CN113447654A - Application of mass spectrometry technology in detecting PSM-E molecular level in urine in preparation of products for early diagnosis of prostate cancer - Google Patents
Application of mass spectrometry technology in detecting PSM-E molecular level in urine in preparation of products for early diagnosis of prostate cancer Download PDFInfo
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Abstract
The invention discloses an application of detecting PSM-E molecular level in urine by a mass spectrometry technology in preparation of a product for early diagnosis of prostate cancer. The urine protein sample is diluted and then directly introduced into a liquid chromatography-electrospray ionization-tandem mass spectrometer for determination, so that the PSM-E content level in urine can be rapidly and accurately detected; the fully-synthesized PSM-E characteristic peptide standard substance is used as an internal standard quantitative analyte, so that errors caused in the pretreatment process of a sample are reduced; the liquid chromatogram tandem mass spectrometry method has the advantages that the selectivity and the accuracy of the method are better improved; the selection and optimization of chromatographic columns and elution conditions improve the chromatographic separation process and shorten the chromatographic analysis time. The invention can realize the quantitative detection of PSM-E in the urine specimen, has simple operation and convenient application, and confirms that the PSM-E content in the urine can be used as a specific biomarker for detecting the prostatic cancer, thereby providing an efficient and feasible technical method for early screening of the prostatic cancer.
Description
Technical Field
The invention relates to the technical field of clinical examination and diagnosis, in particular to application of mass spectrometry technology in detecting PSM-E molecular level in urine in preparation of products for early diagnosis of prostate cancer.
Background
According to the report of '2020 national cancer center annual work report', the incidence rate of prostate cancer in China is the ninth (general population) and the death rate is the eighth (general population). The incidence rate of the prostate cancer in China is increased rapidly, and the incidence age is gradually younger. Furthermore, 60% to 70% of prostate cancer patients are already locally advanced or advanced at the time of initial diagnosis. At present, accurate and convenient prostate cancer screening technology is lacked at home and abroad. Prostate Specific Membrane Antigen (PSMA) is a transmembrane glycoprotein with the functions of hydrolyzing folate and neurotransmitter N-acetyl asparaginase, is highly expressed in prostate cancer, and has been widely noticed in recent years as a diagnostic indicator and a potential therapeutic target for prostate cancer. Recent studies have shown significant intratumoral and intratumoral heterogeneity of PSMA, and conflicting data regarding the biological effects of PSMA on prostate cancer progression have emerged, limiting its use.
Liquid chromatography tandem mass spectrometry (LC-MS/MS) is widely applied to detection of proteins or amino acids in biological matrixes such as blood and the like due to high sensitivity and selectivity. The moderately complex protein sample with high mixing degree needs to be analyzed by LC-MS/MS, and dozens to hundreds of proteins can be identified at one time; in addition to protein identification, if deep analysis of protein sequence information, such as site amino acid mutation, post-translational modification, etc., is required, the sequence fragments of the protein should be scanned as many as possible to obtain a high coverage rate.
LC-MS/MS analysis firstly carries out enzymolysis on a protein solution to obtain a peptide fragment mixture, liquid chromatography separation is carried out on the peptide fragment mixture, such as a C18 reverse phase column of LC, the separated peptide fragment component is ionized by an ion source and then enters a tandem mass spectrometry, and the obtained mass spectrometry data is retrieved and matched with a corresponding database. A signature peptide refers to a polypeptide whose amino acid sequence has unique specificity in the trypsin digestion product of a target protein.
The novel splicing variant PSM-E of the prostate specific membrane antigen as a novel splicing variant of PSMA shows different biological functions and better tumor specificity, and PSM-E is specifically and highly expressed in prostate cancer and shows the biological characteristics of inhibiting the development of the prostate cancer, thereby providing potential for becoming a novel diagnosis and treatment target of the prostate cancer. The prior art (Dianthus superbus, peripheral blood quantitative expression detection analysis of novel prostate specific membrane antigen splice variant PSM-E and clinical significance discussion thereof [ D ]. Zhongshan university, 2009.) specifically discloses that peripheral blood PSM-E detection can be used for diagnosing PCa, PSM-E is superior to PSMA in sensitivity and specificity for diagnosing PCa, PSM-E is superior to PSMA in detection sensitivity and operability.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides the application of mass spectrometry technology in detecting the PSM-E molecular level in urine in the preparation of products for early diagnosis of prostate cancer.
In order to achieve the purpose, the invention is realized by the following scheme:
the first purpose of the invention is to provide the application of the substance for detecting the level of the prostate specific membrane antigen splice variant PSM-E in urine protein in the preparation of products for diagnosing prostate cancer.
The second purpose of the invention is to provide a prostate specific membrane antigen splice variant PSM-E characteristic peptide segment.
It is a third object of the present invention to provide a product for diagnosing prostate cancer.
The invention utilizes liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with proteomics software to determine a characteristic peptide segment capable of identifying and distinguishing PSM-E. And directionally monitoring the polypeptide mixture after proteolysis of the urine sample to be detected through the constructed mass spectrum information of the characteristic peptide segment, thereby realizing quantitative detection of the content of PSM-E in urine. The principle is as follows: the response peak area of the characteristic peptide segment in the mass spectrum and the content of the PSM-E total protein form a linear relation, a standard curve is established between the response peak area of the mass spectrum and the content of the PSM-E total protein, the actual amount of the PSM-E total protein is represented by the spectrum peak area of the peptide segment, and the PSM-E content in the urine sample to be detected is obtained through calculation of the standard curve.
Therefore, the invention claims the application of the substance for detecting the level of the prostate specific membrane antigen splice variant PSM-E in urine protein in the preparation of products for diagnosing prostate cancer.
Preferably, the substance is a substance for detecting the level of prostate specific membrane antigen splice variant PSM-E protein.
Preferably, the level of the prostate specific membrane antigen splice variant PSM-E in the urine protein is detected by detecting the full-length prostate specific membrane antigen splice variant PSM-E and/or the characteristic peptide segment of the prostate specific membrane antigen splice variant PSM-E.
More preferably, the full-length amino acid sequence of prostate specific membrane antigen splice variant PSM-E is set forth in SEQ ID NO: 1 is shown.
More preferably, the amino acid sequence of the PSM-E characteristic peptide fragment of the prostate specific membrane antigen splice variant is as shown in SEQ ID NO: 2, respectively.
SEQ ID NO：2：TILFASWDAEEFGLLGSTEWAEENSRLLQER。
Preferably wherein the substance is for detection by one or more of the following methods: multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, immunohistochemistry, chemiluminescence, radioisotope method, fluorescence (such as immunofluorescence), enzyme labeling, colloidal gold method, biochip detection, and western blotting.
The invention also claims a prostate specific membrane antigen splice variant PSM-E characteristic peptide segment, wherein the amino acid sequence of the prostate specific membrane antigen splice variant PSM-E characteristic peptide segment is shown as SEQ ID NO: 2, respectively.
A product for diagnosing prostate cancer can detect the level of prostate specific membrane antigen splice variant PSM-E in urine protein.
Preferably, the level of the prostate specific membrane antigen splice variant PSM-E in the urine protein is detected by detecting the full-length prostate specific membrane antigen splice variant PSM-E and/or the characteristic peptide segment of the prostate specific membrane antigen splice variant PSM-E.
More preferably, the product comprises an amino acid sequence as set forth in SEQ ID NO: 1, and/or the amino acid sequence of the prostate specific membrane antigen splice variant PSM-E is shown as SEQ ID NO: 2, and 2, showing the characteristic peptide segment of the prostate specific membrane antigen splice variant PSM-E.
Compared with the prior art, the invention has the following beneficial effects:
1. the LC-MS/MS does not need a specific antibody reagent, and has the advantages of high selectivity, high specificity, high sensitivity and the like, and post-transcriptional modification and mutation forms of the protein.
2. Because the mass-to-charge ratio of the measured object is directly measured by the target mass spectrum, the International System of Units (SI) traceability is easily realized by using an isotope or other marker dilution calibration System (including internal standardization and external calibration). The immune method has the problems of non-specific antibody binding, autoantibody interference and the like, so that the traceability of the immune method has a larger challenge.
3. The operation is simple, the result is stable, and the whole process only needs within 2 h.
4. The kit is suitable for early diagnosis of prostate cancer, can be used for early screening of various diseases by one tube of urine specimen, and has strong universality.
5. The application range is wide, the urine sampling is convenient, the device is completely noninvasive, and the device can sample at home. Contamination of the urine sample with DNA, hemoglobin, albumin, lipids, etc. will not affect the results.
The invention carries out directional detection on the polypeptide mixture after the proteolysis of the urine sample to be detected through the constructed mass spectrum information of the characteristic peptide segment, thereby realizing the quantitative detection of the content of PSM-E in the urine. The urine protein sample is diluted and then directly introduced into a liquid chromatography-electrospray ionization-tandem mass spectrometer for determination, and the PSM-E content level in urine can be rapidly and accurately detected. The invention relates to a brand-new method for measuring PSM-E content in urine, which adopts a fully-synthesized PSM-E characteristic peptide standard product as an internal standard quantitative analyte and can reduce errors caused in the pretreatment process of a sample. The selectivity and the accuracy of the method are better improved by applying the liquid chromatography-tandem mass spectrometry. Through the selection and optimization of the chromatographic column and the elution conditions, the chromatographic separation process is better improved, and the chromatographic analysis time is shortened. The invention can realize the quantitative detection of PSM-E in the urine specimen, has simple operation and convenient application, and proves that the PSM-E content in the urine can be used as a specific biomarker for detecting the prostatic cancer, thereby providing an efficient and feasible technical method for early screening of the prostatic cancer.
Drawings
FIG. 1 shows the secondary mass spectrum of PSM-E characteristic peptide.
FIG. 2 shows the amount of PSM-E signature peptide in the urine of healthy and stage I-III prostate cancer patients.
FIG. 3 is an observer operating characteristic (ROC) curve of PSM-E signature peptide as a marker for prostate cancer.
Detailed Description
The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
EXAMPLE 1 Synthesis of a Standard for the PSM-E signature peptide and preparation of a Standard working solution
First, experiment method
1. Synthesis of PSM-E characteristic peptide standard product
The PSM-E characteristic peptide standard product is synthesized by Hangzhou Zhongji peptide biochemistry limited company, and the amino acid sequence of the PSM-E characteristic peptide standard product is as follows: TILFASWDAEEFGLLGSTEWAEENSRLLQER (SEQ ID NO: 2); 5 mg; the purity is 99.5%.
2. Preparation of PSM-E characteristic peptide standard product
By ddH2O diluted BSA standard to 1 ng/. mu.l. Protein markers of 8 concentration gradients were set: the standard was added to the standard wells of a 96-well plate in 0, 1, 2, 4, 8, 10, 12, 14, 16, 18, and 20. mu.l, and less than 20. mu.l of the wells were filled with ultrapure waterMake up to 20. mu.l of water.
TABLE 1 sample application table for standard
Example 2 detection of PSM-E characteristic peptide content in urine
First, proteolysis in urine specimen
The method comprises the following specific steps:
(1) transferring the urine sample to a corresponding ultrafiltration tube with 10KDa, and centrifuging for 20min at 12,000 g;
(2) add 200. mu.l Buffer1(8M urea/100mM Tris-HCl, pH 8.5) to each ultrafiltration tube to dissolve the denatured protein well;
(3) adding 20. mu.l of DTT (100mM) solution, and reacting at 37 ℃ for 2h to reduce the disulfide bond;
(4) adding 20 μ l IAA (500mM) solution, and reacting for 15min at room temperature in dark place;
(5) the protein solution after reductive alkylation at 12,000g was centrifuged for 20min and the bottom solution of the collection tube was discarded. Adding 200uL Buffer1, centrifuging, and repeating for 2 times;
(6) add 200. mu.l Buffer2(8M urea/100mM Tris-HCl, pH 8.0), centrifuge at 12,000g for 20min, discard the bottom solution of the collection tube, repeat 1 time;
(7) adding 200 μ l ammonium bicarbonate solution (25mM), centrifuging at 12,000g for 20min, discarding the solution at the bottom of the collection tube, and repeating for 2 times;
(8) replacing a new collecting pipe, adding 100 mu l of pancreatin solution (0.01 mu g/mu l), and reacting for 14h at 37 ℃;
(9) taking out the ultrafiltration tube, centrifuging for 20min at 12,000g, and collecting peptide fragments after enzymolysis;
(10) adding 100 μ l ammonium bicarbonate solution (25mM) into the ultrafilter tube, centrifuging at 12,000g for 10min, collecting tube bottom solution, and mixing with the previous solution;
(11) and (5) centrifugally drying, and putting on a machine.
Second, LC-MS/MS analysis
The resulting digested polypeptide sample was dried by centrifugation and redissolved in Nano-LC mobile phase A (volume ratio: 0.1% formic acid/water)The bottles were loaded for on-line LCMS analysis. The solubilized sample was applied to a nanobipe C18 pre-column in a volume of 2 μ l (3 μm,
) Then 20. mu.l of mobile phase A was desalted by flushing.
The liquid phase is Easy nLC 1200 nanoliter liquid phase system (ThermoFisher, USA), the sample is desalted and retained on the pre-column, and then separated by analytical column, the specification of the analytical column is C18 reversed phase chromatographic column (Acclaim PepMap RSLC,75 μm × 25cm C18-2 μm)
) Gradient was mobile phase B (volume ratio: 80% acetonitrile, 0.1% formic acid) by volume: from 5% to 38%.
Mass Spectrometry A ThermoFisher Q active system (ThermoFisher, USA) was used in combination with a nanoliter nebulizing Nano Flex ion source (ThermoFisher, USA), the nebulizing voltage was 1.9kV, and the ion transfer tube heating temperature was 275 ℃.
The mass spectrum scanning mode is in an information-Dependent acquisition working mode (DDA, Data Dependent Analysis), the primary mass spectrum scanning resolution is 70000, the scanning range is 350-2000 m/z, and the maximum injection time is 100 ms. At most 20 secondary spectra with charges of 2+ to 5+ are collected under each DDA cycle, and the maximum ion injection time of the secondary mass spectrum is 50 ms. The collision cell energy (high energy collision induced dissociation, HCD) was set to 28eV for all precursor ions and the dynamic exclusion was set to 25 s.
Third, interpretation of results
The PSM-E characteristic peptide fragment sequence and its main reference mass spectrum parameters are shown in Table 2. The secondary mass spectrum of the PSM-E signature peptide is shown in FIG. 1.
Table 2:
example 3 detection of PSM-E characteristic peptide content in urine
First, experiment method
The 132 subjects were selected in total, and the differences in the onset of sex, age, blood pressure, smoking, drug use, etc. among the groups were not statistically significant and were comparable. Serum was collected from 70 healthy subjects (Control), 30 stage I prostate cancer subjects (PCa-I), 51 stage II prostate cancer subjects (PCa-II), and 65 stage III prostate cancer subjects (PCa-III), and the amount of PSM-E signature peptide in urine samples from each subject was determined using the method of example 3.
Second, experimental results
As shown in FIGS. 2 and 3, the concentration of PSM-E signature peptide (SEQ ID NO: 2) in the urine of each group was 0.9831 + -0.8322 ng/ml, 2.4430 + -1.3780 ng/ml,5.5170 + -2.836 ng/ml and 6.8740 + -3.1350 ng/ml, respectively. Wherein, compared with healthy patients, the PSM-E characteristic peptide content in the urine specimen of the prostate patients in stages I-III is obviously increased, and the difference has statistical significance. Using observer operating characteristic curve (ROC) analysis, the area under the ROC curve (AUC) was 0.9424 (95% CI,0.9082 to 0.9765) in each group of specimens, and the difference was statistically significant (p < 0.0001). And the cutoff value of the PSM-E characteristic peptide can be analytically calculated to be 2.249ng/ml (above which is a prostate cancer patient, below which is a healthy person), the sensitivity is 89.92% and the specificity is 90.14%.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Sequence listing
<110> Guangzhou Ji-Shi-Yuan Biotechnology Co., Ltd
Application of mass spectrometry technology in detecting PSM-E molecular level in urine in preparation of products for early diagnosis of prostate cancer
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Claims (10)
1. The application of the substance for detecting the level of the prostate specific membrane antigen splice variant PSM-E in urine protein in the preparation of products for diagnosing prostate cancer.
2. The use according to claim 1, wherein the substance is a substance for detecting the level of prostate specific membrane antigen splice variant PSM-E protein.
3. The use of claim 2, wherein the level of prostate specific membrane antigen splice variant PSM-E in the urine protein is detected as full length prostate specific membrane antigen splice variant PSM-E, and/or a prostate specific membrane antigen splice variant PSM-E signature peptide.
4. The use according to claim 3, wherein the full-length amino acid sequence of the prostate specific membrane antigen splice variant PSM-E is as set forth in SEQ ID NO: 1 is shown.
5. The use according to claim 3, wherein the amino acid sequence of the PSM-E signature peptide fragment of the splice variant of prostate specific membrane antigen is as set forth in SEQ ID NO: 2, respectively.
6. Use according to claim 1, wherein the substance is for detection by one or more of the following methods: multidimensional liquid chromatography tandem mass spectrometry, immunohistochemistry, chemiluminescence, radioisotope, fluorescence, enzyme labeling, colloidal gold, biochip, and western blotting.
7. A prostate specific membrane antigen splice variant PSM-E signature peptide fragment, wherein the amino acid sequence of the prostate specific membrane antigen splice variant PSM-E signature peptide fragment is as shown in SEQ ID NO: 2, respectively.
8. A product for diagnosing prostate cancer, which is capable of detecting the level of prostate specific membrane antigen splice variant PSM-E in urine protein.
9. The product of claim 8, wherein the level of prostate specific membrane antigen splice variant PSM-E in the urine protein is detected as full length prostate specific membrane antigen splice variant PSM-E, and/or a prostate specific membrane antigen splice variant PSM-E signature peptide.
10. The product of claim 8, wherein the product comprises an amino acid sequence as set forth in SEQ ID NO: 1, and/or the amino acid sequence of the prostate specific membrane antigen splice variant PSM-E is shown as SEQ ID NO: 2, and 2, showing the characteristic peptide segment of the prostate specific membrane antigen splice variant PSM-E.
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| CN112480260A (en) * | 2020-12-09 | 2021-03-12 | 福州迈新生物技术开发有限公司 | anti-PSMA protein monoclonal antibody, cell line, preparation method and application thereof |
| CN112501206A (en) * | 2021-02-09 | 2021-03-16 | 百奥赛图(北京)医药科技股份有限公司 | Construction method and application of PSMA (PSMA) gene humanized non-human animal |
| US20210128710A1 (en) * | 2019-11-04 | 2021-05-06 | Inovio Pharmaceuticals, Inc. | Combination therapy to treat brain cancer |
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| US20210128710A1 (en) * | 2019-11-04 | 2021-05-06 | Inovio Pharmaceuticals, Inc. | Combination therapy to treat brain cancer |
| CN112480260A (en) * | 2020-12-09 | 2021-03-12 | 福州迈新生物技术开发有限公司 | anti-PSMA protein monoclonal antibody, cell line, preparation method and application thereof |
| CN112501206A (en) * | 2021-02-09 | 2021-03-16 | 百奥赛图(北京)医药科技股份有限公司 | Construction method and application of PSMA (PSMA) gene humanized non-human animal |
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| CAO, KAI-YUAN， ET AL.: "High expression of PSM-E correlated with tumor grade in prostate cancer: A new alternatively spliced variant of prostate-specific membrane antigen", 《PROSTATE》 * |
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