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Treatment method for goat milk whey protein for studying proteomics

A proteomics and whey protein technology, applied in the field of goat whey whey protein processing for proteomics research, can solve the problems of difficult protein extraction, low-abundance protein easy to miss extraction, etc., to achieve high-pass Quantitative detection, high extraction efficiency, and the effect of easy high-throughput detection

Inactive Publication Date: 2018-08-03
JILIN UNIV
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  • Abstract
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  • Claims
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Problems solved by technology

[0005] Due to the special purpose of proteomics research, other preparation and processing steps of goat milk whey protein for the purpose of dairy product manufacturing and routine physical and chemical analysis are not fully suitable for the preparation and processing of samples for the purpose of proteomics research; Milk samples from different species have different requirements on processing steps and parameters due to their different components. And for the problem that low-abundance proteins with less content are easy to miss extraction, a sample preparation and processing method for goat milk whey proteomics research is provided with simple operation, high extraction rate, strong stability and high throughput

Method used

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  • Treatment method for goat milk whey protein for studying proteomics
  • Treatment method for goat milk whey protein for studying proteomics

Examples

Experimental program
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Effect test

Embodiment 1

[0036] 1) Selection of goat milk samples: Fresh goat milk from healthy Guanzhong dairy goats was selected. In order to eliminate individual differences, goat milk from 30 goats was selected as whole milk samples in this method, and 50ml of goat milk was taken from each goat.

[0037] 2) Sample pretreatment: thaw 50ml goat milk stored at -80°C in a refrigerator at 4°C (or use fresh goat milk), centrifuge at 8000g at 4°C for 20min, carefully remove the upper milk fat layer and the lower layer of casein particles to obtain a crude whey protein solution; the crude whey protein solution was centrifuged at 100,000 g at 4° C. for 1 h to remove casein in the whey, and the supernatant whey was collected.

[0038] 3) Pre-cooling acetone precipitation: Add 5 times the volume of -20°C pre-cooling acetone to the whey liquid obtained in step 2), and mix quickly (this process should be carried out in an ice bath); place overnight at -20°C , discard the supernatant by high-speed centrifugatio...

Embodiment 2

[0046] Compared with Example 1, the second centrifugation condition in step 2) sample pretreatment is 50000g and 2h at 4°C, and the rest of the process and conditions are the same as in Example 1. The number of identified protein species is 357 (see figure 2 ).

Embodiment 3

[0048] Compared with Example 1, the second centrifugation condition in step 2) sample pretreatment is 150,000 g and 0.5 h at 4° C., and the rest of the process and conditions are the same as in Example 1. The number of identified protein species is 360 (see figure 2 ).

[0049] The following examples are for control experiments.

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Abstract

The invention discloses a treatment method for goat milk whey protein for studying proteomics and belongs to the technical field of whey protein extraction and treatment methods. The treatment methodcomprises the following steps of taking a goat milk sample, performing high-speed centrifugation to acquire coarsely separated whey, then performing ultrahigh-speed centrifugation to remove casein, and performing precipitation and purification with precooled acetone to obtain a whey protein sample; adding dithiothreitol and indole-3-acetic acid for reductive alkylation, and adding a trypsin solution for ultrafiltration tube inside enzymolysis incubation to obtain polypeptide enzymatic hydrolysate; and performing desalting treatment on the polypeptide solution, performing centrifugal concentration and drying to dissolve a peptide fragment sample again, and performing direct computer analysis. The treatment method provided by the invention is simple and convenient to operate, high in extraction rate, strong in stability and high in flux, more easily realize high-flux detection of proteomics, particularly to low abundance proteins, and has a better popularization and application prospecton the aspect of goat milk whey protein proteomics study.

Description

technical field [0001] The invention relates to a method for extracting and processing whey protein for the purpose of proteomics analysis, in particular to a sample pretreatment method for goat milk whey proteomics research. Background technique [0002] As an important technical platform in the post-gene era, proteomics is a new field of proteome research, which essentially refers to the study of protein properties on a large scale, including protein expression levels, amino acid sequences, post-translational processing and Protein interaction, to understand the functions of cells, various physiological and biochemical processes, and pathological processes of diseases at the protein level. Proteomics technology can study all the proteins expressed by a set of genes, and the number of protein types identified at one time can be as many as thousands. The pretreatment of samples is the most critical step, which directly determines the success or failure of proteomics experime...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/06C07K1/14C07K1/16
CPCC07K1/14C07K1/16C12P21/06
Inventor 郭明若孙玉雪王翠娜
Owner JILIN UNIV
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