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Application of seminal plasma extracellular vesicle SLC5A12 protein

A technology of 1. SLC5A12 and protein, which is applied in the field of protein detection and can solve the problems of difficult detection and analysis of low-abundance proteins

Active Publication Date: 2021-03-12
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, semen, like other body fluids, contains a large number of high-abundance proteins, making it difficult to detect and analyze low-abundance proteins

Method used

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  • Application of seminal plasma extracellular vesicle SLC5A12 protein
  • Application of seminal plasma extracellular vesicle SLC5A12 protein
  • Application of seminal plasma extracellular vesicle SLC5A12 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1. Enrichment of extracellular vesicles from seminal plasma by differential centrifugation

[0026] 1. Experimental materials:

[0027] Inclusion criteria for NS samples: According to the 2010 World Health Organization guidelines, semen analysis showed normal sperm count (≥15.0 ×10 6 / mL), normal pH value (7.20-8.00), normal sperm motility (32.0%-100%). Three times of centrifugation at 3000rpm for 15 minutes and no sperm detected is considered as azoospermia. NOA inclusion criteria: FSH>12 IU / L and testicular volume less than 12mL or testicular histopathology showed reduced spermatogenesis or no germ cells; exclusion criteria: 1) Known acquired diseases, such as chemotherapy, bilateral cryptorchidism , Testicular torsion; 2) Genetic factors, such as karyotype aberration, y chromosome AZF deletion. The inclusion criteria for OA were a large number of sperm obtained by orchial aspiration or percutaneous epididymis aspiration (PESA); patients with congenital abs...

Embodiment 2

[0036] Example 2. Quantification of sEV protein expression in NS, NOA and OA using TMT marker technology

[0037] 1. Experimental materials:

[0038]A total of 27 sEV samples were detected, including 9 NS, 9 NOA and 9 OA.

[0039] 2. Experimental process:

[0040] (1) After the sEV protein was extracted and digested with trypsin, it was labeled with TMT-10plex. Then, the watersM-class HPLC liquid phase system was used to perform high pH reverse-phase separation on the labeled and mixed peptide samples, and the separated components were separated by Easy nLC 1200 (ThermoFisher) liquid chromatography on-line and analyzed using high-resolution mass spectrometer Orbitrap Fusion Lumos (ThermoFisher) was used for detection.

[0041] (2) Based on the human protein sequence obtained in the Universal Protein Resource Database (UniProt) (2018_07) and using MaxQuant software (1.6.5.0) to search the original mass spectrum file.

[0042] (3) Perseus software was used to conduct one-way...

Embodiment 3

[0044] Example 3. Application of PRM relative quantitative technology to verify sEV differential proteins in NS, NOA and OA groups

[0045] The differential proteins were screened according to the following screening conditions: (1) NS>OA, NOA>OA (2) The differential proteins had specific peptides suitable for PRM experiments. After meeting the above criteria, 10 significantly different sEV proteins and 1 non-differential protein DNM1L were selected as controls for targeting verification.

[0046] 1. Experimental materials:

[0047] A total of 42 sEV samples were validated, including 10 NS, 23 NOA and 9 OA.

[0048] 2. Experimental process:

[0049] (1) Design the specific peptide of the target protein, and entrust China Jietai Company to synthesize the crude isotope-labeled heavy-labeled peptide (heavy).

[0050] (2) sEV protein extraction, trypsin digestion into peptides, adding crude heavy to 600ng of sEV peptides, and then using the PRM mode of Orbitrap Fusion Lumos (Th...

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Abstract

Application of seminal plasma extracellular vesicle SLC5A12 protein belongs to the field of protein detection. In the application, protein carried by sEV of NS, NOA and OA groups is quantitatively analyzed through adopting a TMT labeling technology, inter-group differential protein is identified, the biomarker SLC5A12 is successfully verified through a PRM technology, and the protein can be used for diagnosing and distinguishing non-obstructive azoospermia and obstructive azoospermia.

Description

technical field [0001] The invention belongs to the field of protein detection, and in particular relates to the application of biomarkers for the differential diagnosis of seminal plasma cell extracellular vesicles in obstructive and non-obstructive azoospermia. Background technique [0002] Azoospermia accounts for about 10%-15% of all infertile men, and it is usually divided into two categories: obstructive azoospermia (OA) and non-obstructive azoospermia (NOA). The spermatogenesis function in the testes of OA patients is normal, but the obstruction of the male reproductive tract, such as pathological blockage of the epididymis, vas deferens or ejaculatory duct, or congenital abnormalities lead to sperm transport obstacles, accounting for about 40% of all azoospermia cases. NOA is one of the most serious forms of male infertility caused by testicular failure. Accurate diagnosis and classification of azoospermia is crucial, because the methods of sperm collection and tr...

Claims

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Application Information

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IPC IPC(8): G01N27/62G01N33/68C12N5/076
CPCG01N33/6848G01N33/6893G01N27/62C12N5/061G01N2800/367C12N2509/10
Inventor 郭雪江沙家豪杨晓玉郭曰帅姚礼萍
Owner NANJING MEDICAL UNIV
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