A method for large-scale proteomic identification based on silkworm tissue samples
A technology for proteomics and tissue samples, applied in the field of proteomics, to achieve the effect of shortening sample pretreatment time, reducing detection time, and increasing the number of identifications
Active Publication Date: 2020-06-05
SOUTHWEST UNIV
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However, methods to increase the number of proteomic identifications of silkworm tissue samples have not been reported yet.
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Embodiment 1
[0054] Embodiment 1, the method for carrying out large-scale proteomic identification based on silkworm tissue sample
[0055] (1) Sample pretreatment for silkworm fat body proteomics:
[0056] a. Sample extraction
[0057] 1) Take about 2mg silkworm fat body sample and place it in a 1.5mL centrifuge tube, add 400μL 100mM DTT / 8M urea solution, put it on ice, and grind it with a Kayoudi G50 tissue grinder for 3min;
[0058] 2) Shake with a horizontal shaker for 5 minutes to fully dissolve the sample in the solution;
[0059] 3) Centrifuge at 12000g at 25°C for 15min, take the supernatant and put it in a new centrifuge tube;
[0060] 4) The protein concentration of the sample was detected with a Bradford protein quantification kit.
[0061] b. Sample pretreatment
[0062] 1) Take 300 μg of sample and add it to a 10kDa ultrafiltration tube, take 8M urea solution to make up to 200 μL, blow and aspirate several times with a pipette gun to mix evenly, and place it in a centrifug...
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The invention relates to a method for carrying out large-scale proteomics identification based on a silkworm tissue sample. The method comprises the following steps: pre-treating a domestic silkworm proteomics sample, carrying out graded peptide fragment mass spectrum online detection and constructing a silkworm protein database. By optimizing a protein extraction method, a peptide fragment is divided into 8 grades by adopting a high pH (Potential of Hydrogen) grading method and silkworm fat body samples can be extracted as many as possible, so that the protein identification quantity is improved; a condition that a sample spraying needle is blocked, caused by the fact that a feeding amount is too great, is prevented through optimizing a sample feeding amount and chromatography gradient time; the detection time is shortened through optimizing the chromatography gradient time; a Streamline database containing 21,878 protein sequences is established; the database can be used for identifying more protein quantity and redundant sequences are removed by the database; later-period proteomics data analysis is facilitated. According to the method provided by the invention, a stable and efficient domestic silkworm proteomics identification platform is established and the method has important meaning on silkworm proteomics large-scale identification.
Description
technical field [0001] The invention belongs to the field of proteomics and relates to a method for large-scale proteomic identification based on silkworm tissue samples. Background technique [0002] In recent years, the emergence of high-resolution mass spectrometers has greatly promoted the development of proteomics, and the research on silkworm proteomics has also entered a new stage. However, due to the different methods used in each study, it is difficult to correlate and compare the results of each study, and there are also great differences in the number of proteins identified in each study and the scientific issues explained, which makes it impossible to fully understand the biological characteristics of silkworms. Protein expression across time periods and tissues. Currently, the only animals with a full spectrum of proteins are mice and humans, but both species are mammals. Bombyx mori belongs to Lepidoptera insects, and it goes through four different developmen...
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IPC IPC(8): G01N27/62
CPCG01N33/6848
Inventor 赵萍赵东超张艳董照明夏庆友
Owner SOUTHWEST UNIV