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Hair shaft protein extraction method suitable for mass spectrum identification and application of hair shaft protein extraction method

An extraction method and protein technology, applied in the biological field, can solve problems such as difficulty in protein extraction, protein loss, impact analysis, etc., and achieve the effects of shortening extraction time, simple operation, and improving success rate.

Pending Publication Date: 2021-09-17
上海君谊生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the amount of hair shaft varies from person to person, under special conditions, we can only obtain a limited amount of hair shaft, and it is difficult to extract the protein at this time. If the extraction method and conditions are not suitable, it is easy to cause a large loss of protein , thus affecting the subsequent analysis

Method used

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  • Hair shaft protein extraction method suitable for mass spectrum identification and application of hair shaft protein extraction method
  • Hair shaft protein extraction method suitable for mass spectrum identification and application of hair shaft protein extraction method
  • Hair shaft protein extraction method suitable for mass spectrum identification and application of hair shaft protein extraction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0129] Embodiment 1 Ultrasonic treatment device

[0130] The ultrasonic processing device in this embodiment is used to break up biological samples. figure 1 is a schematic top view of the transducer of the ultrasonic processing device, figure 2 It is a schematic side view of the transducer of the ultrasonic processing device, in which the section shows the ultrasonic generating groove and the connecting hole. The ultrasonic processing device includes a transducer 1 and an ultrasonic generator (not shown). The transducer in this embodiment is a transmitting transducer, and the transducer adopts a piezoelectric crystal vibrator and an amplitude modulator, wherein the ultrasonic generator also includes a control unit, and the control unit is used to control the ultrasonic generation intensity, generation time and working interval pause time. Such as figure 1 , figure 2 As shown, the transducer 1 includes a first end 12 and an opposite end 11 remote from the first end. Th...

Embodiment 2

[0132] Embodiment 2 is applicable to the preparation of hair shaft protein identified by mass spectrometry

[0133] Hair shaft protein preparation comprises the following steps:

[0134] S101, hair shaft sample pretreatment.

[0135] Use an electronic scale to weigh a certain amount of hair sample and place it in a 1.5ml EP tube, add deionized water, vortex, discard the supernatant, repeat three times, wash away the impurities in the hair, and use tweezers to place the hair on the aluminum foil Cut into fragments with a length of 1-8 cm, and then place the cut hair samples in a 1.5 ml centrifuge tube (EP tube).

[0136] Grind the washed hair samples in a liquid nitrogen environment at -36°C to 0°C using a grinder. After starting the set program, the hair sample is basically powdered after grinding, and the sample powder is collected into a new EP tube.

[0137] Table 2 Comparison of the amount of protein extracted by different grinding methods

[0138] grinding...

Embodiment 3

[0156] Example 3 Protein Spectrum Detection

[0157] use as Figure 5 The shown capillary analysis column (capillary chromatography column) suitable for the chromatography-mass spectrometry system performs mass spectrometry analysis on the peptide mixture obtained in Example 2 after the treatment of the hair shaft sample. The characteristics of the capillary analytical column are as described above.

[0158] Install a passive flow splitter in front of the analytical column. The build-up of salt or other residues on the outer surface of the launch tip is eliminated by using a computer-controlled electrospray positioning device to automatically move the launch tip under the cleaning unit.

[0159] use attached Figure 5 The exhaust column platform shown is used for automatic injection analysis of peptides. The autosampler injects the peptides of the sample prepared in Example 2, and passes a split device at the front end of the analysis column to make the flow rate ≤ 1nL / min....

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Abstract

The invention relates to a hair shaft protein extraction method suitable for mass spectrometry detection, which comprises the following steps: a solubilizing solution is added into a sample, ultrasonic treatment is carried out, wherein an ultrasonic treatment device adopted by the ultrasonic treatment comprises a transducer and an ultrasonic generator, a first end of the transducer is connected with the ultrasonic generator, one or more ultrasonic generation tanks are arranged at the opposite end, far away from the first end, of the transducer; and a mixture of the sample and the solubilizing solution is directly put into the ultrasonic generation tanks for ultrasonic treatment; a precipitation solution is added into the sample protein mixture to precipitate protein, and a solubilizing solution is added to redissolve protein precipitate; and an enzyme is added to the mixture for enzyme digestion of the protein to obtain a peptide mixture. According to the extraction method disclosed by the invention, efficient protein extraction is carried out on a sample, and then a mass spectrometer is adopted to carry out high-sensitivity proteomics detection to obtain mass spectrum data which can be further used for SNP site detection and individual identification, so that the problem that individual identification is carried out by taking a hair shaft as a sample can be effectively solved.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a protein extraction method for mass spectrometry detection, in particular to a hair shaft protein extraction method suitable for mass spectrometry identification and its application in ultra-high sensitivity protein spectrum detection and detection. Background technique [0002] DNA is the genetic information carrier of organisms. With the rapid development of molecular biology technology, DNA information analysis is used for species identification or individual identification in many fields. Single nucleotide polymorphism (single nucleotide polymorphism, SNP) refers to the DNA sequence polymorphism caused by a single nucleotide variation at the genome level, and is a new generation of genetic markers that can be used for identification. SNP can be used as a marker for individual identification, and detection methods for SNP sites usually rely on DNA extraction. [0003] Hair, as an e...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N27/62C07K1/30C07K1/14
CPCG01N33/6848G01N27/62C07K1/30C07K1/14
Inventor 戚良
Owner 上海君谊生物科技有限公司
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