Application of mass spectrum technology to detection of molecular level of PSM-E in urine in preparation of product for early diagnosis of prostate cancer

A PSM-E, prostate specific technology, applied in the field of clinical examination and diagnosis, to achieve the effect of strong versatility, reducing errors and wide application range

Active Publication Date: 2021-09-28
广州济士源生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recent studies have demonstrated significant inter- and intra-tumor heterogeneity of PSMA, and conflicting data on the biological effects of PSMA on prostate cancer progression have limited its application.

Method used

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  • Application of mass spectrum technology to detection of molecular level of PSM-E in urine in preparation of product for early diagnosis of prostate cancer
  • Application of mass spectrum technology to detection of molecular level of PSM-E in urine in preparation of product for early diagnosis of prostate cancer
  • Application of mass spectrum technology to detection of molecular level of PSM-E in urine in preparation of product for early diagnosis of prostate cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Synthesis of standard working fluids in Example 1 PSM-E

[0035] First, experimental method

[0036] 1, Synthesis of PSM-E Characteristic Peptide Standards

[0037] The PSM-E feature peptide standard is synthesized via Hangzhou Zhongopeptide Co., Ltd., its amino acid sequence is: TilfaswdaeefllgstewaeenSrlllll (SEQ ID NO: 2); 5 mg; purity 99.5%.

[0038] 2, preparation for PSM-E characteristics peptide standard

[0039] Use DDH 2 O Dilute the BSA standard to 1 ng / μL. Set 8 concentration gradient protein labels: Standard product is added to the standard pores of 96-well cultural sheets by 0, 1, 2, 4, 8, 10, 12, 14, 16, 18, and 20 μL, less than 20 μl Hole with ultrapure water to 20 μL.

[0040] Table 1 Standard product plus sample table

[0041]

Embodiment 2

[0042] Detection of Example 2 wherein the content of the peptide in urine embodiment PSM-E

[0043] A urine specimen proteolysis

[0044] A method as follows:

[0045] (1) The urine samples were transferred to the corresponding 10KDa ultrafiltration tubes, 12,000g 20min centrifugation;

[0046] (2) per each ultrafiltration plus 200μl Buffer1 (8M urea / 100mM Tris-HCl, pH 8.5), sufficiently soluble denatured protein;

[0047] (3) Add 20μl DTT (100mM) solution, 37 ℃ for 2h reduce disulfide bonds;

[0048] (4) Add 20μl IAA (500mM) was dark at room temperature the reaction 15min;

[0049] (5) the protein solution was centrifuged 12,000g reductive alkylation after 20min, the bottom of the collection tube was discarded. Add 200uL Buffer1 centrifugation was repeated twice;

[0050] (6) Add 200μl Buffer2 (8M urea / 100mM Tris-HCl, pH 8.0), 12,000g centrifugation 20min, the bottom of the collection tube was discarded, repeat 1;

[0051] (7) Add 200μl solution of ammonium bicarbonate (25 mM...

Embodiment 3

[0065] Detecting a feature peptide of Example 3 embodiment urine PSM-E

[0066] First, experimental method

[0067] Enrolled 132 subjects, differences among groups of sex, age, blood pressure, smoking, drug use and other leavened dough were not statistically significant, comparable. Collected 70 healthy persons group (Control), the patient 30 Example I prostate carcinoma (PCa-I), 51 Example II prostate carcinoma (PCa-II) and 65 stage III prostatic carcinoma (PCa-III) the method of Example 3 sera, using, the content of each group detected in urine specimens PSM-E characteristics peptide.

[0068] Second, the experiment

[0069] like figure 2 with 3 Shown, PSM-E wherein peptide (SEQ ID NO: 2) in the urine for the concentration of each group were 0.9831 ± 0.8322ng / ml, 2.4430 ± 1.3780ng / ml, 5.5170 ± 2.836ng / ml, 6.8740 ± 3.1350 ng / ml. Which, compared with the healthy group, in urine samples I-III prostate patients PSM-E features peptide were significantly increased, and the dif...

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Abstract

The invention discloses application of A mass spectrum technology to detection of molecular level of PSM-E in urine in preparation of product for early diagnosis of prostate cancer A urine protein sample is diluted and then is directly introduced into a liquid chromatography-electrospray ionization-tandem mass spectrometer for determination, and the content level of the PSM-E in the urine can be rapidly and accurately detected; a fully-synthesized PSM-E characteristic peptide standard substance is adopted as an internal standard quantitative analysis substance, so that errors caused in a sample pretreatment process are reduced; the selectivity and the accuracy of the method are better improved by the liquid chromatography-tandem mass spectrometry; through selection and optimization of a chromatographic column and elution conditions, the chromatographic separation process is improved, and the chromatographic analysis time is shortened. According to the invention, the quantitative detection of the PSM-E in the urine sample can be realized, the operation is simple, the application is convenient, the content of the PSM-E in the urine can be used as a specific biomarker for detecting the prostatic cancer, and an efficient and feasible technical method is provided for early screening of the prostatic cancer.

Description

Technical field [0001] The present invention relates to the field of clinical testing, in particular, to mass spectrometry techniques to detect PSM-E molecular levels in urine in the preparation of early diagnostic prostate cancer products. Background technique [0002] According to the "2020 National Cancer Center Annual Work Report", the incidence of prostate cancer in my country is ranked ninth (total population), and the mortality is ranked eighth (total population). my country's prostate cancer has increased rapid incidence, and the age of onset is gradually emerging. Moreover, 60% to 70% of patients with prostate cancer are already local late or late. At present, there is a lack of accurate and convenient prostate cancer screening techniques at home and abroad. Prostate Specific Membranentigen (PSMA) is a cross-membrane glycoprotein having a hydrolyzed folic acid and neurotransmitter N-acetyl asparamide function, high expression in prostate cancer, which uses it as a diagno...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574G01N33/68C07K14/705G01N30/02G01N30/72
CPCG01N33/57434G01N33/57492G01N33/68C07K14/4748G01N30/02G01N30/72G01N2333/705
Inventor 张娟张甜
Owner 广州济士源生物技术有限公司
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