CN110174459A - Application of the golden cluster in Matrix-assisted laser desorption ionization detection - Google Patents
Application of the golden cluster in Matrix-assisted laser desorption ionization detection Download PDFInfo
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- CN110174459A CN110174459A CN201910515381.XA CN201910515381A CN110174459A CN 110174459 A CN110174459 A CN 110174459A CN 201910515381 A CN201910515381 A CN 201910515381A CN 110174459 A CN110174459 A CN 110174459A
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
- G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry
Abstract
Application the invention discloses golden cluster as matrix in Matrix-assisted laser desorption ionization (MALDI-TOF-MS) detection.The present invention is using golden cluster as matrix, the substances such as amino acid small molecule, cyclodextrin, macrogol are analyzed using Matrix-assisted laser desorption ionization (MALDI-TOF-MS), the result shows that, it is successfully synthesized with the golden cluster that bovine serum albumin(BSA) (BSA) is template, and using golden cluster as matrix, determine the molecular weight of the substances such as amino acid small molecule, cyclodextrin, macrogol respectively using MALDI-TOF-MS, the result of study has widened the use scope of golden cluster.
Description
Technical field
The invention belongs to mass spectrum detection fields, and in particular to golden cluster is in the substance assistant laser desorpted ionized flight time
Application in Mass Spectrometer Method.
Background technique
Matrix Assisted Laser Desorption mass spectrum has Karas and Hillenkamp to propose, because it has efficient ionization
Efficiency, high sensitivity, quickly, it is high-throughput and be easy to the features such as automating, it has also become powerful tool application in point
In terms of analysing polymer, large biological molecule.The basic functional principle of mass spectral analysis (MALDI) are as follows: sample is mixed with matrix first,
Point is on Special sample target, after crystallization to be dried, using pulse type laser irradiating sample and corresponding substrate formed eutectic, base
Mass-energy effectively absorbs the energy of laser and passes to biomolecule, in the process by proton translocation to biomolecule or from life
Object molecule obtains proton, so that biomolecule be made to ionize.
From the working principle of above-mentioned MALDI, we are played non-it can be found that matrix is very crucial in MALDI ionization process
Normal important role, first is that isolation analyte, laser excite matrix first；Second is that the laser of low energy can be absorbed, and by its turn
For the ionization energy of analyte, and then it can achieve parsing and the effect of ionization of analytes；Three are to provide proton, and proton translocation is given
Analyte makes its ionization.It as stroma ground substance, while must satisfy following three conditions: 1) laser can be absorbed；2) energy and sample
Product form crystallite, and cannot be with example reaction；3) efficient ion efficiency.The matrix of existing commercialization is mostly
Organic matter, such as alpha-cyano -4- hydroxycinnamic acid, molecular weight 189；2,5-dihydroxybenzoic acid, molecular weight 154, to polypeptide
It works well with protein ion.
However MALDI is then relatively difficult for the test (< 500Da) of small molecule compound molecular weight, is primarily due to
There is also ionization for matrix itself, it may appear that the molecular ion peak of matrix plus sodium ion plus ammonium ion, fragment peak and matrix
It polymerize peak etc., interference is generated to its analyte testing result, complicated is become to substance-measuring interpretation of result, especially to unknown sample
Analysis then can be extremely difficult.
For the MALDI ionization of small molecule, the relevant researcher of mass spectrum has made a large amount of research and trial.Such as Nanjing
University's teacher's wang project has been combined into Rare earth monocrystal EuF3, characterizes to its structure, and is successfully applied to amino acid, ring paste
The test of the molecular weight of material such as essence, polymer.Cai et al. is using with magnetic metal organic frame (MOFs) nano material
Fe3O4@ZIF-8MNCs determines the molecular weight analytes such as amino acid, quinolines, phenol as material, using negative ion mode,
And it is applied to biological sample analysis.Hong etc. will prepare AgNP/rGO in the graphene oxide of silver nanoparticle deposition to reduction-state
Meet the more empty thin-film materials of nanometer, and is used for small molecule sample analysis.Poly-dopamine is aggregated on magnetic nanoparticle ball by Ma etc.,
Prepare (Fe3O4@PDA NPs) meet material, and for small molecule environmental contaminants (molecular weight: 251.6 to 499.3) sample point
Analysis, and obtain lower detection sensitivity.The matrix that Wang etc. is ionized using graphene as MALDI, and it is successfully applied to amino
The molecular weight of the small-molecule substances such as acid, cyclophosphamide is analyzed.Matrix of these inorganic nano materials as MALDI, makes MALDI can
To be used successfully to the measurement of small molecule molecular weight, the application range of MALDI has been widened, but these nano materials, some needs
Composite material is prepared, synthesis process is complicated.
Gold nanoclusters (golden cluster) have the characteristics such as excellent dimensional effect, optics, electricity, green non-poisonous, it has also become current
Materialogy, chemistry and biology research hotspot.Largely studies have shown that golden cluster has the characteristics that, first is that having good light
Characteristic；Second is that synthesis condition is simple, golden cluster can be directly obtained with a step in the solution, and can have with different templated synthesis
There is the golden cluster of different optical characteristics；Third is that having good biocompatibility.
The present invention synthesizes gold nanoclusters using bovine serum albumin(BSA) as template, inquires into it as matrix for auxiliary laser solution
Inhale the molecular weight that small molecule and polymer are analyzed in ionization time of flight mass spectrometry detection.
Summary of the invention
In order to solve the deficiencies in the prior art, the main object of the present invention is to provide golden cluster in substance assistant laser desorpted electricity
From the application in flight time mass spectrum detection.
The gold cluster detects after being prepared into golden cluster aqueous solution as Matrix-assisted laser desorption ionization, gold
Cluster aqueous solution is prepared as follows:
Under agitation, it is 5mM's that concentration is added in the Bovine Serum Albumin in Aqueous Solution for being 25mg/ml toward mass concentration
HAuCl4Solution mixes, and the NaOH solution that concentration is 1M is then added, mixes, then be placed at 37 DEG C and be incubated for 12h, high speed centrifugation
Separation discards solution, collects precipitating to get golden cluster is arrived；
Into golden cluster plus water is configured to golden cluster aqueous solution, and the volume of golden cluster aqueous solution is Bovine Serum Albumin in Aqueous Solution body
Long-pending twice；
Wherein, Bovine Serum Albumin in Aqueous Solution, HAuCl4The volume ratio of solution and NaOH solution is 125:125:2.
In above-mentioned application, high speed centrifugation revolving speed is 200 revolutions per seconds.
In above-mentioned application, golden cluster is as Matrix-assisted laser desorption ionization detection method specifically:
Object to be detected is configured to the stock solution that concentration is 1mM with water；
It takes the stock solution of determinand in centrifuge tube, golden cluster aqueous solution is added, adding containing mass concentration is 1% trifluoro second
The acetonitrile solution vortex of acid is uniformly mixed, and 1.0 μ L mixed liquor point targets is taken to be analyzed by mass spectrometry after sample drying；Wherein, to
Stock solution, golden cluster aqueous solution and the volume ratio containing the acetonitrile solution that mass concentration is 1% trifluoroacetic acid for surveying object are 1:1:98.
Preferably, the volume ratio containing acetonitrile and trifluoroacetic acid in the acetonitrile solution that mass concentration is 1% trifluoroacetic acid
For 4:1.
In above-mentioned application, golden cluster as matrix small molecule compound the substance assistant laser desorpted ionized flight time
Application in Mass Spectrometer Method is also within protection scope of the present invention.
Preferably, the small molecule compound is one kind of amino acid or cyclodextrin.
In above-mentioned application, Matrix-assisted laser desorption ionization of the golden cluster as matrix in polymer is examined
Application in survey is also within protection scope of the present invention.
Preferably, the polymer is polyethylene glycol.
Compared with prior art, the beneficial effects of the present invention are:
The present invention is using golden cluster as matrix, using Matrix-assisted laser desorption ionization (MALDI-
TOF-MS) substances such as amino acid small molecule, cyclodextrin, macrogol are analyzed, study carefully the result shows that, with bovine serum albumin
White (BSA) is that the golden cluster of template is successfully synthesized, and using golden cluster as matrix, determines ammonia respectively using MALDI-TOF-MS
The molecular weight of the substances such as base acid small molecule, cyclodextrin, macrogol, the result of study have widened the use scope of golden cluster.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is the present invention
Some embodiments for those of ordinary skill in the art without creative efforts, can also basis
These attached drawings obtain other attached drawings.
Fig. 1 is the MALDI-TOF-MS figure of the golden cluster of the present invention and bovine serum albumin(BSA), wherein (a) curve is bovine serum albumin
White mass spectrogram, (b) curve is the mass spectrogram of golden cluster, the equal meson acid of matrix used；
Fig. 2 is that the high-resolution of the golden cluster of the present invention projects electron microscope, wherein (b) figure is the enlarged drawing of (a) figure；
A in Fig. 3) be golden cluster and bovine serum albumin(BSA) (BSA) uv absorption spectra, B) be golden cluster fluorescence excitation with
Launching light spectrogram, wherein (a) curve is the ultra-violet absorption spectrum of bovine serum albumin(BSA), (b) curve is the UV absorption light of golden cluster
Spectrum；
Fig. 4 is the MALDI-TOF-MS figure of the golden cluster of bovine serum albumin(BSA) functionalization, and matrix used is golden cluster；
Fig. 5 is the MALDI-TOF-MS figure of four kinds of kilnitamins (threonine, histidine, alanine, phenylalanine)；
Fig. 6 is the MALDI-TOF-MS figure of two kinds of cyclodextrin (β and γ-CD)；
Fig. 7 is the MALDI-TOF-MS figure of the polyethylene glycol (PEG) (HO- (CH2CH2O) n-H) of different polymerization degree, wherein A
For the mass spectrogram of PEG 2000, B is the mass spectrogram of PEG 42000, and C is the mass spectrogram of PEG 6000, and D is the mass spectrum of PEG 8000
Figure.
Specific embodiment
In order to enable those skilled in the art to more fully understand, technical solution of the present invention is practiced, below with reference to specific
The invention will be further described for embodiment and attached drawing, but illustrated embodiment is not as a limitation of the invention.
Unless otherwise defined, the hereinafter used all technical terms for being and the normally understood meaning of those skilled in the art
It is identical.Technical term used herein is intended merely to the purpose of description specific embodiment, is not intended to the limitation present invention
Protection scope.Unless otherwise specified, it the various raw materials used in the following embodiment of the present invention, reagent, instrument and sets
It is standby to be commercially available by market or be prepared by existing method.
Specifically, the bovine serum albumin(BSA) used in the embodiment of the present invention, HAuCl4It buys in the raw work bioengineering in Shanghai
Co., Ltd；Amino acid, polyethylene glycol purchase are in Aladdin.
It should be noted that using golden cluster in following embodiments as matrix detects object (small molecule compound to be detected
Or polymer) molecular weight, the preparation of specific gold cluster is all made of following method:
5ml mass concentration 25mg/ml Bovine Serum Albumin in Aqueous Solution is weighed, is added dropwise is preinstalled under agitation
5ml, 5mMHAuCl4Solution in, after vortex mixing 1min, be added 80 μ L, 1M NaOH solution, continue vortex mixing 10min,
It is placed in shaking table, 37 DEG C of water-baths are incubated for 12h, are then centrifuged at a high speed with 200 revolutions per seconds of speed and discard solution, collect precipitating, i.e.,
Golden cluster is obtained, is settled to 10ml with water, obtains golden cluster aqueous solution.
In following embodiments, laser desorption ionisation flight time mass spectrum (MALDI-TOF-MS) detection method of use is such as
Under:
It takes the stock solution of 1.0 μ L determinands in the centrifuge tube of 0.5ml, the golden cluster aqueous solution of 1.0 μ L is added, adds 98
The acetonitrile solution vortex containing 1% trifluoroacetic acid of μ L is uniformly mixed, and takes 1.0 μ L mixed liquor point targets, after sample drying, carries out matter
Spectrum analysis.Wherein, the stock solution for being 1mM with water configuration concentration by object to be detected.
The golden cluster being prepared is subjected to MALDI-TOF-MS mass spectrum, transmission electron microscope characterizes it, while studying it
Fluorescent spectroscopic properties.As shown in Figure 1, the golden cluster of expression is general by comparison, it was found that synthesizing molecular weight after golden cluster increases about 5KDa
Containing 25 gold atoms, this is consistent with document report result；From figure 2 it can be seen that golden cluster form is spherical shape, and size
Uniformly, having a size of 4.5 ± 0.3nm；From figure 3, it can be seen that a length of excitation wavelength of maximum absorption wave of golden cluster is 350nm, hair
The a length of 650nm of ejected wave, this is different with individual bovine serum albumin(BSA) (BSA) photoluminescent property.
One, the test of molecular weight
Before for matrix test, we have primarily looked at golden cluster as matrix itself with the presence or absence of interference.Fig. 4 is single
Mass spectrogram when solely there is golden cluster matrix shows gold figure 4, it is seen that no signal peak occurs in the range of tested
Cluster meets the condition as MALDI-TOF-MS matrix.
Embodiment 1
Golden cluster detects amino acid as matrix
Using amino acid as small molecule, assessing golden cluster is the energy that matrix analyzes small molecule molecular weight for MALDI-TOF-MS
Power.Fig. 5 is the mass spectrogram of four kinds of kilnitamins (threonine, histidine, alanine, phenylalanine).It can be sent out from Fig. 5
Existing, matrix interference effect is suppressed, and fragment peak there's almost no, and show higher signal ratio (S/N), resolution ratio
(Resolution), signal strength (Intensity), as shown in table 1.The result shows that the matrix that golden cluster can be used as MALDI is used
In the test of amino acid small molecule molecular weight.
1 four kinds of amino acid testing results of table
Embodiment 2
Golden cluster detects cyclodextrin as matrix
Whether ability is had a wide range of applications to preferably verify golden cluster as matrix, we continue to have investigated golden cluster work
The test of 2 kinds of cyclodextrin (β and γ-CD) molecular weight is used for for matrix, as a result as shown in Figure 6.Golden cluster can be used as the base of MALDI
It is as shown in the table for the Measurement results of cyclodextrin molecular amount for matter.From Table 2, it can be seen that the S/N difference of two kinds of cyclodextrin
For 11 and 9,3 are all larger than, shows that golden cluster can be used as the molecular weight of MALDI matrix test cyclodextrin.
2 two kinds of cyclodextrin testing results of table
| m/z | S/N | Resolution | Intensity | |
| β-CD | 1158.477[M+H]+ | 11 | 4247 | 1240 |
| γ-CD | 1320.598[M+H]+ | 9 | 5285 | 1005 |
Embodiment 3
Golden cluster detects polyethylene glycol as matrix
MALDI-TOF-MS is usually utilized to analyze the molecular weight of some polymer, in order to which nearly golden cluster of an investigation is as matrix
Application range, the present embodiment with the polyethylene glycol (PEG) (HO- (CH2CH2O) n-H) of different polymerization degree be target analytes,
Using golden cluster as matrix, mass spectrometric measurement is carried out, as a result such as Fig. 7-A, B, C, shown in D.Wherein, 2000 PEG, 4000,6000,
8000 range of molecular weight distributions are respectively 1800-2200Da, 3600-4400Da, 5500-7500,7500-8500.It can from figure
With discovery, other than PEG8000, remaining test result molecular weight distribution is PEG 2000:1400-2300 respectively；PEG
4000:3000-5000；PEG 6000:5000-7000.Although 8000 signal resolution of PEG is lower outer, remaining signal is stronger,
And there is good resolution ratio, the PEG signal distributions tested are better than DHB and (are usually used in the one of polymer molecular weight test
Kind of matrix) effect as matrix.
To sum up, it can be widely applied to as matrix substance assistant laser desorpted due to golden cluster provided in an embodiment of the present invention
In ionization time of flight mass spectrometry detection.
Obviously, various changes and modifications can be made to the invention without departing from essence of the invention by those skilled in the art
Mind and range.In this way, if these modifications and changes of the present invention belongs to the range of the claims in the present invention and its equivalent technologies
Within, then the present invention is also intended to include these modifications and variations.Embodiment described above is only to absolutely prove the present invention
And the preferred embodiment lifted, protection scope are without being limited thereto.Those skilled in the art institute on the basis of the present invention
The equivalent substitute or transformation of work, within the scope of the present invention, protection scope of the present invention is subject to claims.
Claims (8)
1. application of the gold medal cluster in Matrix-assisted laser desorption ionization detection.
2. application according to claim 1, which is characterized in that the gold cluster is prepared into after golden cluster aqueous solution auxiliary for matrix
Laser desorption ionisation flight time mass spectrum is helped to detect, the gold cluster aqueous solution is prepared as follows:
Under agitation, it is 5mM's that concentration is added in the Bovine Serum Albumin in Aqueous Solution for being 25mg/ml toward mass concentration
HAuCl4Solution mixes, and the NaOH solution that concentration is 1M is then added, mixes, then be placed at 37 DEG C and be incubated for 12h, high speed centrifugation
Separation discards solution, collects precipitating to get golden cluster is arrived；
Into golden cluster plus water is configured to golden cluster aqueous solution, and the volume of golden cluster aqueous solution is Bovine Serum Albumin in Aqueous Solution volume
Twice；
Wherein, Bovine Serum Albumin in Aqueous Solution, HAuCl4The volume ratio of solution and NaOH solution is 125:125:2.
3. application according to claim 2, which is characterized in that high speed centrifugation revolving speed is 200 revolutions per seconds.
4. application according to claim 2, which is characterized in that golden cluster is as the substance assistant laser desorpted ionized flight time
Mass Spectrometry detection method specifically:
Object to be detected is configured to the stock solution that concentration is 1mM with water；
Golden cluster aqueous solution is added into the stock solution of determinand, adds the acetonitrile solution containing mass concentration for 1% trifluoroacetic acid
It is uniformly mixed, obtains mixed liquor；1.0 μ L mixed liquor point targets are taken to be analyzed by mass spectrometry after sample drying；
Wherein, the stock solution of determinand, golden cluster aqueous solution and the volume ratio containing the acetonitrile solution that mass concentration is 1% trifluoroacetic acid
For 1:1:98.
5. application according to claim 1, which is characterized in that golden cluster as matrix small molecule compound Matrix-assisted
Application in the detection of laser desorption ionisation flight time mass spectrum.
6. application according to claim 5, which is characterized in that the small molecule compound is amino acid or cyclodextrin
It is a kind of.
7. application according to claim 1, which is characterized in that golden cluster as matrix polymer ground substance assistant laser solution
Inhale the application in ionization time of flight mass spectrometry detection.
8. application according to claim 7, which is characterized in that the polymer is polyethylene glycol.
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| CN111175369A (en) * | 2020-02-24 | 2020-05-19 | 陕西师范大学 | MALDI-TOF MS matrix for small molecule detection and application thereof |
| CN112986374A (en) * | 2019-12-13 | 2021-06-18 | 中国科学院大连化学物理研究所 | Laser desorption ionization mass spectrum matrix, detection method, mass spectrum and application of mass spectrum in neurotransmitter detection |
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Application publication date: 20190827 |
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