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Application of mass spectrometry to detect the level of psm-e molecules in urine in the preparation of products for early diagnosis of prostate cancer

A technology for prostate cancer and urine, applied in the field of clinical examination and diagnosis, achieves the effects of wide application, convenient sampling and simple operation

Active Publication Date: 2022-03-22
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 spectrometry to detect the level of psm-e molecules in urine in the preparation of products for early diagnosis of prostate cancer
  • Application of mass spectrometry to detect the level of psm-e molecules in urine in the preparation of products for early diagnosis of prostate cancer
  • Application of mass spectrometry to detect the level of psm-e molecules in urine in the preparation of products for early diagnosis of prostate cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Synthesis of standard product of PSM-E characteristic peptide and preparation of standard working solution

[0035] 1. Experimental method

[0036] 1. Synthesis of PSM-E characteristic peptide standard

[0037] The standard PSM-E characteristic peptide was synthesized by Hangzhou Zhongpeptide Biochemical Co., Ltd., and its amino acid sequence is: TILFASWDAEEFGLLGSTEWAEENSRLLQER (SEQ ID NO: 2); 5 mg; purity 99.5%.

[0038] 2. Preparation of PSM-E Characteristic Peptide Standard

[0039] with ddH 2 O Dilute the BSA standard to 1 ng / μl. Set up 8 concentration gradients of protein labeling products: add the standard products to the standard product wells of the 96-well culture plate according to 0, 1, 2, 4, 8, 10, 12, 14, 16, 18, and 20 μl, less than 20 μl The wells were made up to 20 μl with ultrapure water.

[0040] Table 1 Standard sample addition table

[0041]

Embodiment 2

[0042] The detection of PSM-E characteristic peptide content in embodiment 2 urine

[0043] 1. Proteolysis in urine samples

[0044] The method is as follows:

[0045] (1) Transfer the urine sample to a corresponding 10KDa ultrafiltration tube, and centrifuge at 12,000g for 20min;

[0046] (2) Add 200μl of Buffer1 (8M urea / 100mM Tris-HCl, pH 8.5) to each ultrafiltration tube to fully dissolve the denatured protein;

[0047] (3) Add 20 μl DTT (100 mM) solution, react at 37°C for 2 h to reduce disulfide bonds;

[0048] (4) Add 20 μl of IAA (500 mM) solution, and react at room temperature for 15 min in the dark;

[0049] (5) Centrifuge the protein solution after reductive alkylation at 12,000 g for 20 min, and discard the solution at the bottom of the collection tube. Add 200uL Buffer1 and centrifuge, repeat twice;

[0050] (6) Add 200μl Buffer2 (8M urea / 100mM Tris-HCl, pH 8.0), centrifuge at 12,000g for 20min, discard the solution at the bottom of the collection tube, repea...

Embodiment 3

[0065] The detection of PSM-E characteristic peptide content in embodiment 3 urine

[0066] 1. Experimental method

[0067] A total of 132 research subjects were selected, and there were no statistically significant differences in gender, age, blood pressure, smoking, and drug use among the groups, which were comparable. Data were collected from 70 healthy subjects (Control), 30 patients with stage I prostate cancer (PCa-I), 51 patients with stage II prostate cancer (PCa-II), and 65 patients with stage III prostate cancer (PCa-III). Serum, using the method of Example 3, to detect the content of PSM-E characteristic peptide in the urine samples of each group.

[0068] 2. Experimental results

[0069] like figure 2 and 3 As shown, the concentrations of PSM-E characteristic peptide (SEQ ID NO: 2) in the urine of the above groups were 0.9831±0.8322ng / ml, 2.4430±1.3780ng / ml, 5.5170±2.836ng / ml, 6.8740±3.1350, respectively ng / ml. Among them, compared with the healthy group, th...

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Abstract

The invention discloses the application of mass spectrometry technology to detect the PSM-E molecular level in urine in the preparation of products for early diagnosis of prostate cancer. The urine protein sample of the present invention is directly introduced into a liquid chromatography-electrospray ionization-tandem mass spectrometer for measurement after dilution, which can quickly and accurately detect the content level of PSM-E in urine; the fully synthesized PSM-E characteristic peptide standard is adopted The product is used as an internal standard to quantify the analyte, reducing the error caused in the sample pretreatment process; the liquid chromatography tandem mass spectrometry better improves the selectivity and accuracy of the method; the selection and optimization of the chromatographic column and elution conditions improve the The chromatographic separation process shortens the time of chromatographic analysis. The present invention can realize quantitative detection of PSM-E in urine specimens, has simple operation and convenient application, and clarifies that the content of PSM-E in urine can be used as a specific biomarker for prostate cancer detection, which is an early detection method for prostate cancer. Screening provides an efficient and feasible technical method.

Description

technical field [0001] The invention relates to the technical field of clinical inspection and diagnosis, in particular to the application of mass spectrometry technology to detect the level of PSM-E molecules in urine in preparing a product for early diagnosis of prostate cancer. Background technique [0002] According to the "2020 National Cancer Center Annual Work Report", the incidence of prostate cancer in my country ranks ninth (total population), and the mortality rate ranks eighth (total population). The incidence of prostate cancer in my country is rising rapidly, and the age of onset is gradually younger. In addition, 60% to 70% of prostate cancer patients are locally advanced or advanced when they are first diagnosed. At present, there is a lack of accurate and convenient prostate cancer screening technology at home and abroad. Prostate specific membrane antigen (PSMA) is a transmembrane glycoprotein with the function of hydrolyzing folic acid and the neurotrans...

Claims

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Application Information

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Patent Type & Authority Patents(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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