CN108152094A - A kind of ginseng crude drug MALDI-MSI sample treatments - Google Patents
A kind of ginseng crude drug MALDI-MSI sample treatments Download PDFInfo
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- CN108152094A CN108152094A CN201711146600.9A CN201711146600A CN108152094A CN 108152094 A CN108152094 A CN 108152094A CN 201711146600 A CN201711146600 A CN 201711146600A CN 108152094 A CN108152094 A CN 108152094A
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- Prior art keywords
- sample
- crude drug
- ginseng crude
- maldi
- msi
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
- G01N27/64—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode using wave or particle radiation to ionise a gas, e.g. in an ionisation chamber
Abstract
The invention discloses a kind of ginseng crude drug MALDI MSI sample treatments.The present invention includes the following steps：A. medicinal material is sliced；B. sample water logging；C. sample is fixed；D. embedding freezing；E. sample sections；F. melt mounting；G. matrix and detection are sprayed.The problems such as it is brittle that the method for the present invention solves ginseng crude drug's sample pre-treatments, easy fragmentation, method of the invention melt that mounting process is simple, quick, effect is good, can reduce background interference problem, effectively enhance the signal strength of Mass Spectrometer Method.
Description
Technical field
The present invention relates to laboratory sample process field, more particularly to a kind of ginseng crude drug MALDI-MSI sample treatments.
Background technology
Ginseng is the root or rhizome of araliaceae ginseng plant ginseng Panax ginseng C.A.Mey..Ginseng is praised from ancient times
For King of Herbs, the effect of nature and flavor are sweet, slight bitter, flat, have and reinforce vital energy, and veins takes off admittedly reinforces the spleen to benefit the lung, and promotes the production of body fluid, tranquilizing the mind.With
It to be taken off in physically weak, spleen eating less, cold weak pulse of limb, the deficiency syndrome of the lung is breathed with cough and heart failure and cardiogenic shock.The primary chemical of ginseng
Ingredient includes panaxoside, polysaccharide, amino acid and a small amount of volatile oil.Modern pharmacological studies have shown that ginseng, which has, strengthens body pair
The resistance of adverse factor enhances body function, the pharmacological activity such as antitumor, anti-inflammatory, anti-oxidant.Ginseng is the name for enjoying great fame long
One of your Chinese medicine and Chinese herbal medicine most widely used in the world.In recent years, ginseng be field of Chinese medicines most study medicinal material it
One and the preferable model medicinal material of all kinds of researchs of Chinese medicine, therefore develop sample treatment of the ginseng under New Technical Condition and have
Important meaning.
In recent years, substance assistant laser desorpted ionized mass spectrum image（Matrix-Assisted Laser Desorption/
Ionization-Mass Spectrometric Imaging, abbreviation MALDI-MSI）Technology has obtained tremendous development.MALDI-
MSI advantages are not needing to that both can a variety of target molecules have been analyzed and positioned, and energy with target molecule progress isolated or purified
Unknown molecular or other relevant molecules in enough samples to detection carry out spatial distribution positioning, and the molecular range of detection may include
Drug, metabolin, polypeptide or median size protein molecule etc..MALDI-MSI has become tracer tissue marker object and hair at present
One of powerful of existing marker.In recent years, MALDI-MSI starts the research applied to Chinese herbal medicine.Pass through MALDI-MSI
Technology can observe directly the distribution of important chemical composition and content image situation in Chinese medicine, and then can reflect indirectly
The quality condition of medicine.Thus the method for development and application MALDI-MSI technologies detection ginseng crude drug is significant.
In the entire detection process of MALDI-MSI, the sample pretreatment process of MALDI-MSI is that influence mass spectrum image is true
The most important link of reality and accuracy is the extremely critical step of whole operation step 1, fresh animal tissue
MALDI-MSI sample pre-treatments have more ripe method.Ginseng crude drug is usually dry sample, if with dry ginseng medicine
Material sample carries out sample pre-treatments, and sample ductility is poor, easy fragmentation, is extremely difficult to keep the effect of its integrality, in addition,
Dry medicinal material sample is more difficult to carry out melting mounting processing.Therefore, if with existing flesh tissue method, applied to ginseng crude drug
Sample treatment, it is difficult to reach ideal detection result.There is presently no the MALDI-MSI sample treatment sides of preferable ginseng
Method.Therefore, the MALDI-MSI sample treatments for developing ginseng crude drug have important research and application value.
Invention content
The technical problems to be solved by the invention are to have overcome the deficiencies of the prior art and provide a kind of ginseng crude drug MALDI-MSI
Sample treatment solves easy fragmentation before ginseng crude drug's sample treatment, easy fragmentation, it is difficult to the problem of keeping integrality, with
And ginseng crude drug's sample is on MALDI-MSI targets are transferred to, and melts that mounting process steps complexity is cumbersome, and Mass Spectrometer Method signal is weak to ask
Topic.
The technical solution adopted in the present invention is that the present invention includes the following steps：
A. medicinal material is sliced：After ginseng crude drug's sample freezen protective, the root of ginseng crude drug is taken, by 1.5 cm thicks, by people
Ginseng medicinal material sample is cut into fan-shaped section；
B. sample water logging：Sample disc after freezing is cleaned, is dried, added in liquid-transfering gun into sample disc 15-25 microlitres go from
Sub- water is put into the ginseng crude drug's sample cut；
C. sample is fixed：Ginseng crude drug's sample disc is placed in freezing-microtome, is freezed, is fixed on ginseng crude drug's sample
In sample disc；
D. embedding freezing：With liquid-transfering gun, deionized water is added dropwise into ginseng crude drug's sample of -60 DEG C of freezings, until deionized water
Until ginseng crude drug's sample is completely covered, after continue to freeze；
E. sample sections：Ginseng crude drug's sample by step D processing is sliced, the temperature of the slice of the freezing-microtome
Degree control is -15 DEG C of tool temperature, -5 DEG C of sample disc temperature, and slice thickness is 14-30 microns；
F. melt mounting：Ginseng crude drug's sample of slice is transferred to on the conductive glass plate conducting surface that alcohol is cleaned and is dried, is used
Finger is heated in glass back, makes peripheral one layer of ice thaw and ginseng crude drug's sample is made closely to paste on a glass, then take out
Vacuum drying；
G. spray matrix and detection：Ginseng crude drug's sample that step F is handled well is subjected to spray matrix, is then carried out with MALDI-MSI
Detection and analysis.
Further, the freezen protective temperature of ginseng crude drug's sample in step A is -20 DEG C to -80 DEG C.
Further, the deionized water in step B further includes Milli-Q water, distilled water.
Further, the continuation cooling time in step D is 5-10min.
Further, in the time that glass back heats it is 3-8s with finger described in step F.
Further, drying time is vacuumized as 15-25min in step F.
Further, matrix used in step G be N- (1- naphthalenes)-ethylene diamine hydrochloride, N- (1- naphthalenes)-hydrochloric acid second
The N- (1- naphthalenes)-ethylene diamine hydrochloride is dissolved in purity in 50% methanol, to use MALDI- by a concentration of 10mg/mL of diamines
MSI is detected analysis.
The beneficial effects of the invention are as follows：
1. the reagent of the present invention is cheap, it is easy to get, operating procedure is simple, and effect is good；
2. making ginseng crude drug's sample that there is some ductility and flexibility by water process, solve ginseng crude drug's sample pre-treatments
It is easily rupturable, the problems such as easy fragmentation, sample is made easily to keep its integrality；
3. it is simple to melt mounting process, effect is good, and can both samples of Ginseng be transferred on MALDI-MSI targets by not needing to double faced adhesive tape, be carried
It is high to melt mounting quality and melt mounting efficiency；
4. background interference problem can be effectively reduced by being handled by the method for the present invention, the mass signal intensity of detection is enhanced.
Description of the drawings
Fig. 1 is the operating process schematic diagram of the present invention；
Fig. 2 is the dicing effect of the different freezing matrix effects of the embodiment of the present invention 1（a）Generic media effect；(b) matrix of the present invention
Effect；
Fig. 3 is that the embodiment of the present invention 1 melts mounting design sketch；
Fig. 4 is the mass spectrogram of the embodiment of the present invention 1:(a) the mass spectrum effect of this method；(b) the mass spectrum effect of conventional method；
Fig. 5 is the mass spectrogram of the embodiment of the present invention 1；
Fig. 6 is the embodiment of the present invention 1 in karyoplasmic ratio（m/z）658.90 mass spectrum striograph；
Fig. 7 is the embodiment of the present invention 1 in karyoplasmic ratio（m/z）833.51 mass spectrum striograph；
Fig. 8 is the embodiment of the present invention 1 in karyoplasmic ratio（m/z）995.43 mass spectrum striograph；
Fig. 9 is the embodiment of the present invention 1 in karyoplasmic ratio（m/z）1143.39 mass spectrum striograph；
Figure 10 is the mass spectrogram of the embodiment of the present invention 2:(a) the mass spectrum effect of this method；(b) the mass spectrum effect of conventional method；
Figure 11 is the mass spectrogram of the embodiment of the present invention 2；
Figure 12 is the embodiment of the present invention 2 in karyoplasmic ratio（m/z）659 mass spectrum striograph；
Figure 13 is the embodiment of the present invention 2 in karyoplasmic ratio（m/z）833 mass spectrum striograph；
Figure 14 is the embodiment of the present invention 2 in karyoplasmic ratio（m/z）995 mass spectrum striograph；
Figure 15 is the embodiment of the present invention 2 in karyoplasmic ratio（m/z）1113 mass spectrum striograph；
Figure 16 is the embodiment of the present invention 2 in karyoplasmic ratio（m/z）1143 mass spectrum striograph.
Specific embodiment
The present invention is used for the pre-treatment of MALDI-MSI for current ginseng crude drug, and sample ductility is poor, easy fragmentation,
Be extremely difficult to keep the effect of its integrality, in addition, melt mounting step complexity cumbersome for conventional method, Mass Spectrometer Method signal compared with
It is weak, it is difficult to reach ideal detection result.In view of the above-mentioned problems, design the MALDI-MSI sample treatments for ginseng crude drug
Method.
Specific embodiment one：
The MALDI-MSI sample treatments of ginseng crude drug, step are as follows：
A. medicinal material is sliced：The root of ginseng crude drug's sample is taken, ginseng crude drug's sample is 4 years raw ginseng crude drugs, by 1.5 lis
Meter Hou Du is cut into fan-shaped section；
B. sample water logging：The sample disc of freezing is cleaned, is dried, adds in 20 microlitres of deionized waters into sample disc with liquid-transfering gun,
It is put into the ginseng crude drug's sample cut；
C. sample is fixed：Sample disc is placed in freezing-microtome, is freezed, ginseng crude drug's sample is made to be fixed on sample disc
In；
D. embedding freezing：With liquid-transfering gun, deionized water is added dropwise into ginseng crude drug's sample after freezing, until deionized water is by people
After joining until medicinal material sample is completely covered, continue freezing 5 minutes；
E. sample sections：Ginseng crude drug's sample by step D processing is sliced, adjusts the slice of the freezing-microtome
Temperature, tool temperature be -15 DEG C, sample disc temperature be -5 DEG C, slice thickness be 20 microns；
F. melt mounting：Ginseng crude drug's sample of slice is transferred to on the conductive glass plate conducting surface that alcohol is cleaned and is dried, is used
Finger is heated in glass back, makes peripheral one layer of ice thaw and ginseng crude drug's sample is made closely to paste on a glass, then take out
Vacuum drying 20 minutes；
G. spray matrix and detection：Spray N- (1- naphthalenes)-ethylene diamine hydrochloride matrix, N- (1- naphthalenes)-ethylene diamine hydrochloride it is a concentration of
The N- (1- naphthalenes)-ethylene diamine hydrochloride is dissolved in purity in 50% methanol by 10mg/mL, is detected point with MALDI-MSI
Analysis.
Interpretation of result
As shown in Figures 1 to 9, in the present embodiment, ginseng crude drug's sample that the present invention is handled, sample are not easily broken, rich to prolong
After malleability easily keeps its integrality, melts that mounting process is simple, and effect is good, and the application present invention carries out sample treatment, MALDI-MSI
The mass signal of detection significantly increases.
Specific embodiment two：
The MALDI-MSI sample treatments of ginseng crude drug, step are as follows：
A. medicinal material is sliced：The root of ginseng crude drug's sample is taken, ginseng crude drug's sample is 8 years raw ginseng crude drugs, by 1.5 lis
Meter Hou Du is cut into fan-shaped section；
B. sample water logging：The sample disc of freezing is cleaned, is dried, adds in 19 microlitres of deionized waters into sample disc with liquid-transfering gun,
It is put into the ginseng crude drug's sample cut；
C. sample is fixed：Sample disc is placed in freezing-microtome, is freezed, ginseng crude drug's sample is made to be fixed on sample disc
In；
D. embedding freezing：With liquid-transfering gun, deionized water is added dropwise into ginseng crude drug's sample after freezing, until deionized water is by people
After joining until medicinal material sample is completely covered, continue freezing 5 minutes；
E. sample sections：Ginseng crude drug's sample by step D processing is sliced, adjusts the adjusting of the freezing-microtome
The temperature of slice, tool temperature are -15 DEG C, and sample disc temperature is -5 DEG C, and slice thickness is 20 microns；
F. melt mounting：Ginseng crude drug's sample of slice is transferred to on the conductive glass plate conducting surface that alcohol is cleaned and is dried, is used
Finger is heated in glass back, makes peripheral one layer of ice thaw and ginseng crude drug's sample is made closely to paste on a glass, then take out
Vacuum drying；
G. spray matrix and detection：Spray N- (1- naphthalenes)-ethylene diamine hydrochloride matrix, N- (1- naphthalenes)-ethylene diamine hydrochloride it is a concentration of
The N- (1- naphthalenes)-ethylene diamine hydrochloride is dissolved in purity in 50% methanol by 10mg/mL, is detected point with MALDI-MSI
Analysis.
Interpretation of result
As shown in Fig. 1 and Figure 10 to Figure 16, in the present embodiment, after carrying out samples of Ginseng processing using the present invention, MALDI-MSI
The mass signal intensity of detection significantly increases.
Although the embodiment of the present invention is described with practical solution, the limit to meaning of the present invention is not formed
It makes, for those skilled in the art, is all to the modification of its embodiment and with the combination of other schemes according to this specification
Obviously.
Claims (8)
1. a kind of ginseng crude drug MALDI-MSI sample treatments, which is characterized in that include the following steps：
A. medicinal material is sliced：After ginseng crude drug's sample freezen protective, the root of ginseng crude drug is taken, by 1.5 cm thicks, by people
Ginseng medicinal material sample is cut into fan-shaped section；
B. sample water logging：Sample disc after freezing is cleaned, is dried, added in liquid-transfering gun into sample disc 15-25 microlitres go from
Sub- water is put into the ginseng crude drug's sample cut；
C. sample is fixed：Ginseng crude drug's sample disc is placed in freezing-microtome, is freezed, is fixed on ginseng crude drug's sample
In sample disc；
D. embedding freezing：With liquid-transfering gun, deionized water is added dropwise into ginseng crude drug's sample of -60 DEG C of freezings, until deionized water
After until ginseng crude drug's sample is completely covered, continue to freeze；
E. sample sections：Ginseng crude drug's sample by step D processing is sliced, the temperature of the slice of the freezing-microtome
Degree control is -15 DEG C of tool temperature, -5 DEG C of sample disc temperature, and slice thickness is 14-30 microns；
F. melt mounting：Ginseng crude drug's sample of slice is transferred to on the conductive glass plate conducting surface that alcohol is cleaned and is dried, is used
Finger is heated in glass back, makes peripheral one layer of ice thaw and ginseng crude drug's sample is made closely to paste on a glass, then take out
Vacuum drying；
G. spray matrix and detection：Ginseng crude drug's sample that step F is handled well is subjected to spray matrix, is then carried out with MALDI-MSI
Detection and analysis.
2. a kind of ginseng crude drug MALDI-MSI sample treatments according to claim 1, it is characterised in that：In step A
Ginseng crude drug's sample freezen protective temperature be -20 DEG C to -80 DEG C.
3. a kind of ginseng crude drug MALDI-MSI sample treatments according to claim 1, it is characterised in that：In step B
Deionized water further include Milli-Q water, distilled water.
4. a kind of ginseng crude drug MALDI-MSI sample treatments according to claim 1, it is characterised in that：In step D
Continuation cooling time be 5-10min.
5. a kind of ginseng crude drug MALDI-MSI sample treatments according to claim 1, it is characterised in that：In step F
In the time that glass back heats it is 3-8s with finger.
6. a kind of ginseng crude drug MALDI-MSI sample treatments according to claim 1, it is characterised in that：In step F
Vacuumize drying time as 15-25min.
7. a kind of ginseng crude drug MALDI-MSI sample treatments according to claim 1, it is characterised in that：In step G
Matrix used is N- (1- naphthalenes)-ethylene diamine hydrochloride.
8. a kind of ginseng crude drug MALDI-MSI sample treatments according to claim 7, it is characterised in that：In step G
A concentration of 10mg/mL of N- (1- naphthalenes)-ethylene diamine hydrochloride, it is 50% that the N- (1- naphthalenes)-ethylene diamine hydrochloride is dissolved in purity
In methanol, analysis is detected with MALDI-MSI.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109655521A (en) * | 2019-01-28 | 2019-04-19 | 中国地质科学院水文地质环境地质研究所 | Based on Accelerator mass spectrometry14The method for fast measuring of C |
| CN112747980A (en) * | 2020-12-23 | 2021-05-04 | 鉴甄检测技术（上海）有限公司 | Method for making frozen slices of Chinese medicinal cinnamon |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109655521A (en) * | 2019-01-28 | 2019-04-19 | 中国地质科学院水文地质环境地质研究所 | Based on Accelerator mass spectrometry14The method for fast measuring of C |
| CN112747980A (en) * | 2020-12-23 | 2021-05-04 | 鉴甄检测技术（上海）有限公司 | Method for making frozen slices of Chinese medicinal cinnamon |
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