CN107884467A - Improve the method and product of Mass Spectrometer Method glycosyl crystallization - Google Patents
Improve the method and product of Mass Spectrometer Method glycosyl crystallization Download PDFInfo
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- CN107884467A CN107884467A CN201711054357.8A CN201711054357A CN107884467A CN 107884467 A CN107884467 A CN 107884467A CN 201711054357 A CN201711054357 A CN 201711054357A CN 107884467 A CN107884467 A CN 107884467A
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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
- G01N27/626—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 heat to ionise a gas
- G01N27/628—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 heat to ionise a gas and a beam of energy, e.g. laser enhanced ionisation
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/26—Mass spectrometers or separator tubes
- H01J49/34—Dynamic spectrometers
- H01J49/40—Time-of-flight spectrometers
Abstract
The invention provides a kind of method by improving glycosyl sample secondary crystallization, is included in acetonitrile solution and is separately added into trifluoroacetic acid solution, DHB matrix to prepare matrix solution, and the sample solution for then adding special ratios carries out point sample crystallization on chip.Present invention also offers the mass spectrum bearing calibration for the accuracy rate for improving Mass Spectrometer Method bio-target molecule, it is included on the chip of the hydrophilic region positioning hole with multiple vertical cross arrangements, weep hole exterior domain, cent(e)ring hole, checking correction hole, standby correction hole, point sample glycosyl sample crystallization respectively, point sample matrix solution simultaneously crystallizes, and the glycosyl sample of correction hole position is bombarded by mass spectrum, it is corrected with the mass spectral results to sample to be tested.Present invention also offers the chip for being applicable above-mentioned mass spectrum correction and detection.The present invention improves matrix solution formula, can obtain the preferable primary crystallization of crystal habit and secondary crystallization effect, and its bearing calibration helps to obtain more stable and accurate Mass Spectrometer Method result.
Description
Technical field
The present invention relates to one kind MALDI-TOF Mass Spectrometer Methods are being improved by improving biological specimen crystallization and correcting mode just
The method of true rate, energy Mass Spectrometer Method glycosyl, belongs to mass spectrum detection field.
Background technology
MALDI-TOF-MS (MALDI-TOF MS) technology turns into current protein group
Learn the classical technology in research.During the successful application of the technology, suitable sample-pretreating method is played primary and closed
The effect of key.Only suitable sample-pretreating method is combined with suitable organic substrate, could successfully be realized to core
The precise Identification of acid and protein and other.Choosing for matrix, solvent, salt (metal ion) and sample preparation methods
Select be maldi analysis high polymer success or failure key factor.The condition of optimization is sample molecule and matrix is formed uniformly altogether
Crystallization.MALDI methods analysis one of macromolecular, important key is the suitable matrix of selection.As a result show, the preferable base of effect
Matter only has several.Water solubility synthesis macromolecule such as polyethylene glycol (PEG), polypropylene glycol often arise in the MALDI researchs of early stage
In, because the matrix of analysis polypeptide can also be applied to them, can be from macromolecule and base for other macromolecule
The thinking of some selection matrix is found in the dissolubility of matter.In general, select that during matrix matrix and high molecular polarity should be made
Than more consistent, there is compatibility each other.
Micro-array chip is characterized by high density arrays.Microarray technology is exactly to utilize molecule Hybridization principle, is made while quilt
The sample and microarray hybridization compared, by detecting hybridization signal intensities and data processing, them is changed into different specimens
The abundance of specific gene, so as to compare the difference of the gene expression dose of different specimens comprehensively.Micro-array chip is equal because of its height
One property, structural stability, amount of samples is few and high flux and as developing a most rapid part in chip field.Microarray is not
Only there is extensive purposes in biological heredity field, also have potential use in terms of other quantitative and relative quantitative assay
On the way.
Micro-array chip micropore and surrounding hydrophilic and hydrophobic qualitative difference, preferably limits sample because its pore size is identical
Scope residing for product so that the region analyzed is consistent, and makes it possible quantitative analysis.Mass spectrum is into figure because it is without mark
Note, without separation, by carrying out the quantitative analysis of component in mixture to the analysis of image, make multi-component while examine
Survey is possibly realized, therefore carries out multi-component Simultaneous Quantitative Analysis into figure using mass spectrum in micro-array chip, is that one kind has extensively
The quantitative analysis tech of general application prospect.Application of the mass spectrum into figure in quantitative analysis is largely depended in micro-array chip
In the homogeneity of sample crystallization, the quality in being gathered due to mass spectrometric data is discriminated against, and the peak height or peak area of mass spectrogram can not conducts
The foundation of the quantitative analysis of sample, therefore how to obtain reliably, stable quantitative data is just particularly important.
In application MALDI-TOF MS, generally by biological specimen and a kind of low molecule amount inorganic compound solution of saturation
(being referred to as matrix) carries out mixing and is added on target plate, and sample after matrix cocrystallization with foring with the sample of matrix parcel framework after dry
This solid precipitates.For sample matrix crystalline solid through laser emission, matrix absorbs energy, energy accumulation and rapid heat production from laser,
So that host crystal distils, make sample adsorption, electric charge transfer occurs between matrix and sample and causes ionized sample molecule, ion
Identical kinetic energy is obtained under accelerating field, time-of-flight detector is entered after high pressure accelerates, focused on.During according to ion flight
Between difference carry out analysis and draw ion mass-to-charge ratio (m/z) and ion peak value, form quality collection of illustrative plates, detection accuracy is high.Pass through
Software analysis compares, and screens and determines specific finger-print, so as to realize differentiation to objective microbe kind or bacterial strain and
Identification.MALDI-TOF MS can be used for analyzing polytype sample, including organic molecular solution, nucleic acid, protein and whole
Individual microorganism, wherein gene, protein and microorganism are at present in the most widely used project in mass spectrometry clinical testing laboratory.
The ionizable relative molecular mass of substance assistant laser desorpted ionized mass spectrum (MALDI-TOF MS) ion gun be 100~
1000 000 biomolecule, acted on by high-tension electricity and short laser pulse, allow host crystal literization, make matrix and sample molecules
Gasification enters mass spectrometric gas phase, and the characteristics of it is most prominent is that quasi-molecular ionization is very strong, big to the dosis tolerata of impurity in sample,
Direct Analysis nucleic acid, protein can provide effectively through mixture caused by ionization for clinical examination Plays thing Quality Research
Technical guarantee, it is the first choice of the reference method of clinical examination.Therefore the sample crystal habit and the direct shadow of quality on chip
Ring mass spectrographic verification result.
In the sample crystal habit and matter quantifier elimination for improving chip, existing research emphasis is how to improve chip
Quality.For example, Chinese granted patent 201410090967.3, " preparing the alternate micro-array chip of hydrophobe and its for matter
The method of spectrum imaging quantitative analysis " provides one kind by setting hydrophobic and hydrophilic region to prepare detection chip on chip,
Wherein using screen printing technique by hydrophobic polymer (dimethyl silicone polymer or polymethyl methacrylate) according to design
For good template brush on electro-conductive glass, wherein applying area is hydrophobic region, and clear area is as reserved hydrophilic area.Then, use is hydrophilic
Material (such as aqueous solution of sample system) is coated with clear area, forms hydrophilic area and the hydrophobic region at homogeneous interval.This method passes through profit
With screen printing technique come design template shape, the area that left some space in advance in hydrophilic area (also known as " being left white processing "), it is therefore desirable to
Special printing equipment and operation software, while target plate will definitely be stood when being coated with hydrophobic region.Meanwhile screen printing technique
Precision is low, and the boundary of close and distant aqua region can not reach micron accuracy.Furthermore, it is desirable to by the testing mixture system aqueous solution equably
Hydrophilic area is layered on, chip baking and curing, 60 degrees Celsius of solidification 2h in baking oven is placed in, adds production time and cost.
Chinese granted patent 201110401165.6, " be enriched with to biological sample and the method for desalination purification processing " are public
A kind of method that the detection target plate with closed pattern surface is prepared using hydrophobic and water wetted material has been opened, including by base
Constructed on bottom and be used as barrier with larger-diameter polymer coating (such as polymethyl methacrylate, polystyrene or photoresist)
Circle area, is then attached in whole substrate using fluorine-containing monolayer or evaporated metal layer as hydrophobic layer.Because the hydrophobic layer can not
Above-mentioned barrier circle area is adhered to, therefore target plate is immersed in organic solvent and is ultrasonically treated, barrier circle area can be removed.Most
Afterwards, then by the polymer coating round area's internal modifications small diameter concentric circles, it is (fluorine-containing so as to obtain hydrophobic outer ring
Monolayer or evaporated metal layer)-hydrophilic centre circle-hydrophobic inner ring (polymer coating) concentric circles mass spectrum target plate, because
This this method is also known as " barrier coating ".However, this method constructs the closed pattern of hydrophobe-hydrophile-hydrophobic region in substrate
Surface, it is necessary to carry out two step hydrophobic treatments, i.e., using fluorine-containing reagent at 100~250 DEG C 1~5 hour of heat growth, work
Skill is complicated, and process takes.Because this method needs the spacing of accurate control polymer coating twice, spacing is too small cause outer ring and
Inner ring is connected, simultaneously because being mutated different substrate surfaces respectively using two kinds of different hydrophobic materials, what is obtained is hydrophobic
The contact angle of area's drop is too small, causes hydrophobic effect poor, have impact on the application of common lab.
Chinese patent application 200610023671.5, disclose " a kind of low-abundance protein target previous step desalination and enrichment
Method ", wherein by hydrophobic polymer (polymethyl methacrylate, polyethylene, polystyrene, polyvinyl fluoride etc.) in advance in target
The sample cell middle body of plate is coated, and then carries out albumen point sample so that protein sample is enriched on hydrophobic polymer layer.
Then, matrix solution is added into point sample area so that pollutant and inorganic salts in protein sample are spread out, and finally obtain sample
With the crystallization of matrix uniform and delicate.Because this method is that protein sample directly is added into hydrophobic layer, by adding excessive matrix
Solution is purified, although objectively there is certain purification effect, causes the waste of protein sample.Meanwhile if manually
Operation in the aperture of each Kapton films, takes 0.2 μ l hydrophobic polymers solution points, and the precision of 0.2 μ l solution is difficult
With control, the hydrophobic homogeneity of orifice surface is influenceed；If be automatically brought into operation, it is necessary to configure corresponding point sample equipment, increase cost, prepare
Complexity, it is not appropriate for being not easy the detection and application of trace protein sample prepared.
Due in the first research of above-mentioned target plate, or target plate surface does not have hydrophilic-hydrophobic difference, (such as Chinese patent
200610023671.5), cause crystal habit poor, or form the coating of hydrophilic-hydrophobic difference, but hydrophobe boundary can not reach
It is too small to micron accuracy or liquid-drop contact angle, but preparation process is excessively complicated, it is impossible to and save time and cost (such as Chinese patent
201410090967.3rd, patent 201110401165.6), or extra device and inspection software are needed, or need excessive
Precious albumen sample detected, the degree of accuracy that these result in Mass Spectrometer Method sample peak is low, and signal to noise ratio is low, and baseline is high.
It is immediate in the prior art, inventor formerly before submit Chinese patent (CN201710539756, for flying
Row time mass spectrum detects the preparation method of the general-purpose chip of albumen and nucleic acid；CN201710539893, for flight time mass spectrum
Detect the general-purpose chip of albumen and nucleic acid) in, it is proposed that improve sample crystal habit and matter by preparing new mass spectrum chip
Amount, the chip mainly include the hydrophily loading wells and hydrophobic pores outskirt that body surfaces have micro- permutation arrangement, hydrophilic points
Sample hole covers 150-800nm hydrophilic film, and hydrophobic pores outskirt covers 150nm-2 μm of hydrophobic film, its surface water droplet
120 ° of contact angle ＞, wherein the hydrophilic film is special silica oxides film, zinc-oxide film, aluminum oxide film
Film etc., hydrophobic pores outskirt carry out processing by silane coupler and form hydrophobic film.The invention to a certain extent can
Improve the crystalline quality of sample.However, the research emphasis of the invention essentially consists in the mass spectrum chip for providing a kind of dual-purpose type, joint
The adaptor chip of particular design, designed by neck different on adapter, multiple chips can be placed, nucleic acid and egg can be realized
Bai Butong samples while sample detection, save mass spectrum enabling lockup.Because the research emphasis of the invention has not focused on pin
To in the Mass Spectrometer Method of single sample, how to improve the crystalline quality of single sample, thus the present inventor still need as
Studied on basis.
In addition, in addition to gene, albumen and microorganism, research protein glycosylation can not only be deepened to glycoprotein
The understanding of biological function, and occur to inquiring into disease, find that disease markers and developing new drug are significant.Immune ball
Albumen (English full name, IgG), also known as antibody are a kind of important glycoprotein, are mainly distributed in blood plasma (or serum).It is by two
The less light chain of bar identical molecular weight and two larger heavy chains of molecular weight are formed by disulfide bond, the glycosyl position of antibody
It is that N- connections are sugared in heavy chain FC fragments.IgG glycosylation is dived for the cytotoxicity and the inflammatory such as anti-inflammatory, proinflammatory for adjusting IgG
Power is very crucial.Contact between autoimmune state and the specific glycosylation pattern of IgG antibody is suffering from rheumatoid joint
Be observed in the patient of scorching and a little autoimmunity vasculitises, wherein reported with the galactosylation of the reduction of IgG antibody and
Sialylated correlation.The focus of current research is turned into the analysis of IgG glycosyl structures.Tao Lei et al. (《Pharmaceutical Analysis magazine》,
O. 11th in 2011) analyze influence of the glycosyl excision to its structure and function in IgG1 type monoclonal antibodies, the results showed that after glycosyl excision
The circular dichroism spectra of antibody changes, and antigen binding capacity declines, and external CDC activity is basic to disappear.
But rich in polyhydroxy and chromophore is substantially free of in glycosyl structure, in properties such as electroneutral so that carbohydrate analysis
Extremely difficult, labyrinth detection is even more abnormal difficult.Therefore, it is necessary to which quick, simple and accurate mode analyzes glycosyl knot
The technology of structure, and carry out glycosyl analysis by various methods.In order to carry out glycosyl analysis, it is necessary to first from included in biological sample
Glycosyl is isolated and purified in product.Usually the glycosyl on glycoprotein is cut away and analyzed after isolating and purifying.Because glycosyl is in itself
There is no chromophoric group, and it is not easy to ionize on mass spectrograph, in order to isolate and purify with can be more during Structural Identification
Glycosyl is effectively detected, the general method for carrying out column front derivation.Mainly glycosyl is marked for this method, makes on glycosyl band
Ultraviolet or fluorophor, the sensitivity of detection is improved, while hydrophobic grouping on glycosyl band can be made again, reduce the polarity of glycosyl,
Glycosyl is set to be retained on reverse-phase chromatographic column, beneficial to the separation of glycosyl.The reagent master of mark is performed the derivatization to glycosyl at present
To include the method for 2-AB labeled derivative glycosyls.
Chinese patent application 201410844142.6, denomination of invention " quick complete detection monoclonal antibody N glycosylation sites
The method of upper oligosaccharides " discloses cuts off oligosaccharides by enzyme digestion reaction, then marks oligosaccharides using 2-AB solution, leads to after purifying oligosaccharides
Cross LC- fluorescence-ESI-MS analyses.However, this method needs to be purified by aminopropyl solid-phase extraction column, column purification step is crossed
It is complex and expensive, therefore it is unsuitable for batch processing excision glycosyl and 2-AB marks and purifying.In addition, Tao Lei et al. is simultaneously public
The method for being cut off from antibody or albumen or determining glycosyl is opened.However, this method is also required to chromatographic column or crosses post purifying, cause
High flux or batch processing are unable to, therefore limits batch processing excision glycosyl and 2-AB marks and purifying.
Chinese patent application 200610084289.5, denomination of invention " glycosyl device for excising " are disclosed for from aqueous slkali
Cut off the device of glycosyl, including reactive tank and the ion exchange column that isolates and purifies.However, the device is adapted only to from composite carbon aquation
Glycosyl is cut off in compound, and whole device includes complicated component parts, therefore it is also unsuitable for batch processing excision glycosyl and 2-AB
Mark and purifying.
In addition, the general step for marking glycosyl using 2-AB reagents is:(1) glycosyl is cut off from glycoprotein by glycosidase；
(2) glycosyl of acquisition is purified；(3) 2-AB marks glycosyl；(4) purifying of glycosyl after marking, that is, two traditional steps
Method of purification.Wherein, analyzed for the glycosyl of serum or blood plasma IgG, also need IgG purification procedures.Therefore for serum (or
Blood plasma) IgG glycosyl analysis, equivalent to need purified three times, wherein glycosyl must be purified before mark, otherwise occur
Impurity disturbs, and causes to cut sugar not exclusively, therefore two step method has complex steps and may cause the loss of glycosyl product.Together
When, at some in the prior art, need to change buffer solution before digestion, otherwise cause to cut anase activity dying down, it is insufficient to cut sugar,
So as to it cannot be guaranteed that the sensitivity and the degree of accuracy of testing result.
In view of MALDI-TOF MS can be used for analyzing polytype sample, including organic molecular solution, nucleic acid, protein
And whole microorganism, wherein gene, protein and microorganism is at present in the most widely used item in mass spectrometry clinical testing laboratory
Mesh, while glycosyl or glycosyl be as potential research emphasis, therefore, it is necessary to which one kind is by improving sample on existing chip basis
This crystallization condition is to improve the method for MALDI-TOF Mass Spectrometer Method bio-target molecule accuracys rate.
The content of the invention
One of principle of the invention is, MALDI-TOF Mass Spectrometer Method biological targets are improved by improving sample crystallization condition
The method of molecule accuracy rate, this method are not directed to how to improve existing mass spectrum chip, but by groping and optimizing mass spectrum core
The crystallization condition of piece, to improve the level of crystallization to bio-target molecule on chip, so as to make full use of existing mass spectrum
Chip, to reduce testing cost.
The two of the principle of the invention are, for glycosyl target molecule on chip crystallographic property, there is provided a kind of glycosyl target molecule
Generic crystallization method.
The three of the principle of the invention are, on the basis of sample crystallization condition is improved, it is further provided a kind of sample mass spectrum
The bearing calibration of detection, so as to determine precondition to improve MALDI-TOF Mass Spectrometer Method bio-target molecule accuracys rate.
Therefore, first purpose of the invention is to provide a kind of method for improving glycosyl sample primary crystallization, and step includes：
(1) using the acetonitrile of deionized water and high-efficiency liquid chromatographic-grade according to 1：1 volume ratio mixing, adds volume fraction
For 0.1% trifluoroacetic acid solution, mixed solution is obtained；
(2) weigh 50mg DHB matrix (DHB) fully to be dissolved with mixed solution, DHB substrate concentrations are
50mg/mL；
(3) DHB matrix and citric acid diamine solution are taken, according to 9：1 volume ratio is well mixed, and produces matrix solution.
(4) 0.5 μ L-1 μ L matrix solutions and 0.5 μ L-1 μ L samples are chosen, point sample crystallization is carried out on chip.
In one embodiment, 50mg DHB is added wherein in step 2, and 3- is shaken by 2000-3000rpm
10min, 8000-12000rpm centrifuge 3-10min, obtain DHB solution.
In other embodiments, 0.5 μ L samples are wherein chosen in step 4 and 0.75 μ L matrix solutions are crystallized.
In any of the above-described embodiment, aforesaid operations are selected from 100,000 grades of cleanliness factors of operation room, 20-25 DEG C of environment temperature,
Carried out under the conditions of ambient humidity 20-30%.
Second purpose of the invention is to provide a kind of mass spectrum core for the accuracy rate that can improve Mass Spectrometer Method bio-target molecule
Piece, the chip include：The chip body being made up of silicon materials or glass or titanium alloy, its surface have multiple vertical cross arrangements
Hydrophilic region positioning hole, weep hole exterior domain, cent(e)ring hole, checking correction hole, standby correction hole；
Wherein, positioning hole has water-wet behavior, for titrating matrix solution or sample；Weep hole exterior domain, surface have
Hydrophobic property；The structure for the close and distant water spacer in micro-array chip surface that positioning hole and hole exterior domain are formed, can be with auxiliary liquid sample
This contraction is condensed upon in hydrophilic region, concentrates sample crystallization, and standard circular is presented, and improves Ionization Efficiency；
Cent(e)ring hole and checking correction hole, for correcting the titration location verification correction hole of protein standard, for verifying
Calibration result；
Standby correction hole, as standby, cent(e)ring hole or checking correction hole are if there is exception, available backup correction
Hole.
In one embodiment, positioning hole is (4-8) × (4-8) vertical crisscross arrangement, center on chip
Correction hole, checking correction hole and standby correction hole in the chips between position vertical array, quantity is 1 or 2.In a tool
In body embodiment, the quantity in cent(e)ring hole, checking correction hole and standby correction hole is 1.
In another embodiment, the hydrophilic film of the chip hydrophily positioning hole covering 150-800nm, hydrophobicity
Hole outskirt covers 150nm-2 μm of hydrophobic film, and 120 ° of its surface water droplet contact angle ＞ is in a specific embodiment, described
Hydrophilic film is silica oxides film, zinc-oxide film, aluminum oxide film etc., and hydrophobic pores outskirt is even by silane
Connection agent carries out processing and forms hydrophobic film.In another embodiment, the silane coupler be selected from vinyl silanes,
Amino silane or dimethyldichlorosilane.
3rd purpose of the invention is to provide a kind of mass spectrum correction of accuracy rate for improving Mass Spectrometer Method bio-target molecule
Method, step include：
(1) as described above, preparing matrix solution, and the protein standard substance for configuring target molecule sample to be measured and correction is molten
Liquid；
(2) in hydrophilic region positioning hole, point 0.5-1 μ L bio-target molecule sample solutions, formed after volatilizing naturally and once tied
It is brilliant；
(3) in cent(e)ring hole, checking correction hole, point 0.5-1 μ L correction protein standard substance solution, formed after volatilizing naturally
Primary crystallization；
(4) respectively in hydrophilic region positioning hole, cent(e)ring hole, checking correction hole, on primary crystallization surface, point 0.5-1 μ
L matrix solutions, secondary crystallization is formed after volatilizing naturally；
(5) bombard cent(e)ring hole position respectively by Laser time-flight MS, verify the calibration samples albumen of correction hole
Standard solution, obtain the spectrogram of protein standard substance peak molecular weight；
(6) when two peak molecular weight deviations of protein standard substance are less than 200PPM, as correction is effective
(7) after correcting successfully, you can carry out Mass Spectrometer Method to the bio-target molecule sample to be measured of hydrophilic region positioning hole.
In one embodiment, 0.5 μ L samples are chosen and 0.75 μ L matrix solutions is crystallized.
In another embodiment, the bio-target molecule is glycosyl sample to be tested, the calibration samples protein standard substance
Solution is that protein standard substance P14R (synthetic pepitide) and ACTH fragment 18-39 (human) standard is more
Peptide mixed liquor.
In any of the above-described embodiment, the mass spectrum, parameter setting is as follows：
Turing mode:linear；
Mass Range:1000-4000；
Max Laser Rep Rate:20.0；
Power:80；
Profiles:50；
Shots:10。
Vacuum threshold：As vacuum ＜ 5E-6, start to detect；
In a preferred embodiment, the MALDI-TOF mass spectrums are CLIN-TOF-II flight time mass spectrums.
4th purpose of the invention is to protect the standard that can improve Mass Spectrometer Method bio-target molecule used in the above method
The mass spectrum chip of true rate.
Brief description of the drawings
Fig. 1 is biological specimen primary crystallization figure；
Fig. 2 is matrix solution and sample secondary crystallization comparison diagram, wherein the μ L samples of (a) 0.5 μ L matrix solutions+0.5, (b) 1
The μ L samples of μ L matrix solutions+0.5, the μ L samples of (c) 0.75 μ L matrix solutions+0.5.
Fig. 3 is improved microarray Mass Spectrometer Method chip surface structural representation, including：(1) hydrophilic region positioning hole；
(2) weep hole exterior domain；(3) cent(e)ring hole；(4) correction hole is verified；(5) standby correction hole；
Fig. 4 is 3 protein standard substance pattern detection mass spectrograms；
Fig. 5 is that 3 glycosyl samples to be tested detect mass spectrogram；
Technique effect
1st, laser mass spectrometry of the invention detection is to be based on microarray Mass Spectrometer Method chip, and its micro-array chip surface texture increases
Correction up hole, mass spectra peak Mass accuracy is improved, so as to improve the accuracy of glycosyl Mass Spectrometer Method；
2nd, the present invention proposes a kind of preferable matrix solution formula, and cleanliness factor ambient temperature and humidity condition, makes biological specimen
Primary crystallization form is preferable；
3rd, the present invention proposes that a kind of preferable matrix solution and sample volume match, and makes matrix solution with sample in chip list
Evenly, testing result is more preferably for face cocrystallization.
4th, it is proposed by the present invention by improving crystal habit and bearing calibration, 3 clinical glycosyl samples are chosen, detection is accurate
Rate reaches 100%；
5th, the present invention improves matrix solution formula, can obtain the preferable primary crystallization of crystal habit and secondary crystallization effect
Fruit, and its bearing calibration can aid in acquisition and more stablize and accurate Mass Spectrometer Method result
Specific embodiment
Below in conjunction with drawings and Examples, the present invention will be further described.
The preparation of embodiment one, matrix solution
The matrix solution main component is DHB, adds a certain proportion of acetonitrile, accelerates the volatilization of matrix primary crystallization,
Quickly form homogeneous intact primary crystallization.
The preparation steps of matrix solution are as follows：
(1) using the acetonitrile of deionized water and high-efficiency liquid chromatographic-grade according to 1：1 volume ratio mixing, adds volume fraction
For 0.1% trifluoroacetic acid solution, mixed solution is obtained；
(2) weigh 50mg DHB matrix (DHB) fully to be dissolved with mixed solution, DHB substrate concentrations are
50mg/mL；
(3) DHB matrix and citric acid diamine solution are taken, according to 9：1 volume ratio is well mixed, and produces matrix solution.
Above-mentioned DHB specification will can choose SIGMA brands in content more than 99%.
Above-mentioned DHB powder 50mg is weighed with electronic balance of the precision more than 0.001g, preferably
Ground, the present embodiment weigh DHB 50.05mg.
Above-mentioned lemon acid diamine powder 0.010-0.020mg is weighed with electronic balance of the precision more than 0.001g, can
To choose Chinese medicines group brand, analyze pure (AR), concentration ＞ 99%.
Above-mentioned deionized water can choose thermo Fisher brands, specification 100ml deionized water；Acetonitrile can select
The silent winged generation that brand of purchase match, high-efficiency liquid chromatographic-grade (HPLC).
Embodiment two, biological specimen primary crystallization form
According to substance assistant laser desorpted ionized principle, the bioprotein sample (P14R reference polypeptides) of selection standard,
In the protein sample solution that (1) hydrophilic region positioning hole, point 0.5-1 μ L prepare, formed once after volatilizing naturally
Crystallization；
(2) cent(e)ring hole, the protein sample solution that point 0.5-1 μ L are prepared, primary crystallization is formed after volatilizing naturally；
(3) correction hole is verified, the protein sample solution that point 0.5-1 μ L are prepared, primary crystallization is formed after volatilizing naturally；
As Fig. 1 is shown, left figure is the crystallization figure of hydrophilic region positioning hole, the crystallization figure of correction hole centered on right figure.Two
Regular circle is presented in crystal shape, and the mellow and full such as jade in surface, quality rule is homogeneous, and crystal is respectively careful to growing, for ideal
Protein crystal form.
It should be pointed out that embodiment one and two should be in 100,000 grades of cleanliness factors of operation room, 20-25 DEG C of environment temperature, ring
Carried out under the conditions of the humidity 20-30% of border.
Embodiment three, by secondary crystallization, determine the optimal proportion of matrix and sample solution
According to substance assistant laser desorpted ionized principle, the chemical example or biological sample [protein standard substance of selection standard
P14R (synthetic pepitide) or/and ACTH fragment 18-39 (human)] mixed according to suitable concn proportioning,
Respectively in the μ L protein solutions of 3 holes midpoint 0.5,0.75,1, primary crystallization is formed after volatilizing naturally；
Then on primary crystallization surface, 0.75 μ L matrix solutions is put, secondary crystallization is formed after volatilizing naturally；
The contrast of secondary crystallization is as shown in Figure 2.Wherein, the crystallization of 0.5 μ L and 1 μ L matrix solutions is irregular, uneven thickness
Even, middle empty, surrounding is thick.Therefore when laser bombardment sample, sample peak poor accuracy, noise is high, and baseline is high.
And 0.75 μ L matrix solutions are regular, thickness is uniform, and crystal orientation growth is careful, and quality is intensive, it is contemplated that when laser bangs
When hitting sample, sample peak degree of accuracy 200PPM, noise S/N >=3.
Thereby determine that and crystallized from 0.5 μ L samples and 0.75 μ L matrix solutions, be preferred plan.
Protein standard substance corrects before example IV, glycosyl pattern detection
(1) before glycosyl pattern detection protein standard substance pretreatment
Because the m/z values of theoretical glycosyl are all smaller, and protein standard substance (P14R (synthetic pepitide) and
ACTH fragment 18-39 (human)) there is the m/z values (M/Z=of suitable glycosyl detection correction in reference polypeptide mixed liquor
1535.84 and 2467.70), and purchase facilitate it is cheap.Selected protein standard substance is bought by sigma official websites
The protein standard substance P14R (synthetic pepitide) that m/z values are 1535.84 and the protein standard that m/z values are 2467.70
Product ACTH fragment 18-39 (human).
(2) pre-process
In the cent(e)ring hole of microarray Mass Spectrometer Method chip and checking correction hole, 0.5 μ L protein standard substances, natural wind are put
After dry, then 0.75 μ L matrix solutions are put, after volatilizing naturally, microarray Mass Spectrometer Method chip is loaded into adaptor chip, sample introduction, put
Enter CLIN-TOF-II flight time mass spectrums sample room.
CLIN-TOF-II flight time mass spectrum parameters are set：
Mass Range:1000-4000；
Max Laser Rep Rate:20.0；
Power:60；
Profiles:50；
Shots:10。
Vacuum threshold：
As vacuum ＜ 5E-6, start to detect；
(3) albumen mark product correct
Firstth, the protein standard substance of laser bombardment cent(e)ring hole position.
2 standard items peak molecular weights of protein standard substance spectrogram are corrected, wherein the molecular weight deviation at each peak is less than
200PPM, you can.
Secondth, the protein standard substance of laser bombardment checking correction hole position
2 standard items peak molecular weights of protein standard substance spectrogram are verified, wherein the molecular weight deviation at each peak is less than
200PPM, as correction are effective.
(4) instrumental correction
Firstth, the protein standard substance of laser bombardment cent(e)ring hole position
2 standard items peak molecular weights of protein standard substance spectrogram are corrected, wherein the molecular weight deviation at each peak is less than
200PPM, you can.
Secondth, the protein standard substance of laser bombardment checking correction hole position
2 standard items peak molecular weights of protein standard substance spectrogram are verified, wherein the molecular weight deviation at each peak is less than
200PPM, as correction are effective, see accompanying drawing 4.
After correcting successfully, instrument Power values 60 are adjusted to i.e. detectable glycosyl sample after 80s.
Embodiment five, glycosyl pattern detection
Preparation：
3 glycosyl samples are to be prepared by 3 pooled plasmas by identical method.Plasma sample passes through：(1) with parent
Pillar with IgG is by isolating and purifying to obtain IgG；(2) glycosyl is cut off from IgG glycoprotein by glycosidase；(3) to the sugar of acquisition
Base is purified；(4) 2-AB marks glycosyl；(5) purifying of glycosyl after marking.The glycosyl of the IgG in blood plasma is analyzed：
The matrix solution prepared, (the known notable glycosyl peak m/z values of 2 often occurred are for 3 glycosyl samples
1606.484th, 1768.626), the microarray Mass Spectrometer Method chip cleaned up.
(2) glycosyl sample point sample：
3 hydrophilic region positioning holes and cent(e)ring hole, checking correction hole are chosen in microarray Mass Spectrometer Method chip surface,
0.5 μ L glycosyl sample solutions of point, after volatilizing naturally, cent(e)ring hole and the μ L matrix solutions of checking correction hole point 0.75,3 hydrophilic
The μ L matrix solutions of zone location hole point 0.75, after volatilizing naturally, microarray Mass Spectrometer Method chip is loaded into adaptor chip, entered
Sample, it is put into CLIN-TOF-II flight time mass spectrums sample room.
(3) CLIN-TOF-II flight time mass spectrum parameters are set：
Turing mode:linear；
Mass Range:1000-4000；
Max Laser Rep Rate:20.0；
Power:80；
Profiles:50；
Shots:10。
(d) vacuum threshold works as vacuum ＜ 5E-6, starts to detect；
(5) sample collection
The glycosyl sample of laser bombardment hydrophilic region positioning hole, gather 3 glycosyl sample mass spectrograms；Partial detail view is shown in accompanying drawing
5。
(6) spectrum analysis
Glycosyl theoretical value database progress glycosyl sample analysis from Beijing Yixin Bochuang Biotechnology Co., Ltd. obtains
It is identical with glycosyl data base theory value to go out the glycosyl actual measurement m/z values of 3 samples, as a result all correct, the i.e. actual measurement of glycosyl sample
As a result accuracy rate 100%.
It is as follows by glycosyl theoretical value database analysis probation report：
Table 3
| Theoretical m/z values | 1606.484 | 1768.626 |
| No. 1 actual measurement m/z value | 1607.981 | 1769.637 |
| No. 2 actual measurement m/z values | 1607.322 | 1768.883 |
| No. 3 actual measurement m/z values | 1607.442 | 1768.82 |
………………………………………………
It is reported above to draw, 3 glycosyl samples, every 4 notable mass spectra peaks of example, detection range 1000-4000, wherein two
Individual peak, each peak m/z values are consistent with theoretical value, accuracy rate of testing result 100%.
Wherein, as shown in figure 5, can quickly, clearly, accurately be judged according to specific glycosyl m/z theoretical values table
Corresponding sugar-type.
In summary, the present invention improves matrix solution formula, can obtain the preferable primary crystallization of crystal habit and secondary
Crystallization effect, and its bearing calibration can aid in acquisition and more stablize and accurate Mass Spectrometer Method result.
Claims (9)
1. a kind of method for the crystallization for improving Mass Spectrometer Method glycosyl sample, step include：
(1) using the acetonitrile of deionized water and high-efficiency liquid chromatographic-grade according to 1：1 volume ratio mixing, adding volume fraction is
0.1% trifluoroacetic acid solution, obtains mixed solution；
(2) weigh 50mg DHB matrix (DHB) fully to be dissolved with mixed solution, DHB substrate concentrations are
50mg/mL；
(3) DHB matrix and citric acid diamine solution are taken, according to 9：1 volume ratio is well mixed, and produces matrix solution.
(4) 0.5 μ L-1 μ L matrix solutions and 0.5 μ L-1 μ L samples are chosen, point sample crystallization is carried out on chip.
2. the method for claim 1 wherein the DHB that 50mg is added in step 2, and 3- is shaken by 2000-3000rpm
10min, 8000-12000rpm centrifuge 3-10min, obtain DHB solution.
3. the method for claim 1 wherein choose 0.5 μ L samples and 0.75 μ L samples are crystallized in step 4.
4. the method for claim 3, wherein aforesaid operations are selected from 100,000 grades of cleanliness factors of operation room, 20-25 DEG C of environment temperature, environment
Carried out under the conditions of humidity 20-30%.
5. the mass spectrum chip of the accuracy rate that can improve Mass Spectrometer Method glycosyl target molecule used in claim 1-4 method,
The chip includes：The chip body being made up of silicon materials or glass or titanium alloy, its surface have multiple vertical cross arrangements
Hydrophilic region positioning hole, weep hole exterior domain, cent(e)ring hole, checking correction hole, standby correction hole；
Wherein, positioning hole has water-wet behavior, for titrating matrix solution or sample；Weep hole exterior domain, surface have hydrophobic
Characteristic；The structure for the close and distant water spacer in micro-array chip surface that positioning hole and hole exterior domain are formed, can be received with auxiliary liquid sample
Contracting is condensed upon in hydrophilic region, concentrates sample crystallization, and standard circular is presented, and improves Ionization Efficiency；
Cent(e)ring hole and checking correction hole, for correcting the titration location verification correction hole of protein standard, for verifying correction
Effect；
Standby correction hole, as standby, cent(e)ring hole or checking correction hole are if there is exception, available backup correction hole.
6. the chip of claim 5 wherein positioning hole is (4-8) × (4-8) vertical crisscross arrangement on chip, center
Correction hole, checking correction hole and standby correction hole in the chips between position vertical array, quantity is 1 or 2.
7. the chip of claim 6, wherein chip hydrophily positioning hole covering 150-800nm hydrophilic film, hydrophobic
Property hole outskirt cover 150nm-2 μm of hydrophobic film, 120 ° of its surface water droplet contact angle ＞.
8. the chip of claim 7, wherein the hydrophilic film is silica oxides film, zinc-oxide film, oxidation
Aluminium film etc., hydrophobic pores outskirt carry out processing by silane coupler and form hydrophobic film.
9. the chip of claim 10, wherein the silane coupler is selected from vinyl silanes, amino silane or dimethyl dichloro
Silane.
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