% B for 4 min, 40% - 100% B for 3 min, 100% B for 2 min; sample loading volume: 3 μl. Mass spectrometry detection parameters, data acquisition mode: PRM acquisition mode, top 20 fragment spectra; MS2 resolution (200 m / z): 30,000; MS2 AGC target: 2e 5 , maxIT: 70 ms; normalized collision energy (NCE): 28%; isolation window: 1.6 m / z.

[0072] Adjust the mass spectrometry parameters to enable rapid acquisition and detection of the sample according to the PRM ion list.

[0073] Use Skyline to analyze the data measured by the mass spectrometer, and use internal or external standards to perform relative or absolute quantification of the target ion fragments. Then, based on the quantification results of the ion fragments, qualitatively or quantitatively analyze the sample. Conventional immunoassay or protein interaction protocols can also detect the target modification. In this example, only the PRM protocol is described in detail.

[0074] The single oxidized proline of type V collagen α2 (COL5A2, Uniprot ID: P05997) described in this example can also be used as a therapeutic target for pancreatic cancer. Different forms of protein or small molecule drugs can be prepared targeting the proline mono-oxidation site of COL5A2.

[0075] Detection verification set: Three healthy samples and three samples from clinically diagnosed pancreatic cancer patients (not samples in the analysis set of Example 1) were used as research objects for verification, and the detection results were consistent with the known results.

[0076] Example 3 Application of single oxidized proline in COL1A2 protein in the diagnosis of pancreatic cancer

[0077] Use Skyline to analyze the measured mass spectrometry data to obtain the target peptide segment AGEDGH P GKPGR; EGPVGL P GIDGR; GDGGPPGMTGF P GAAGR chromatographic retention time. Combine this retention time with the divalent and trivalent parent ions of the target peptide segment and their corresponding daughter ions to construct the PRM ion list for acquisition during mass spectrometry detection. (See Table 2. Ion list. Each parent ion can correspond to multiple monovalent, divalent, and trivalent daughter ions. For ease of detection, only the daughter ion fragments with relatively high abundances are listed in this example)

[0078] Table 4. AGEDGH P Divalent and trivalent parent ions of the GKPGR peptide segment and their corresponding daughter ions

[0079]

[0080] Table 5. EGPVGL P Divalent and trivalent parent ions of the GIDGR peptide segment and their corresponding daughter ions

[0081]

[0082] Table 6. GEAGAAG PThe 2-valent and 3-valent parent ions of the AGPAGPR peptide and their corresponding daughter ions

[0083]

[0084] The collected samples are processed according to "I. Tissue sample processing" in Example 1. The liquid phase mass spectrometry conditions described in Example 2 are used for detection. The mass spectrometry parameters are adjusted so that the samples can be quickly collected and detected according to the PRM ion list. The data measured by the mass spectrometer are analyzed using skyline, and the target ion fragments are relatively or absolutely quantified using internal or external standards. Then, based on the quantitative results of the ion fragments, the samples are qualitatively or quantitatively analyzed. Determine whether the patient has pancreatic cancer under the premise of qualitative or quantitative analysis of the sample, or use it as a criterion for its diagnosis.

[0085] The mono-oxidized proline in type I collagen α2 (COL1A2, Uniprot ID: P08123) described in this example can also be used as a therapeutic target for pancreatic cancer. Drugs in different forms such as proteins or small molecules can be prepared targeting the mono-oxidized proline site in COL1A2.

[0086] Detection validation set: 3 healthy samples and 3 clinically confirmed pancreatic cancer patient samples (not belonging to the samples in the analysis set of Example 1) were used as research objects for validation, and the detection results were consistent with the known results.

[0087] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a detection reagent for amino acid site modification in collagen in the preparation of a pancreatic cancer detection product, characterized in that, The modification is proline mono-oxidation modification in the α2 chain of type V collagen and / or proline mono-oxidation modification in the α2 chain of type I collagen; The proline is any one or more of the three sites at positions 578, 593, and 659 in the α2 chain of type V collagen; the Uniprot ID of the protein: P05997; the position of the protein and the peptide it belongs to are: proline at position 578 and its peptide GLTGN P GVQGPEGK; proline at position 593 and its peptide LGPLGA P GEDGR; proline at position 659 and its peptide DGEVGPSGPVGP P GLAGER; The proline is any one or more of the three sites at positions 147, 471, and 789 in the α2 chain of type I collagen; the protein: P08123; the positions in the protein and the peptide segments to which they belong are: proline at position 147 and the peptide segment AGEDGH P GKPGR; proline at position 471 and the peptide segment EGPVGL P GIDGR; proline at position 789 and the peptide segment GDGGPPGMTGF P GAAGR.

2. Use of the detection reagent for amino acid site modification in collagen according to claim 1 in the preparation of a pancreatic cancer detection product, characterized in that, The application is the application of a detection reagent for amino acid site modification in collagen in the preparation of a pancreatic cancer diagnostic kit.

3. Use of the detection reagent for amino acid site modification in collagen according to claim 2 in a pancreatic cancer detection product, characterized in that, The kit includes a cell / tissue sample processing reagent, a protein extraction reagent, a peptide digestion reagent, and a mass spectrometry detection auxiliary reagent.

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