CN105352778B - A kind of preparation processing method for marine protein group study sample - Google Patents
A kind of preparation processing method for marine protein group study sample Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a kind of preparation processing methods for marine protein group study sample.It is homogenized after lysate and protease inhibitors is added in marine organisms, is centrifuged, is taken supernatant and sediment；Sediment is subjected to cryogenic pulverization, ultrahigh speed low-temperature centrifugation obtains extracting solution of protein；Extracting solution of protein is subjected to acetone precipitation twice again, to protein group Sample Purification on Single, then through restoring, being alkylated, comprehensive type, concentration and the higher proteomics sample of purity are obtained in being organized from marine organisms.Present invention process is reasonable, operation is reliable, preparation science, have a wide range of application, recycle protein group to greatest extent in the case where bringing little damage to protein, and special optimization is carried out to the shotgun protein group Liquid Chromatography-Tandem Mass Spectrometry measurement based on isotope labelling absolute quantitation, the correlative study to carrying out marine protein matter group has greater significance.
Description
Technical field
The present invention relates to being tested or analysis of material by means of measuring the chemically or physically property of material, especially a kind of use
The preparation processing method of Yu Haiyang bioprotein group study sample.
Background technology
Proteomics is phase in the early 1990s, the new term proposed first by Marc Wikins and scholars, substantially
Refer to it being the feature for studying protein in extensive level, includes the expression of protein, the modification after translation, albumen
With protein-interacting etc., occur about disease thus to obtain on protein level, the entirety of the processes such as cell metabolism and it is complete
The understanding in face.Importantly, genome is the entity of quite stable, and protein group is by the interaction with genome
Change constantly occurs.One life entity is in the different piece of its body and the different phase of life cycle, protein expression
There may be huge differences.The entirety for the protein that one life entity is possessed in its whole life cycle, or more
In small scale, certain types of cell is known respectively as in the entirety for undergoing the protein possessed when specific type stimulation
The protein group of this life entity or cell type.With the completion of human genome sketch, present many scholars start to explore
How gene and protein by interacting form other oroteins.
Most of existing proteomics research is carried out to terrestrial life, the research report of rare marine organisms.And
For marine organisms due to its special living environment, the complexity and tolerance of protein are different from terrestrial life, cause to make at present
With the general procedure method of proteomics research during carrying out proteomics research to marine organisms, usually along with
The denaturation of protein group, loss of activity and protein recovery are low.Therefore, research and development are a kind of studies for marine protein group
The preparation processing method of sample is particularly important.
Invention content
A kind of the object of the present invention is to provide operations reliable, preparation science, protein breakdown are small and the rate of recovery is high is used for
The preparation processing method of marine protein group study sample.
The technical solution adopted by the present invention to solve the technical problems is：One kind studying sample for marine protein group
The preparation processing method of product, it is characterised in that：It passes through following process steps：
(1) sample pretreatment：It takes marine organisms in centrifuge tube, lysate is added, 1 ︰ 2~5 (m/v) of solid-liquid ratio is added
Protease inhibitors makes its final concentration of 0.5~1.2mM, and after fully being broken up with tissue homogenizer, centrifugation takes supernatant and precipitation
Object, supernatant are tissue solubility holoprotein solution；
(2) Membrane protein extraction：By step（1）In obtained sediment be resuspended using Tris-Hcl buffer solutions, re-suspension liquid into
Row cryogenic pulverization, centrifugation collect supernatant, obtain the extracting solution of histocyte memebrane protein；
(3) acetone precipitation：By step（1）, the supernatant that obtains in step (2) merge, then in combined supernatant
The precooling acetone soln of 4~6 times of its volume is added, concussion mixes, and 2~4h is precipitated under the conditions of -20 DEG C, and precipitation is collected in centrifugation；
(4) secondary acetone precipitation：The pre- cold acetone that 3~5 times of its volume is added in the precipitation obtained in step (3) is molten
Liquid, concussion mix, and 1~2h is precipitated under the conditions of -20 DEG C, and precipitation is collected in centrifugation；By above-mentioned steps repetitive operation 1~2 time；Merge
Precipitation；
(5) total protein extraction：After precipitation after the merging obtained in step (4) is dried at normal temperatures, it is dissolved in cracking
In liquid, 1 ︰ 2~5 (m/v) of solid-liquid ratio, room temperature acts on 2~4h；Centrifugation, takes supernatant；Supernatant centrifuges again, abandons precipitation, takes
Clear liquid；Merge supernatant to get the total protein solution of tissue；
(6) it restores, be alkylated：The total protein solution obtained in step (5) is quantitatively weighed as sample, its volume 3 is added
The NH of~4 times of 50mmol/L4HCO3Then the DTT of 0.5mol/L is added to final concentration of 3~4mmol/L in solution, room temperature is incubated
Educate 30~50min；Then 0.5mol/L iodoacetamides are added to final concentration of 55~90mmol/L, room temperature is protected from light incubation 20
~50min；The NH of 50mmol/L is used again4HCO3It rinses 1~3 time, acetonitrile dehydration is lyophilized, preserves, obtain marine protein group
Sample.
Marine organisms in the step (1) are sargassum, undaria pinnitafida or seaweed.
The step (1), the lysate in step (5) formula be：50mM Tris-HCl(pH 7.4)、150mM
NaCl, 1%NP-40 and 0.1%SDS are mixed or are commercially available lysate RLT Buffer.
Protease inhibitors in the step (1) is PMSF or Cocktail.
The formula of Tris-Hcl buffer solutions in the step (2) is：50mM Tris-HCL, 1mM EGTA and 1mM
PMSF is mixed, and it is 7.4 to adjust pH value.
Cryogenic pulverization in the step (2) is that liquid nitrogen grinding is crushed or is placed on ice chest, ultrasonication.
The ultrasonication is control Ultrasonic Cell Disruptor power 50~100w, ultrasonic 0.8s, closes 0.8s, ultrasonication
1~5min of time.
Preparation processing method provided by the present invention for marine protein group study sample is to give birth to ocean
Object is homogenized after lysate and protease inhibitors is added, and is centrifuged, is taken supernatant and sediment；Sediment progress low temperature is broken
Broken, ultrahigh speed low-temperature centrifugation obtains extracting solution of protein；Extracting solution of protein is subjected to acetone precipitation twice again, to protein group
Sample Purification on Single, then through restoring, being alkylated, comprehensive type, concentration and the higher albumen of purity are obtained in being organized from marine organisms
Matter group imitates product.The method that the present invention uses cryogenic pulverization is crushed thoroughly, and does not damage the natural structure of protein；This hair
It is bright that pollution of nucleic acid and outer source ion can effectively be avoided to introduce, to ensure the purity of protein example.Final product of the present invention
Isotope labelling carries out Liquid Chromatography-Tandem Mass Spectrometry analysis after being particularly suitable for pancreatin hydrolysis, is suitable for carrying out the two-way electricity of protein
Swimming, high performance liquid chroma- tography（HPLC）And the proteomics researches such as protein group Mass Spectrometric Identification；Present invention process is reasonable, operation
Reliably, preparation science has a wide range of application, and recycles protein group to greatest extent in the case where bringing little damage to protein,
And the shotgun protein group Liquid Chromatography-Tandem Mass Spectrometry measurement based on isotope labelling absolute quantitation is carried out especially excellent
Change, the correlative study to carrying out marine protein matter group has more great meaning.
Specific implementation mode
With reference to embodiment, the present invention will be further described.
Embodiment 1
A kind of preparation processing method for marine protein group study sample passes through following process steps：
(1) sample pretreatment：It takes sargassum in centrifuge tube, lysate is added, protease is added in 1 ︰ 3 (m/v) of solid-liquid ratio
Inhibitor PMSF makes its final concentration of 1.0mM, and after fully being broken up with tissue homogenizer, 4 DEG C of temperature is controlled using refrigerated centrifuge,
Rotating speed 10000g is controlled, centrifuges 20min, it is tissue solubility holoprotein solution to take supernatant and sediment, supernatant；Its
In, lysate is to use：50mM Tris-HCl (pH 7.4), 150mM NaCl, 1%NP-40 and 0.1%SDS are mixed；
(2) Membrane protein extraction：By step（1）In obtained sediment be resuspended using Tris-Hcl buffer solutions, re-suspension liquid into
Row liquid nitrogen grinding is broken, then refrigerated centrifuge is used to control 4 DEG C of temperature, controls rotating speed 10000g, centrifuges 15 min, in collection
Clear liquid obtains the extracting solution of histocyte memebrane protein；Wherein, Tris-Hcl buffer solutions are to use：50mM Tris-HCL、1mM
EGTA and 1mM PMSF are mixed, and it is 7.4 to adjust pH；
(3) acetone precipitation：By step（1）, the supernatant that obtains in step (2) merge, then by the supernatant after merging
The middle precooling acetone soln that 5 times of its volume is added, concussion mix, and 3h are precipitated under the conditions of -20 DEG C, then control using refrigerated centrifuge
4 DEG C of temperature controls rotating speed 10000g, centrifuges 15min, collects precipitation；
(4) secondary acetone precipitation：The precooling acetone soln of 4 times of its volume will be added in the precipitation obtained in step (3), shake
Mixing is swung, 1.5h is precipitated under the conditions of -20 DEG C, then 4 DEG C of temperature is controlled using refrigerated centrifuge, controls rotating speed 10000g, centrifugation
15min collects precipitation；By above-mentioned steps repetitive operation 2 times；Merge precipitation；
(5) total protein extraction：After precipitation after the merging obtained in step (4) is dried at normal temperatures, it is dissolved in cracking
In liquid, 1 ︰ 3 (m/v) of solid-liquid ratio, room temperature acts on 3h；Centrifugal Machine Control rotating speed 10000g is used again, is centrifuged 15min, is taken supernatant；
Supernatant uses Centrifugal Machine Control rotating speed 10000g again, centrifuges 15min, abandons precipitation, take supernatant；Merge supernatant to get
The total protein solution of tissue；Wherein, lysate is to use：50mM Tris-HCl (pH 7.4), 150mM NaCl, 1%NP-40 and
0.1%SDS is mixed；
(6) it restores, be alkylated：The total protein solution obtained in step (5) is quantitatively weighed as sample, its volume is added
The NH of 3.5 times of 50mmol/L4HCO3Then the DTT of 0.5 mol/L is added to final concentration of 3.5mmol/L in solution, room temperature is incubated
Educate 45min；Then 0.5mol/L iodoacetamides are added to final concentration of 70mmol/L, room temperature, which is protected from light, is incubated 45min；It uses again
The NH of 50mmol/L4HCO3It rinses 2 times, acetonitrile dehydration is lyophilized, preserves, obtains marine protein group and imitate product.
The preparation processing method for the marine protein group study sample that the present embodiment is provided, rational technology, behaviour
Make reliably, preparation science has a wide range of application, and recycles protein to greatest extent in the case where bringing little damage to protein
Group, and the shotgun protein group Liquid Chromatography-Tandem Mass Spectrometry measurement based on isotope labelling absolute quantitation has been carried out especially
Optimization.
Embodiment 2
A kind of preparation processing method for marine protein group study sample passes through following process steps：
(1) sample pretreatment：It takes undaria pinnitafida in centrifuge tube, lysate is added, protease is added in 1 ︰ 5 (m/v) of solid-liquid ratio
Inhibitor C ocktail makes its final concentration of 1.2mM, and after fully being broken up with tissue homogenizer, temperature is controlled using refrigerated centrifuge
4 DEG C, rotating speed 8000g is controlled, centrifuges 15min, it is tissue solubility holoprotein solution to take supernatant and sediment, supernatant；
Wherein, lysate using the production and sales of Sheng Gong biotech firms lysate RLT Buffer；
(2) Membrane protein extraction：By step（1）In obtained sediment be resuspended using Tris-Hcl buffer solutions, re-suspension liquid into
Row is placed on ice chest, controls Ultrasonic Cell Disruptor power 100w, ultrasonic 0.8s, closes 0.8s, ultrasonication 1min, is then used low
4 DEG C of warm Centrifugal Machine Control temperature controls rotating speed 15000g, centrifuges 5min, collects supernatant, obtains carrying for histocyte memebrane protein
Take liquid；Wherein, Tris-Hcl buffer solutions are to use：50mM Tris-HCL, 1mM EGTA and 1mM PMSF are mixed, and are adjusted
It is 7.4 to save pH；
(3) acetone precipitation：By step（1）, the supernatant that obtains in step (2) merge, then by the supernatant after merging
The middle acetone soln that the 6 times of precoolings of its volume are added, concussion mix, and 2h are precipitated under the conditions of -20 DEG C, then control using refrigerated centrifuge
5 DEG C of temperature controls rotating speed 15000g, centrifuges 5min, collects precipitation；
(4) secondary acetone precipitation：The acetone soln of 5 times of precoolings of its volume, shake will be added in the precipitation obtained in step (3)
Mixing is swung, 1h is precipitated under the conditions of -20 DEG C, then 5 DEG C of temperature is controlled using refrigerated centrifuge, controls rotating speed 15000g, centrifuge 5min,
Collect precipitation；By above-mentioned steps repetitive operation 1 time；Merge precipitation；
(5) total protein extraction：After precipitation after the merging obtained in step (4) is dried at normal temperatures, it is dissolved in cracking
In liquid, 1 ︰ 5 (m/v) of solid-liquid ratio, room temperature acts on 2h；Centrifugal Machine Control rotating speed 15000g is used again, is centrifuged 5min, is taken supernatant；
Supernatant uses Centrifugal Machine Control rotating speed 15000g again, centrifuges 5min, abandons precipitation, take supernatant；Merge supernatant to get group
The total protein solution knitted；Wherein, lysate using the production and sales of Sheng Gong biotech firms lysate RLT Buffer；
(6) it restores, be alkylated：The total protein solution obtained in step (5) is quantitatively weighed as sample, its volume 4 is added
The NH of 50mmol/L again4HCO3Then the DTT to final concentration of 3mmol/L of 0.5 mol/L is added in solution, incubation at room temperature
30min；Then 0.5mol/L iodoacetamides are added to final concentration of 90mmol/L, room temperature, which is protected from light, is incubated 20min；It uses again
The NH of 50mmol/L4HCO3It rinses 3 times, acetonitrile dehydration is lyophilized, preserves, obtains marine protein group and imitate product.
The preparation processing method that the present embodiment is provided, rational technology, operation is reliable, and preparation science has a wide range of application,
Recycle protein group to greatest extent in the case where bringing little damage to protein.
Embodiment 3
A kind of preparation processing method for marine protein group study sample passes through following process steps：
(1) sample pretreatment：It takes seaweed in centrifuge tube, lysate is added, protease suppression is added in 1 ︰ 2 (m/v) of solid-liquid ratio
Preparation PMSF makes its final concentration of 0.5mM, and after fully being broken up with tissue homogenizer, 4 DEG C of temperature, control are controlled using refrigerated centrifuge
Rotating speed 10000g processed centrifuges 15min, and it is tissue solubility holoprotein solution to take supernatant and sediment, supernatant；Wherein,
Lysate uses：50mM Tris-HCl (pH 7.4), 150mM NaCl, 1%NP-40 and 0.1%SDS are mixed；
(2) Membrane protein extraction：By step（1）In obtained sediment be resuspended using Tris-Hcl buffer solutions, re-suspension liquid into
Row is placed on ice chest, controls Ultrasonic Cell Disruptor power 50w, ultrasonic 0.8s, closes 0.8s, ultrasonication 5min, is then used low
4 DEG C of warm Centrifugal Machine Control temperature controls rotating speed 12000g, centrifuges 10min, collects supernatant, obtains histocyte memebrane protein
Extracting solution；Wherein, Tris-Hcl buffer solutions are to use：50mM Tris-HCL, 1mM EGTA and 1mM PMSF are mixed, and are adjusted
It is 7.4 to save pH；
(3) acetone precipitation：By step（1）, the supernatant that obtains in step (2) merge, then by the supernatant after merging
The middle precooling acetone soln that 4 times of its volume is added, concussion mix, and 4h are precipitated under the conditions of -20 DEG C, then control using refrigerated centrifuge
0 DEG C of temperature controls rotating speed 12000g, centrifuges 10min, collects precipitation；
(4) secondary acetone precipitation：The precooling acetone soln of 3 times of its volume will be added in the precipitation obtained in step (3), shake
Mixing is swung, 2h is precipitated under the conditions of -20 DEG C, then 0 DEG C of temperature is controlled using refrigerated centrifuge, controls rotating speed 12000g, centrifugation
10min collects precipitation；By above-mentioned steps repetitive operation 2 times；Merge precipitation；
(5) total protein extraction：After precipitation after the merging obtained in step (4) is dried at normal temperatures, it is dissolved in cracking
In liquid, 1 ︰ 2 (m/v) of solid-liquid ratio, room temperature acts on 4h；Centrifugal Machine Control rotating speed 12000g is used again, is centrifuged 10min, is taken supernatant；
Supernatant uses Centrifugal Machine Control rotating speed 12000g again, centrifuges 10min, abandons precipitation, take supernatant；Merge supernatant to get
The total protein solution of tissue；Wherein, lysate using the production and sales of Sheng Gong biotech firms lysate RLT Buffer；
(6) it restores, be alkylated：The total protein solution obtained in step (5) is quantitatively weighed as sample, its volume 3 is added
The NH of 50mmol/L again4HCO3Then the DTT to final concentration of 4mmol/L of 0.5 mol/L is added in solution, incubation at room temperature
50min；Then 0.5mol/L iodoacetamides are added to final concentration of 55mmol/L, room temperature, which is protected from light, is incubated 50min；It uses again
The NH of 50mmol/L4HCO3It rinses 1 time, acetonitrile dehydration is lyophilized, preserves, obtains marine protein group and imitate product.
The preparation processing method for the marine protein group study sample that the present embodiment is provided, rational technology, behaviour
Make reliably, preparation science recycles protein group in the case where bringing little damage to protein to greatest extent, and to being based on
The shotgun protein group Liquid Chromatography-Tandem Mass Spectrometry measurement of isotope labelling absolute quantitation is especially optimized.
Claims (7)
1. a kind of preparation processing method for marine protein group study sample, it is characterised in that：It passes through following work
Skill step：
(1) sample pretreatment：It takes marine organisms in centrifuge tube, lysate is added, protease is added in 1 2~5m/v of ︰ of solid-liquid ratio
Inhibitor makes its final concentration of 0.5~1.2mM, and after fully being broken up with tissue homogenizer, centrifugation takes supernatant and sediment, on
Clear liquid is tissue soluble protein solution；
(2) Membrane protein extraction：By step（1）In obtained sediment be resuspended using Tris-Hcl buffer solutions, re-suspension liquid carries out low
Temperature is broken, and centrifugation collects supernatant, obtains the extracting solution of histocyte memebrane protein；
(3) acetone precipitation：By step（1）, the supernatant that obtains in step (2) merge, be then added in combined supernatant
The precooling acetone soln of 4~6 times of its volume, concussion mix, and 2~4h is precipitated under the conditions of -20 DEG C, and precipitation is collected in centrifugation；
(4) secondary acetone precipitation：The precooling acetone soln of 3~5 times of its volume will be added in the precipitation obtained in step (3), shake
Mixing is swung, 1~2h is precipitated under the conditions of -20 DEG C, precipitation is collected in centrifugation；By above-mentioned steps repetitive operation 1~2 time；Merge precipitation；
(5) total protein extraction：After precipitation after the merging obtained in step (4) is dried at normal temperatures, it is dissolved in lysate,
1 2~5m/v of ︰ of solid-liquid ratio, room temperature act on 2~4h；Centrifugation, takes supernatant；Supernatant centrifuges again, abandons precipitation, takes supernatant；It closes
And supernatant is to get the total protein solution of tissue；
(6) it restores, be alkylated：The total protein solution obtained in step (5) is quantitatively weighed as sample, 3~4 times of its volume is added
50mmol/L NH4HCO3Then the DTT to final concentration of 3~4mmol/L of 0.5mol/L, incubation at room temperature 30 is added in solution
~50min；Then 0.5mol/L iodoacetamides are added to final concentration of 55~90mmol/L, room temperature be protected from light incubation 20~
50min；The NH of 50mmol/L is used again4HCO3It rinses 1~3 time, acetonitrile dehydration is lyophilized, preserves, obtain marine protein group and imitate
Product.
2. a kind of preparation processing method for marine protein group study sample according to claim 1, special
Sign is：Marine organisms in the step (1) are sargassum, undaria pinnitafida or seaweed.
3. a kind of preparation processing method for marine protein group study sample according to claim 1, special
Sign is：The step (1), the lysate in step (5) formula be：50mM Tris-HCl pH7.4、150mM
NaCl, 1%NP-40 and 0.1%SDS are mixed or are commercially available lysate RLT Buffer.
4. a kind of preparation processing method for marine protein group study sample according to claim 1, special
Sign is：Protease inhibitors in the step (1) is PMSF or Cocktail.
5. a kind of preparation processing method for marine protein group study sample according to claim 1, special
Sign is：The formula of Tris-Hcl buffer solutions in the step (2) is：50mM Tris-HCL, 1mM EGTA and 1mM
PMSF is mixed, and it is 7.4 to adjust pH value.
6. a kind of preparation processing method for marine protein group study sample according to claim 1, special
Sign is：Cryogenic pulverization in the step (2) is that liquid nitrogen grinding is crushed or is placed on ice chest, ultrasonication.
7. a kind of preparation processing method for marine protein group study sample according to claim 6, special
Sign is：The ultrasonication is control Ultrasonic Cell Disruptor power 50~100w, ultrasonic 0.8s, closes 0.8s, ultrasonication
1~5min of time.
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| CN110308228A (en) * | 2019-08-09 | 2019-10-08 | 陈溪 | A kind of analysis method of sea cucumber protein group |
| CN111635921A (en) * | 2020-05-27 | 2020-09-08 | 中国医学科学院基础医学研究所 | Sample preparation method for proteomics analysis |
| CN113185571A (en) * | 2021-04-20 | 2021-07-30 | 江苏越红生物科技有限公司 | Protein extraction and preparation method thereof |
| CN114539348B (en) * | 2022-01-25 | 2024-04-05 | 四川省农业科学院水稻高粱研究所 | Protein extraction method for fermented grain microorganism macro proteomics detection |
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| EP2297184A1 (en) * | 2008-05-21 | 2011-03-23 | Daedalus Innovations Llc | Solubilization of proteins in reverse micelles by extraction from a solid support |
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| CN104877004A (en) * | 2015-04-29 | 2015-09-02 | 广西壮族自治区药用植物园 | Extraction method of fructus momordicae leaf total protein for proteomics analysis |
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| EP2297184A1 (en) * | 2008-05-21 | 2011-03-23 | Daedalus Innovations Llc | Solubilization of proteins in reverse micelles by extraction from a solid support |
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| CN104710520A (en) * | 2013-12-13 | 2015-06-17 | 中国科学院大连化学物理研究所 | Method for extracting proteins from marine microalga lipid droplet |
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