CN104597106A - Method for measuring activity of glutamic-pyruvic transaminase based on DART-MS/MS - Google Patents
Method for measuring activity of glutamic-pyruvic transaminase based on DART-MS/MS Download PDFInfo
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Abstract
The invention relates to a method for measuring activity of glutamic-pyruvic transaminase based on DART-MS/MS. The method comprises the following steps: (1) DART-MS/MS detection; (2) an activity unit of glutamic-pyruvic transaminase; (3) a computing formula of the activity unit of glutamic-pyruvic transaminase; and (4) measurement of activity of glutamic-pyruvic transaminase. By adopting DART-MS/MS as detection means, the method can directly detect a product aminopropionic acid in the enzyme reaction process. The method has the characteristics of rapid detection, high detection sensitivity, strong interference rejection, accuracy in quantification and the like. In the detection process, 2, 4-dinitrophenylhydrazine is not added, so that pollution and interference are avoided. The method is simple and convenient to operate, rapid to detect and convenient to popularize and apply.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of method adopting Direct Analysis in Real Time ion gun (DART) to measure Pancreas Glutamate-Pyruvate Transaminase Vigor in silkworm.
Background technology
Alanine is the principal ingredient in silk, its content is more than 30%, and the content of alanine is very low in the Major Foods mulberry leaf of silkworm, silkworm in order to provide more alanine to fibroin, by the glutamic-pyruvic transaminase in body by other amino acid converting be alanine.So the Pancreas Glutamate-Pyruvate Transaminase Vigor in Silkworm, Bombyx mori affects the synthesis of fibroin, and then affect the output of silk.Therefore, develop quick, efficient, sensitive novel detection technique to the vigor detecting glutamic-pyruvic transaminase in silkworm, the assessment of silk yield and the development of this silkworm industry to be had great significance.
Traditional glutamate-pyruvate transaminase determination method is reitman-frankel method
[1-4], the method adopts glutamic acid and Sodium Pyruvate effect to form Sodium L-alaninate and a-ketoglutaric acid, and after having reacted, remaining Sodium Pyruvate and 2,4 one dinitrophenylhydrazine effects, generate dinitro benzene track, in the basic conditions in brown.Then spectrophotometer is adopted to detect.But there is following problem in the method, first reaction substrate ketoglutaric acid easily and DNPH react the phenylhydrazone nitre quinone generating rufous, interference detection results, need the method adopting other loaded down with trivial details to correct.Meanwhile, DNPH is inflammable and explosive substances, requires higher in storage.In addition, the nitrophenyl hydrazine generated after adopting reitman-frankel method reaction and phenylhydrazone nitre quinone difficult treatment, very easily cause environmental pollution.Finally, the detecting device that the method adopts is spectrophotometer, and detection sensitivity is lower.
Direct Analysis in Real Time (DirectAnalysis in Real Time) is called for short DART, a kind of Thermal desorption and ionization techniques, DART can in seconds analyze the compound being present in gas, liquid, solid or material surface, thus to sample nondestructive consumption qualitative and quantitative analysis.In the present invention, the vigor adopting DART to detect glutamic-pyruvic transaminase as ion gun still belongs to the first time under study for action.By our detection scheme, can direct-detection resultant of reaction alanine, compared with traditional reitman-frankel method, in testing process, do not need product to carry out chromogenic reaction with other compositions again, simplify test experience step, in testing process, it also avoid the use of DNPH, solve the problem of producing thing interference and environmental pollution.Simultaneously the method, detects compared to traditional reitman-frankel method spectrophotometer, its detection sensitivity is higher, reappearance is better, quantitatively quicker.Therefore, the method has obvious advantage than traditional Lai Shi detection method, is suitable for replacing Lai Shi detection method to apply.
[1] Liu's limit, Li Qingfeng, Xu Fangguo, Wang Yingchun. in tussah different cultivars, the determination and analysis of posterior silkgland cells number and gpt activity. silkworm industry science, 2003,29 (2): 200-202.
[2] Xu Jingbo, Zhang Shu. the moon Serum of Common Carp gpt activity and the active mensuration suppressed. loose distant academic periodical, 1991,4,4-8.
[3] Chen Chen, Huang Feng, Shu Qiuyan, Zhang Li, Zhou Yanping. Conjugated linoleic acid is on the impact of Growth of Grass Carps Ctenopharyngodon Idellus, muscular components, glutamic-oxalacetic transaminease and gpt activity. hydrobiont journal, 2010,34 (3), 647-651.
[4] Chen Junhui, Tao Li, Li Jun, etc. Biochemistry Experiment (third edition). Science Press .2006,190-210
Summary of the invention
The technical matters solved: the invention provides a kind of method measuring Pancreas Glutamate-Pyruvate Transaminase Vigor in silkworm based on DART-MS/MS, using DART as ion gun in mensuration, can direct-detection product alanine, quantitatively more sensitive and accurate, do not need to add 2 in Simultaneously test reaction, the use of 4-dinitrophenylhydrazine, avoids interference and environmental pollution.
Technical scheme: a kind of method measuring Pancreas Glutamate-Pyruvate Transaminase Vigor in silkworm based on DART-MS/MS, comprise the steps: that (1) DART-MS/MS detects: adopt DART ion gun and triple level Four bar mass spectrum to detect alanine under positive ion mode or negative ion mode, carrier gas is nitrogen, heating-up temperature is 200-500 DEG C, Gridvoltage scope is 100-400V, air pressure is 0.2-0.5MPa, detection mode is reaction monitoring pattern (SRM), collision voltage is 0-200eV, and collision air pressure is 0-5mTorr; (2) Pancreas Glutamate-Pyruvate Transaminase Vigor unit: Pancreas Glutamate-Pyruvate Transaminase Vigor is defined as the enzyme liquid of 1mL at 38 ± 1 DEG C, water-bath 10min, generate the amount of 1 μm of ol alanine as 1 enzyme activity unit, unit of activity is U/mL; (3) Pancreas Glutamate-Pyruvate Transaminase Vigor computing formula:
(4) Pancreas Glutamate-Pyruvate Transaminase Vigor measures: mensuration group: fully mixed according to the volume ratio of 1:1:1:2 by the phosphate buffer of the glutamic acid solution of Bombyx mori posterior silkgland tissue fluid and 5mmol/L, 4mmol/L Sodium Pyruvate, pH=7.4,10min is reacted in 38 ± 1 DEG C of thermostat water baths, put into 70 DEG C of water-bath 30min-1h, the reactant liquor obtained is carried out DART detection according to institute in step (1) to condition; Blank group: adopt the Bombyx mori posterior silkgland tissue fluid liquid in ultrapure water replacement mensuration group, other conditions are consistent with mensuration group reacts, and then the reactant liquor obtained is carried out DART detection according to institute in step (1) to condition.
Described heating-up temperature is preferably 500 DEG C.
Described Gridvoltage ranges preferably from 100V.
Described air pressure is preferably 0.5MPa.
Described collision voltage is preferably 10eV.
Described collision air pressure is preferably 1.5mTorr.
Beneficial effect: the present invention adopts DART-MS/MS as detection means, can product alanine in direct-detection enzyme reaction process, the method have detect fast, detection sensitivity is high, the features such as antijamming capability is strong and quantitatively accurate.The present invention does not need to add DNPH and avoids pollution and interference in testing process.The method is easy and simple to handle, detection is quick, is easy to be extended and applied.
Accompanying drawing explanation
Fig. 1 is under ion mode, the first mass spectrometric figure of alanine;
Fig. 2 is the daughter ion CID mass spectrogram of mass-to-charge ratio 90.0 under positive ion mode;
Fig. 3 is alanine typical curve.
Specific embodiments
Key instrument: TSQ Quantum Access MAX triple level Four bar LC-MS instrument (Thermo Fisher Scientific company of the U.S.), DART ion gun (American I onSense company), EL20pH counts (plum Teller-Tuo benefit Instrument Ltd. of Switzerland), AH-S2, AH-S8 digital display thermostat water bath (Community of Jin Tan County Medical Devices Co., Ltd.).
Instrument index: TSQ Quantum Access MAX triple level Four bar LC-MS instrument, first mass spectrometric quality of scanning scope is 50-400, and second order ms daughter ion quality of scanning scope is 50-300.
Typical curve:
(1) DART-MS/MS detects: adopt DART ion gun and triple level Four bar mass spectrum to detect alanine in the positive-ion mode, carrier gas is nitrogen, heating-up temperature is 500 DEG C, Grid voltage scope is 100V, air pressure is 0.5MPa, obtain first mass spectrometric figure (see Fig. 1) and second order ms figure (see Fig. 2) after scanning, thus determine that ion pair is
with ultrapure water, the alanine solution of 5mmol/L is diluted to 2.5,1.25,0.63 and 0.31mmol/L respectively, then according to matrix solution: the volume ratio of alanine=4:1 is configured to alanine standard solution, detect under SRM pattern, collision voltage is 10eV, collision air pressure 1.5mTorr, each measurement of concetration 6 times, is depicted as typical curve by acquired results, typical curve is Y=112433804X-4306759 (see Fig. 3), R
2=0.997.
(2) Pancreas Glutamate-Pyruvate Transaminase Vigor unit: Pancreas Glutamate-Pyruvate Transaminase Vigor is defined as the enzyme liquid of 1mL at 38 ± 1 DEG C, water-bath 10min, generate the amount of 1 μm of ol alanine as 1 enzyme activity unit, unit of activity is U/mL.
(3) Pancreas Glutamate-Pyruvate Transaminase Vigor computing formula:
Embodiment 1
Mensuration group: by after five ages second day the glutamic acid solution of Bombyx mori posterior silkgland tissue fluid and 5mmol/L, 4mmol/L Sodium Pyruvate, pH=7.4 phosphate buffer fully mix according to the ratio of 1:1:1:2,10min is reacted in 38 ± 1 DEG C of thermostat water baths, put into 70 DEG C of water-bath 30min-1h, the reactant liquor obtained is carried out DART detection according to institute in step (1) to condition.
Blank group: the ultrapure water of 0.2mL is replaced the enzyme liquid in mensuration group, and other conditions are consistent with mensuration group reacts, then carries out DART detection according to institute in step (1) to condition by the reactant liquor obtained.
Bring mass spectroscopy value into computing formula, obtaining Pancreas Glutamate-Pyruvate Transaminase Vigor in silkworm is 0.71U/mL.
Embodiment 2
Mensuration group: the phosphate buffer of the glutamic acid solution of the 3rd day Bombyx mori posterior silkgland tissue fluid after five ages and 5mmol/L, 4mmol/L Sodium Pyruvate, pH=7.4 is fully mixed according to the ratio of 1:1:1:2,10min is reacted in 38 ± 1 DEG C of thermostat water baths, put into 70 DEG C of water-bath 30min-1h, the reactant liquor obtained is carried out DART detection according to institute in step (1) to condition.
Blank group: the ultrapure water of 0.2mL is replaced the enzyme liquid in mensuration group, and other conditions are consistent with mensuration group reacts, then carries out DART detection according to institute in step (1) to condition by the reactant liquor obtained.
Bring mass spectroscopy value into computing formula, obtaining Pancreas Glutamate-Pyruvate Transaminase Vigor in silkworm is 0.76U/mL.
Embodiment 3
Mensuration group: the phosphate buffer of the glutamic acid solution of the 4th day Bombyx mori posterior silkgland tissue after five ages and 5mmol/L, 4mmol/L Sodium Pyruvate, pH=7.4 is fully mixed according to the ratio of 1:1:1:2,10min is reacted in 38 ± 1 DEG C of thermostat water baths, put into 70 DEG C of water-bath 30min-1h, the reactant liquor obtained is carried out DART detection according to institute in step (1) to condition.
Blank group: the ultrapure water of 0.2mL is replaced the enzyme liquid in mensuration group, and other conditions are consistent with mensuration group reacts, then carries out DART detection according to institute in step (1) to condition by the reactant liquor obtained.
Bring mass spectroscopy value into computing formula, obtaining Pancreas Glutamate-Pyruvate Transaminase Vigor in silkworm is 0.82U/mL.
Claims (6)
1. measure a method for Pancreas Glutamate-Pyruvate Transaminase Vigor in silkworm based on DART-MS/MS, it is characterized in that comprising the steps:
(1) DART-MS/MS detects: adopt DART ion gun and triple level Four bar mass spectrum to detect alanine under positive ion mode or negative ion mode, carrier gas is nitrogen, heating-up temperature is 200-500 DEG C, Grid voltage scope is 100-400V, air pressure is 0.2-0.5MPa, detection mode is reaction monitoring pattern (SRM), and collision voltage is 0-200eV, and collision air pressure is 0-5mTorr;
(2) Pancreas Glutamate-Pyruvate Transaminase Vigor unit: Pancreas Glutamate-Pyruvate Transaminase Vigor is defined as the enzyme liquid of 1mL at 38 ± 1 DEG C, water-bath 10min, generate the amount of 1 μm of ol alanine as 1 enzyme activity unit, unit of activity is U/mL;
(3) Pancreas Glutamate-Pyruvate Transaminase Vigor computing formula:
(4) Pancreas Glutamate-Pyruvate Transaminase Vigor measures:
Mensuration group: the phosphate buffer of the glutamic acid solution of Bombyx mori posterior silkgland tissue fluid and 5mmol/L, 4mmol/L Sodium Pyruvate, pH=7.4 is fully mixed according to the volume ratio of 1:1:1:2,10min is reacted in 38 ± 1 DEG C of thermostat water baths, put into 70 DEG C of water-bath 30min-1h, the reactant liquor obtained is carried out DART detection according to institute in step (1) to condition;
Blank group: adopt the Bombyx mori posterior silkgland tissue fluid liquid in ultrapure water replacement mensuration group, other conditions are consistent with mensuration group reacts, and then the reactant liquor obtained is carried out DART detection according to institute in step (1) to condition.
2. a kind of method measuring Pancreas Glutamate-Pyruvate Transaminase Vigor in silkworm based on DART-MS/MS according to claim 1, is characterized in that described heating-up temperature is 500 DEG C.
3. a kind of method measuring Pancreas Glutamate-Pyruvate Transaminase Vigor in silkworm based on DART-MS/MS according to claim 1, is characterized in that described Grid voltage scope is 100V.
4. a kind of method measuring Pancreas Glutamate-Pyruvate Transaminase Vigor in silkworm based on DART-MS/MS according to claim 1, is characterized in that described air pressure is 0.5MPa.
5. a kind of method measuring Pancreas Glutamate-Pyruvate Transaminase Vigor in silkworm based on DART-MS/MS according to claim 1, is characterized in that described collision voltage is 10eV.
6. a kind of method measuring Pancreas Glutamate-Pyruvate Transaminase Vigor in silkworm based on DART-MS/MS according to claim 1, is characterized in that described collision air pressure is 1.5mTorr.
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| CN106970168A (en) * | 2017-05-03 | 2017-07-21 | 山东出入境检验检疫局检验检疫技术中心 | The method that DART MS methods quickly determine the amino acid in black garlic |
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Application publication date: 20150506 Assignee: Silkworm pharmaceutical factory affiliated to Sericulture Institute, Chinese Academy of Agricultural Sciences Assignor: JIANGSU University OF SCIENCE AND TECHNOLOGY Contract record no.: X2020980007222 Denomination of invention: A method for determination of alanine aminotransferase activity in Bombyx mori based on dart-ms / MS Granted publication date: 20170623 License type: Common License Record date: 20201029 |
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Assignee: Silkworm pharmaceutical factory affiliated to Sericulture Institute, Chinese Academy of Agricultural Sciences Assignor: JIANGSU University OF SCIENCE AND TECHNOLOGY Contract record no.: X2020980007222 Date of cancellation: 20201222 |