FFPE reference product for proteome quality control, and preparation method and application thereof
A protein and reference product technology, applied in the biological field, can solve the problems of not being able to simulate clinical samples well, lack of uniformity of tissue FFPE samples, and unable to meet the requirements of qualitative and quantitative evaluation of proteomes, etc.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0092] Select single-species sample sample1: human-100%. During the extraction process of each batch of clinical proteome samples, a piece of paraffin tissue sample can be taken for protein extraction of single-species samples, which can be used as the quality of protein extraction for parallel FFPE cohort samples. Standard control product:
[0093] 1) Soak the FFPE slices in a xylene bottle jar in a fume hood to ensure that the tissue is fully soaked below the liquid level, and replace with a new jar after 5-10 minutes each time for a total of three dewaxing; then place them in 100%, 75% %, 50% ethanol for rehydration after dewaxing, 5~10min each time;
[0094] 2) Use a small amount of PBS to soak the surface of the FFPE tissue for about 2 minutes and then remove it. Use a clean blade to scrape the tissue and transfer it to a 1ml EP tube. Add 4% SDS dissolved in 100mM TrisHCl (pH=8.5) lysate and place in an ice water bath Ultrasonic cracking, ultrasonic power 25%, ultrasonic...
Embodiment 2
[0100] Take the FFPE standard plate, which contains 4 groups of different samples. Two samples, sample1 and sample4, are FFPE standard products prepared from pure 100% E.coli and human cells respectively. After protein extraction and enzymatic hydrolysis, the peptides can be directly mixed into Different ratios are used for quality control of mass spectrometry quantitative detection methods; sample2 and sample3 are cell lines of two species mixed in different ratios, and the theoretical sample2:sample3:sample4= can be formed under the condition of equal detection of human cell proteome The preset theoretical ratio of 1:2:4 can simultaneously control the whole process from FFPE protein extraction to mass spectrometry quantitative detection:
[0101] Sample1: human-0% + E.coli-100%
[0102] Sample2: human-25% + E.coli-75%
[0103] Sample3: human-50% + E.coli-50%
[0104] Sample4: human-100% + E.coli-0%.
[0105] 1) Dewaxing and rehydration: Soak the FFPE slices in a xylene ja...
Embodiment 3
[0124] Take the FFPE standard plate, which contains two groups of three different samples. Through the preset quantitative ratio, the quantitative ratio of human protein proteome between Sample1 and Sample2 is 1:1, and the quantitative ratio of E.coli proteome is 1:2. , the quantitative ratio of yeast proteome is 4:1; take two standard samples for proteome preparation, extraction and enzymatic hydrolysis, and mass spectrometry detection. The specific process is as follows:
[0125] Sample1: human-65% + E.coli -15% + Yeast-20%
[0126] Sample2: human-65% + E. coli -30% + Yeast-5%.
[0127] 1) Dewaxing and rehydration: Soak the FFPE slices in a xylene jar in a fume hood to ensure that the tissue is fully immersed below the liquid surface. Replace the jar with a new one after 5-10 minutes each time, and perform dewaxing three times in total; Rehydrate after dewaxing in 100%, 75%, 50% ethanol, 5-10 minutes each time.
[0128] 2) Ultrasonic lysis: Use a small amount of PBS to soa...
PUM
| Property | Measurement | Unit |
|---|---|---|
| length | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More