CN109521104A - It is a kind of for an identification method for emerald green historical relic adhesive - Google Patents
It is a kind of for an identification method for emerald green historical relic adhesive Download PDFInfo
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- CN109521104A CN109521104A CN201811212167.9A CN201811212167A CN109521104A CN 109521104 A CN109521104 A CN 109521104A CN 201811212167 A CN201811212167 A CN 201811212167A CN 109521104 A CN109521104 A CN 109521104A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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Abstract
The invention belongs to historical relic sci-tech protection technical fields, and in particular to a kind of for an identification method for emerald green historical relic adhesive.This method carries out pre-treatment to sample first, will be dissolved in buffer after treated sample solid-phase extraction column desalination, vacuum drying later；Centrifugal treating collects supernatant, carries out efficient liquid phase chromatographic analysis detection, subsequently carries out Tandem Mass Spectrometry Analysis；Finally selection albumen database is retrieved.The accurate identification for the ancient times point kingfisher historical relic adhesive that seriously degradation, content are extremely low may be implemented using kingfisher historical relic adhesive identification method proposed by the present invention of putting, it is possible to identify the precise information of adhesive source animal type.The accuracy of identification and sensitivity significantly improve.
Description
Technical field
The invention belongs to historical relic sci-tech protection technical fields, and in particular to a kind of for an identification side for emerald green historical relic adhesive
Method.
Background technique
Kingfisher, i.e., emerald green plumage, the plumage of kingfisher.Point kingfisher is the combination of the smithcraft and feather technique of Chinese tradition, first with golden or
The metal of gold-plating is made into the pedestal of different pattern, then the beautiful blue feather in kingfisher back is embedded on pedestal, each to be made
Kind implements.With a historical relic for emerald green technique production, reflecting feel is good, beautiful in colour.The emerald green technique of point originates from the Warring states, Qing Dynasty Kangxu,
Yongzheng, Qianrong reach peak period, and late Qing Dynasty is still popular to the Republic of China, contain superb technical level and immortal artistic value.
The basic structure of the emerald green historical relic of point is made of three parts: emerald green plumage, pedestal (such as pileum, hair hairpin as its mark
Deng), by both of the above combine adhesive used.Suitable adhesive is selected, effective combine with the emerald green plumage of guarantee and pedestal is
The key of the emerald green technique of point.However, the information of adhesive used in conventional point kingfisher technique can not pass on so far.
Accurate identification is carried out to ancient times adhesive remaining in existing point kingfisher historical relic using method appropriate, can be point
Emerald green technology history and Protective strategy provide basic information.Existing identification method is infra-red sepectrometry, is contained according in sample
Functional group, infer the emerald green adhesive type of point.This identification method needs to collect visible adhesive sample.However, the emerald green text of point
Adhesive used in object is easily degraded during long-term preservation.Remaining ancient times gluing agent content is extremely low in the existing emerald green historical relic of point,
Often without visible marks.Allow to be collected into and meet the sample that infra-red sepectrometry identification requires, qualification result can only also be given
Wherein contain animal glue out, the precise information of adhesive source animal type can not be provided.
Summary of the invention
Goal of the invention: it is a kind of for an identification method for emerald green historical relic adhesive, to solve conventional point kingfisher adhesive identification skill
Art can not detect the point kingfisher adhesive sample of no visible marks, and fubaritic adhesive source animal type precise information is asked
Topic.
Technical solution: a kind of for an identification method for emerald green historical relic adhesive, comprising the following steps:
Step 1: sample pre-treatments, the pre-treating method are as follows: extract and separation after containing a gel sample for emerald green adhesive
Product successively carry out decolourizing-dehydration-keep the temperature-being dehydrated again-and digest-be dried；
Step 2: after step 1 treated sample solid-phase extraction column desalination, vacuum drying, it is dissolved in buffer solution A；From
Heart processing, collects supernatant, carries out efficient liquid phase chromatographic analysis detection, eluent is buffer solution B；The buffer solution A is 2% second
Nitrile, 0.1% formic acid；The buffer solution B is 98% acetonitrile, 0.1% formic acid；
Step 3: Tandem Mass Spectrometry Analysis；
Step 4: database retrieval.
Preferably, sample discoloration method are as follows: be cut into the painted areas of the gel containing a little emerald green historical relic sample extraction object more
A about 1mm2Fritter, decolourized 2 times with the acetonitrile of the ammonium hydrogen carbonate of 25mM and 50% respectively, each 1h.
Preferably, the acetonitrile of 500 μ L 100% is selected when dehydration.
Preferably, the self-control 2cm C18 of high performance liquid chromatography efficient liquid phase chromatographic analysis condition: is added with autosampler
In column and 10cm C18 column (75 μm of interior diameter)；The rate that sample is added is that 15 μ L/min continue 4min, and elution rate is
400nL/min continues 91min, and eluate concentration increases linearly to 35%, after then increasing linearly to 80% in 5min from 2%
Continue 8min, is eventually returned to 2% and continues 2min.
Preferably, Tandem Mass Spectrometry Analysis condition: mass resolution 60,000；Tandem Mass Spectrometry Analysis uses collision-induced solution
From mode, standard collision energy is 35%, and fragment ion is detected with linear ion hydrazine；10 highest Ion Countings of abundance exist
5000 or more parent ion enters second mass analysis；Electron spray voltage is 1.5kV；Ion is prevented using automatic growth control
Trap overfill accumulates 1 × 10 in an ion trap4A ion generates second order ms；Mass charge ratio range is 350-2,000Da.
Preferably, use MASCOT software using chordate animal to carry out in the Uniprot albumen database that type limits
Retrieval；Retrieval parameter are as follows: trypsase mode allows accidentally to cut number 2, and urea methylation is fixed modification, deaminated, be oxidized to can
Take as an elective course decorations, parent ion allowable error 20ppm, fragment ion allowable error 0.1Da；Method of quality control is that false positive rate controls
1% or less；Identification of Fusion Protein only may be implemented when identifying two specific peptide fragments；All peptide fragments identified are all used
BLAST is retrieved again in irredundant NCBI albumen database, to determine the specificity of sequence.
Advantageous effects: using it is proposed by the present invention put emerald green historical relic adhesive identification method may be implemented seriously to degrade,
The accurate identification of the extremely low ancient times point kingfisher historical relic adhesive of content, it is possible to identify the precise information of adhesive source animal type.Mirror
Fixed accuracy and sensitivity significantly improves.
Detailed description of the invention
The mass spectrogram that Fig. 1 is a little bovine collagen albumen alpha-1 (I) chain specificity peptide fragment GEGGPQGPR in emerald green historical relic,
Fig. 2 is a little bovine collagen albumen alpha-1 (III) chain specificity peptide fragment GGPGGPGPQGPAGK in emerald green historical relic
Mass spectrogram,
The mass spectrum that Fig. 3 is a little bovine collagen albumen alpha-2 (I) chain specificity peptide fragment IGQPGAVGPAGIR in emerald green historical relic
Figure.
Specific embodiment
Specific identification case one: a kind of for an identification method for emerald green historical relic adhesive:
The painted areas of gel containing a little emerald green historical relic sample extraction object is cut into multiple about 1mm by 12Fritter, use respectively
The ammonium hydrogen carbonate of 25mM and 50% acetonitrile decolourize 2 times (each 1h), and the acetonitrile of 500 μ L 100% is then added for being dehydrated.
By sample in 56 DEG C of heat preservation 60min in the ammonium bicarbonate buffers of the 25mM containing 200 μ L dithiothreitol (DTT)s (10mM), to cut
Disulfide bond in disconnected sample.Then the ammonium bicarbonate buffers by sample in the 25mM containing 200 μ L iodoacetamides (55mM) exist
It is protected from light heat preservation 45min at room temperature, the cysteine in sample is alkylated.Then, sample is cleaned 2 with 25mM ammonium hydrogen carbonate
It is secondary, it is dehydrated again with 500 μ L acetonitriles.Sample is placed in trypsin solution, and (concentration 10ng/ μ L, solvent 25mM ammonium hydrogen carbonate are slow
Fliud flushing) in 37 DEG C overnight enzymatic hydrolysis, be then added 5% formic acid terminate enzymatic hydrolysis.Finally, being examined after sample nitrogen drying in mass spectrum
It is dissolved in front of survey in 0.1% trifluoroacetic acid of 3 μ L.
2 after sample solid-phase extraction column desalination, vacuum drying, will be dissolved in 200 μ L buffer solution A (2% acetonitrile, 0.1% first
Acid).After being centrifuged 10min at 20000g, collect supernatant (sample concentration about μ g/ μ L).By 10 μ L supernatant automatic samplings
Device is added in the self-control 2cm C18 column and 10cm C18 column (75 μm of interior diameter) of high performance liquid chromatography.Sample be added rate be
15 μ L/min continue 4min, and elution rate is that 400nL/min continues 91min, eluent for buffer solution B (98% acetonitrile, 0.1%
Formic acid), concentration increases linearly to 35% from 2%, continues 8min after then increasing linearly to 80% in 5min, is eventually returned to
2% continues 2min.
3 samples successively pass through electron spray and tandem mass spectrum, mass resolution 60,000 after high performance liquid chromatography.String
Join mass spectral analysis and use collision induced dissociation mode, standard collision energy is 35%, and fragment ion is detected with linear ion hydrazine.10
A highest Ion Counting of abundance enters second mass analysis in 5000 or more parent ion.Electron spray voltage is 1.5kV.Using
Automatic growth control prevents ion trap overfill, accumulates 1 × 10 in an ion trap4A ion generates second order ms, mass-to-charge ratio
Range is 350-2,000Da.
4 use MASCOT software 2.3.02 version (to limit by type of chordate animal in irredundant Uniprot albumen database
System) in retrieval high performance liquid chromatography-tandem mass combination generate initial data.Search parameter are as follows: trypsase mode allows
Accidentally cut number 2, urea methylation is fixed modification, deaminated, be oxidized to optional modification, parent ion allowable error 20ppm, fragment from
Sub- allowable error 0.1Da.Method of quality control is that false positive rate controls below 1%.Only identifying two specific peptides
Identification of Fusion Protein may be implemented when section.All peptide fragments identified are all examined in irredundant NCBI albumen database with BLAST again
Rope, to determine the specificity of sequence.
Test result analysis: table 1 is 9 spies using technique successful identification point kingfisher sample adhesive source bovine collagen
Anisotropic peptide fragment list.Fig. 1-3 is the Mass Spectrometric Identification of peptide fragment GEGGPQGPR, GGPGGPGPQGPAGK, IGQPGAVGPAGIR respectively
Result figure illustrates that qualification result is reliable.
Table 1
Claims (6)
1. a kind of for an identification method for emerald green historical relic adhesive, it is characterised in that: the following steps are included:
Step 1: sample pre-treatments, the pre-treating method are as follows: extract and separation after containing the gel sample of emerald green adhesive according to
Secondary decolourize-dehydration-keeps the temperature-being dehydrated again-and digests-be dried；
Step 2: after step 1 treated sample solid-phase extraction column desalination, vacuum drying, it is dissolved in buffer solution A；At centrifugation
Reason collects supernatant, carries out efficient liquid phase chromatographic analysis detection, eluent is buffer solution B；The buffer solution A is 2% acetonitrile,
0.1% formic acid；The buffer solution B is 98% acetonitrile, 0.1% formic acid；
Step 3: successively pass through electron spray and Tandem Mass Spectrometry Analysis；
Step 4: use MASCOT software using chordate animal to be retrieved in the Uniprot albumen database that type limits
Analysis.
2. according to claim 1 a kind of for an identification method for emerald green historical relic adhesive, it is characterised in that: sample decoloration
Method are as follows: the painted areas of the gel containing a little emerald green historical relic sample extraction object is cut into multiple about 1mm2Fritter, use respectively
The ammonium hydrogen carbonate of 25mM and 50% acetonitrile decolourize 2 times, each 1h.
3. according to claim 1 a kind of for an identification method for emerald green historical relic adhesive, it is characterised in that: dehydration
The acetonitrile of Shi Xuanyong 100%.
4. according to claim 1 a kind of for an identification method for emerald green historical relic adhesive, it is characterised in that: efficient liquid phase
Chromatographiccondition are as follows: be added with autosampler in the self-control 2cm C18 column and 10cm C18 column of high performance liquid chromatography；Sample
The rate of addition is that 15 μ L/min continue 4min, and elution rate is that 400nL/min continues 91min, and eluate concentration is linear from 2%
Increase to 35%, continue 8min after then increasing linearly to 80% in 5min, is eventually returned to 2% and continues 2min.
5. according to claim 1 a kind of for an identification method for emerald green historical relic adhesive, it is characterised in that: tandem mass spectrum
Analysis condition: mass resolution 60,000；Tandem Mass Spectrometry Analysis uses collision induced dissociation mode, and standard collision energy is
35%, fragment ion is detected with linear ion hydrazine；10 highest Ion Countings of abundance enter two in 5000 or more parent ion
Grade mass spectral analysis；Electron spray voltage is 1.5kV；Ion trap overfill is prevented using automatic growth control, accumulates 1 in an ion trap
×104A ion generates second order ms；Mass charge ratio range is 350-2,000Da.
6. according to claim 1-5 a kind of for an identification method for emerald green historical relic adhesive, it is characterised in that:
The retrieval parameter of retrieval analysis are as follows: trypsase mode allows accidentally to cut number 2, and urea methylation is fixed modification, deaminated, oxygen
Turn to optional modification, parent ion allowable error 20ppm, fragment ion allowable error 0.1Da；Method of quality control is false positive rate
Control is below 1%；Identification of Fusion Protein only may be implemented when identifying two specific peptide fragments；All peptide fragments identified are all
It is retrieved again in irredundant NCBI albumen database with BLAST, to determine the specificity of sequence.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810877871X | 2018-08-03 | ||
| CN201810877871 | 2018-08-03 |
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| CN109521104A true CN109521104A (en) | 2019-03-26 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105273061A (en) * | 2015-10-09 | 2016-01-27 | 东阿阿胶股份有限公司 | Common polypeptide in animal glue and application thereof in detection |
| CN106770825A (en) * | 2016-12-22 | 2017-05-31 | 东阿阿胶股份有限公司 | A kind of composition for detecting donkey hide derived components content in animal glue, kit and its detection method |
| WO2018058023A2 (en) * | 2016-09-26 | 2018-03-29 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Methods for identifying proteins that bind ligands |
-
2018
- 2018-10-17 CN CN201811212167.9A patent/CN109521104A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105273061A (en) * | 2015-10-09 | 2016-01-27 | 东阿阿胶股份有限公司 | Common polypeptide in animal glue and application thereof in detection |
| WO2018058023A2 (en) * | 2016-09-26 | 2018-03-29 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Methods for identifying proteins that bind ligands |
| CN106770825A (en) * | 2016-12-22 | 2017-05-31 | 东阿阿胶股份有限公司 | A kind of composition for detecting donkey hide derived components content in animal glue, kit and its detection method |
Non-Patent Citations (5)
| Title |
|---|
| HUIJUAN MAI等: "Investigating the materials and manufacture of Jinzi: The lining of Futou (Chinese traditional male headwear) from the Astana Cemeteries, Xinjiang, China", 《JOURNAL OF CULTURAL HERITAGE》 * |
| HUIYUN RAO等: "Proteomic identification of adhesive on a bone sculpture-inlaid wooden artifact from the Xiaohe Cemetery, Xinjiang, China", 《JOURNAL OF ARCHAEOLOGICAL SCIENCE》 * |
| SOPHIE DALLONGEVILLE等: "Identification of Animal Glue Species in Artworks Using Proteomics: Application to a 18th Century Gilt Sample", 《ANALYTICAL CHEMISTRY》 * |
| ZHANYUN ZHU等: "Mass Spectrometric Identification of Adhesive Utilized in a Tian-tsui Tiara of the mid-Qing Dynasty (1776–1839 CE) in the Collection of the Tang Clan Folk Museum", 《STUDIES IN CONSERVATION》 * |
| 应天翼: "使用胰蛋白酶进行胶内酶解", 《生物产业技术》 * |
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