N<15> stable isotope labeling method for biological protein quantifying and tracing

A technology of stable isotope and labeling method, which is applied in the field of N15 stable isotope labeling of biological protein quantification and tracing, can solve the problems of low labeling efficiency of primary cell culture, expensive SILAC reagents, and high test cost, and achieves short test period and high cost. The effect of practical value and low test cost

Inactive Publication Date: 2016-11-16
广州微因生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 2. The operation is cumbersome
SILAC quantitative process includes: cell culture→protein extraction→immunoprecipitation→electrophoretic separation→enzymatic digestion→mass spectrometry detection→quantitative detection, the operation is complicated, the labeling efficiency of primary cell culture is low, and it is inconvenient to implement
[0007] 3. High test cost
SILAC reagents are very expensive
[0008] 4. Long test cycle

Method used

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  • N&lt;15&gt; stable isotope labeling method for biological protein quantifying and tracing
  • N&lt;15&gt; stable isotope labeling method for biological protein quantifying and tracing
  • N&lt;15&gt; stable isotope labeling method for biological protein quantifying and tracing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1 (N 15 Preparation of bacterial culture medium)

[0056] N for quantification and tracking of biological proteins described in this example 15 Stable isotope labeling method, specifically N 15 The preparation method of stable isotope labeling medium, comprises the following steps:

[0057] 1) Use only N 15 (excluding N 14 ) of the inorganic medium to cultivate the yeast;

[0058] Here, Pichia pastoris x-33 and basal salt medium (M9 mother liquor) are used;

[0059] 2) using the glass bead breaking method to lyse the yeast cells;

[0060] 3) Using protein semihydrolysis (acid hydrolysis) for N 15 Crude extraction of yeast protein;

[0061] 4)N 15 The preparation of bacterial culture medium, promptly will step 3) the N that produces after rough extraction 15 Peptone is used as the basic raw material of the medium, replacing the conventional N 14 Peptone is used for cultivation, and then adding inorganic salt substances without N to make the final N ...

Embodiment 2

[0071] Embodiment 2 (N 15 Preparation of fungal culture medium)

[0072] The difference from Example 1 is that step 1) of this example uses Cystogenic yeast, Saccharomyces cerevisiae and basal salt medium (M9 mother liquor), and step 2) uses liquid nitrogen grinding method to lyse yeast cells, and step 3 ) using the yeast autolysis method (using its own enzymatic hydrolysis protein) to carry out N 15 The crude extraction of yeast protein, final step 4) makes N 15 Fungal culture medium.

[0073] N 15 Preparation of fungal culture medium

[0074]

[0075] Note: ① FeSO4.7H2O, Cacl2.2H2O, MgSO4.7H2O are made into high-concentration mother liquor, and added to the medium according to the amount after sterilization;

[0076] ②Glucose is made into mother liquor, and added to the medium according to the amount after sterilization.

[0077] The type of inorganic salt can adjust its composition and proportion according to the difference of specific fungal species. This impleme...

Embodiment 3

[0078] Embodiment 3 (N 15 Preparation of plant medium)

[0079] Different from embodiment 1 is the step 4) of present embodiment finally makes N 15 Plant medium, the specific steps are as follows:

[0080] 1) Use only N1 5 (excluding N 14 )'s inorganic medium to cultivate the yeast; Pichia x-33 and basal salt medium (M9 mother liquor) can be used here;

[0081] 2) using the glass bead breaking method to lyse the yeast cells;

[0082] 3) Using protein semihydrolysis (acid hydrolysis) for N 15 Crude extraction of yeast protein;

[0083] 4)N 15 Preparation of plant medium: the peptone of the following medium comes from step 3) in N 15 Crude extraction of yeast protein.

[0084] N 15 Preparation of plant medium:

[0085]

[0086] Note: ①FeSO 4 .7H 2 O. Cacl 2 .2H 2 O. KH 2 PO 4 , Na—EDTA, MgSO 4 .7H 2 O is made into a high-concentration mother solution, which is added to the medium according to the amount after sterilization.

[0087] ②Glucose is made into m...

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Abstract

The invention discloses an N<15> stable isotope labeling method for biological protein quantifying and tracing. The method comprises the steps that yeast is utilized for converting an N<15> inorganic salt into biological protein, wherein when the culture passage number of the yeast reaches 6 or above, 98% or above of protein extracted from the yeast is N<15> protein; the high-purity N<15> protein extracted from the yeast is converted into N<15> yeast peptone, the N<15> yeast peptone is utilized for replacing conventional N<14> yeast peptone to be used for culturing multiple organisms, and specific N<15> isotope labeling is provided for biological protein labeling quantifying and tracing. The method can be applied to more organism varieties and is easy and convenient to operate, low in experimental cost and short in test cycle, and people can conduct proteome analysis on a large number of samples in an efficient and low-cost mode conveniently.

Description

technical field [0001] The present invention relates to the technical field of protein stable isotope labeling and quantification, in particular to a kind of N 15 Stable isotope labeling method. Background technique [0002] Quantitative analysis of proteins in vivo has become an important way to study protein functions and seek disease protein markers and drug targets. At present, the protein quantification technology is mainly the mass spectrometry quantitative method of isotope labeling. The isotope metabolic labeling method is one of the isotope labeling methods and is currently the preferred method for protein quantification. It is incorporated into the cell culture medium in the form of isotope elements or isotope-labeled amino acids, and completes protein isotope labeling during the process of cell growth and metabolism. Among the products of the isotope metabolic labeling method, the Stable Isotope Kit for Mammalian Cells (SILAC) is widely used in the quantitative ...

Claims

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Application Information

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IPC IPC(8): C12P21/00C07K1/13G01N33/68
CPCC12P21/00C07K1/13G01N33/68G01N2458/15
Inventor 肖传乐李影影罗峰陈龙谢尚潜
Owner 广州微因生物科技有限公司
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