CN108845023A - A kind of laser desorption ionisation Mass Spectrometry detection method - Google Patents
A kind of laser desorption ionisation Mass Spectrometry detection method Download PDFInfo
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- 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
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- 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 laser desorption ionisation Mass Spectrometry detection methods, include the following steps：(1) efficient light absorption material is prepared, as load sample substrate；(2) sample to be tested is added dropwise to load sample substrate, and keeps sample to be tested dry；(3) laser desorption ionisation mass spectral analysis is carried out.Use efficient light absorption material as the load sample substrate of laser desorption ionisation, it can effectively realize organic and biomolecule desorption ionisation, it is nearly free from fragment ion, it can be realized the Mass Spectrometer Method and analysis to the various samples including carbohydrate, amino acid, metabolin, drug molecule etc., there is good signal-to-noise ratio, repeatability, high sensitivity and high-salt tolerance.By means of the present invention, it without complicated preparation of samples process, without adding organic substrate, is interfered at low quality end without background peaks, provides a kind of general, simple, efficient, quick, sensitive soft ionization detection method for various biomolecules.
Description
Technical field
It is specifically a kind of using efficient light absorption material as load sample substrate the present invention relates to analytical technique of mass spectrum field
Laser desorption ionisation Mass Spectrometry detection method.
Background technique
Matrix solid-dispersion (matrix-assisted laser desorption/ionization,
MALDI) since the end of the eighties in last century is suggested, extensive development has been obtained.Since MALDI has high sensitivity, standard
The features such as exactness is high, detection speed is fast and mass range is wide has spy in measurement large biological molecule and high polymer application aspect
Different superiority has become the strong work of detection and identification polypeptide, protein, polysaccharide, nucleotide and high polymer in recent years
Tool.The key of MALDI analysis is the introduction of the organic substrate that can effectively absorb laser, and matrix is absorbed with after analyte cocrystallization
The energy of laser pulse simultaneously passes to analyte, enables analyte desorption and ionizes.Currently used matrix is main
It is small molecule acid, including alpha-cyano -4- hydroxycinnamic acid (CHCA), sinapic acid (SA) and 2,5-dihydroxybenzoic acid (DHB) etc..
Although matrix can promote desorption and the ionization process of biomolecule, but also sample preparation and analysis are procedure complicated, and
And the selection of matrix is extremely important to analysis result, needs to select suitable matrix according to the property of analyte.Introduce matrix
Brought one more important question is that the strong background peaks as caused by matrix ion can low quality range (<500Da) make
At severe jamming, so that MALDI is difficult to use in the analysis of small-molecule substance.On the other hand, matrix and sample are easy to occur not
It is homogeneously crystallized, so that MALDI analysis is less reproducible.
In recent years, obtaining rapidly without matrix laser desorption ionization (LDI) technology for load sample substrate is done using inorganic material
Development, basic principle be using base material absorb laser energy cause fast temperature rise, transfer energy to analysis
Object promotes the thermal desorption and subsequent ionization of analyte.Currently, a variety of inorganic material have been successfully applied to no matrix laser solution
In adion mass spectral analysis, 1999, Jing Wei et al. used load of the porous silicon as laser desorption ionization for the first time
Sample substrate effectively eliminates organic substrate bring background interference；2004, Minjia Yuan et al. was delivered using mesoporous
WO3-TiO2Laser desorption ionization Mass Spectrometry detection method of the material as load sample substrate；2007, Yoshinao Wada et al.
Use the porous aluminas with platinum or gold plating as load sample substrate；2012, Cheng Jin hair tonic table graphene matrix was in base
The patent of application in the detection of matter assisted laser desorption ionisation flight time mass spectrum；2016, Feng Yu Qi et al. delivered black squama
The patent of application in Matrix-assisted laser desorption ionization.These inorganic material are concentrated mainly on nonmetallic
Field can be effectively reduced interference caused by upshift signal.But there are still such as easily contaminated, production costs for most of material
The disadvantages such as height, preparation process are complicated, service life is short, ionizing efficiency is low.It has been confirmed that for no matrix laser desorption ionization
Base material all have the properties such as high specific surface area and certain UV absorption substantially, can rapidly absorb laser energy
And being converted into heat is the whether successful key factor of base material.
In the recent period, Zhong Minlin et al. proposes the method using ultrafast pulsed laser regulation metal surface photo absorption performance, utilizes
Picosecond or femtosecond pulse in metal surface prepare micron and nanometer composite structure, by regulating and controlling the parameter of pulse laser and swashing
Relative motion between light beam and metal surface can shape characteristic to micro nano structure and scale feature regulate and control, finally may be used
Realize the efficient absorption in wide spectral range, whole process is flexibly controllable, and processing cost is low, high-efficient.At present not yet
Reported the application for being analyzed by mass spectrometry efficient light absorption metal material as laser desorption ionization substrate.
Summary of the invention
It is low, easy to solve ionizing efficiency existing for the existing inorganic material as laser desorption ionisation load sample substrate
The problems such as molecular fragment, high production cost, preparation process are complicated, service life is short is generated, the present invention provides a kind of efficient light absorption
Method of the material as load sample substrate in laser desorption ionization.Use efficient light absorption material attached as laser desorption in this method
The load sample substrate of ionization fast and effeciently absorbs ultraviolet laser energy and fast by efficient light absorption material without adding matrix
Speed converts thereof into heat, promotes desorption and the ionization process of analyte, to realize the accurately and rapidly mass spectrum to analyte
Detection.
The present invention includes the following steps：
Step (1) prepares efficient light absorption material, as load sample substrate；Efficient light absorption material can be added by femtosecond pulse
Work obtains, and contains micro-/nano compound structure with high light absorptive.
Absorptivity of the efficient light absorption material in ultraviolet, visible light and infrared band (250~2500nm)>70%, efficiently inhale
Luminescent material can be by including but is not limited to that copper, titanium, aluminium, magnesium or alloy (such as stainless steel) are made, can also be by nonmetallic materials system
At.
Sample to be tested is added dropwise to the load sample substrate by step (2), and keeps the sample to be tested dry.Preferably, herein
Before, organic solvent can be used to clean load sample substrate, such as use methanol or ethyl alcohol, cleaning method is ultrasonic cleaning 5
Minute.
Water, methanol or alcohol solvent can be used and sample to be tested is made into sample solution, sample solution is taken to drop to the height
Light absorbent surface is imitated, and is spontaneously dried in air.Sample to be tested includes but is not limited to amino acid, carbohydrate, drug molecule, generation
Thank to object, molecular weight ranges are 100~2000Da.Sample to be tested is complex sample or biological sample, and complex sample includes mixture,
Such as biological samples such as onions extracting liquid and yeast cells.Biological sample includes biological cell sample.Biological cell sample can be with
It is directly added dropwise on the efficient light absorption material and carries out laser desorption ionisation mass spectral analysis.Biological sample can also use extraction
Solvent is taken to extract biological cell.
Extractant can be the mixture of water, methanol and acetonitrile.
Biological sample is the clasmatosis product generated after being crushed using Ultrasonic Cell Disruptor to biological cell.
Step (3) carries out laser desorption ionisation mass spectral analysis, and such as sample to be tested is placed in mass spectrograph and carries out laser
Desorption ionisation mass spectral analysis, mass spectrograph can be MALDI-TOF mass spectrograph.Mass spectrometric analysis method according to standard laser solution
Adion Mass Spectrometer Method program.
Further, the present invention can also include step (4), be cleaned by ultrasonic the load sample base using organic solvent and water
Bottom, and make the load sample substrate drying, remain next use.Organic solvent includes methanol, ethyl alcohol.
Compared with prior art, the beneficial effects of the invention are as follows
(1) it can be realized without adding matrix to effective desorption of many kinds of substance and ionization.The present invention is using efficient
Substrate molecule is eliminated in load sample substrate of the light absorbent as laser desorption ionisation, the effect of can effectively replace organic substrate
Bring signal interference realizes the accurate analysis to small-molecule substance.
(2) low background peaks interference.The efficient light absorption material itself that the present invention uses is nearly free from background peaks, in low quality
End avoids the various background interferences of itself bring of the nano materials such as nano particle almost without background peaks interference.
(3) due to enormously simplifying the operating process of point sample without adding matrix, without according to different analytes come excellent
Change matrix used, it is versatile, it can be achieved that substance efficient, quick detection.
(4) repeatability of analysis is improved.Due to avoiding existing with the uneven cocrystallization of matrix without adding matrix
As substantially increasing the repeatability of analysis.
(5) ionizing efficiency is high, no fragment peak.The light absorbent that the present invention uses has pore structure abundant, efficiently purple
The features such as outer laser absorption ability and high photothermal conversion efficiency, can effective adsorption analysis object molecule, absorb ultraviolet laser,
Hole inner wall temperature rapid increase is induced, so that the thermal desorption process of analyte be greatly facilitated, realizes the height to various substances
Sensitivity analysis.Simultaneously because its effective UV absorption ability, substantially reduces the laser energy deposited in determinand molecule,
Molecular fragment can be effectively reduced.
(6) efficient light absorption material surface has hydrophobicity, and aqueous solution will be present in surface in the form of droplet, and will not be spread
It opens.Compared with general surface, hydrophobic surface will generate better signal to the sample in aqueous solution.
(7) it can be realized effective ionization to neutral saccharide compound.The present invention can effectively solve neutral carbohydrate chemical combination
The low problem of object ionizing efficiency, without performing the derivatization the quick detection and identification that can carry out carbohydrate.
(8) the base material preparation process used is simple, at low cost.The efficient light absorption material that the present invention uses passes through a step
Ultrafast laser etching can be prepared by, and preparation is simple, and processing cost is low, high-efficient, can by changing laser parameter and sweep parameter
To produce a plurality of types of micron and nanometer composite structures, there is structural robustness, be suitable for being mass produced.
(9) the base material structural stability used is strong, and service life is long.Metal material mechanical stability with higher
And chemical stability, the micro nano structure being formed in situ on metal have enough durabilities, can store in air several
Change without structure in a month.Long-term recycling may be implemented by carrying out ultrasonic cleaning to surface.
Detailed description of the invention
Fig. 1 is the figure that the efficient light absorption material processed on copper is fixed on commercialization MALDI target plate；
Scanning electron microscope image of the Fig. 2 for efficient light absorption material obtained on copper, absorptance 95%；Wherein a is uniform
Micron hole configurations, b is the nanoscale features for being covered on micrometer structure surface；
Fig. 3 is the photo figure that sample solution (use water as solvent) drops to efficient light absorption material surface obtained on copper；
Fig. 4 is that conventional MALDI is respectively adopted, and uses DHB as matrix (a) and uses efficient light absorption material obtained on copper
The mass spectrogram for doing LDI (b) the analysis glucose of load sample substrate compares (molecular ion peak 203.05 [M+Na]+,219.03[M+K
]+)；
Fig. 5 is to do substrate using efficient light absorption material obtained on copper to analyze lactose (365.12 [M+Na of molecular ion peak
]+,381.09[M+K]+), maltotriose (molecular ion peak 527.16 [M+Na]+,543.13[M+K]+), maltopentaose (molecule
Quasi-molecular ions 851.26 [M+Na]+,867.25[M+K]+), beta-cyclodextrin (molecular ion peak 1157.38 [M+Na]+,1173.33[M+
K]+) and gamma-cyclodextrin (molecular ion peak 1319.44 [M+Na]+,1335.40[M+K]+) mixture the attached electricity of laser desorption
From mass spectrogram；
Fig. 6 is to do substrate using efficient light absorption material obtained on copper to analyze 1nmol (a), 100fmol (b) and 20fmol
(c) lactose (molecular ion peak 365.12 [M+Na]+,381.09[M+K]+) laser desorption ionization mass spectrogram.
Fig. 7 is attached for the laser desorption for doing substrate analysis of amino acid mixture using efficient light absorption material obtained on stainless steel
Ionogen spectrogram, Pro in figure:Proline, Glu:Glutamic acid, Phe:Phenylalanine, Arg:Arginine, His:Histidine, Tyr:
Tyrosine, Trp:Tryptophan；
Fig. 8 is the laser desorption ionization matter done substrate using efficient light absorption material obtained on copper and analyze onions extracting liquid
Spectrogram.Molecular ion peak 203.06,219.05,381.09,543.15,705.20,867.24 and 1029.31 is respectively in figure
[C6H12O6+Na]+,[C6H12O6+K]+,[C12H22O11+K]+,[C18H32O16+K]+,[C24H42O21+K]+,[C30H52O26+K]+With
[C36H62O31+K]+；
Fig. 9 is the laser solution done substrate using efficient light absorption material obtained on copper and analyze four kinds of brewing yeast cell samples
Adsorb ionogen spectrogram.Wherein a：Yeast cells group；b：Yeast cells breakdown products；c：Yeast cell extract (extracts molten
Liquid is methanol/acetonitrile/water 40/40/20)；d：Yeast cell extract (extraction solution is methanol/water 80/20).
Specific embodiment
In the following description, multiple and different aspects of the invention will be described, however, for common skill in the art
For art personnel, the present invention can be implemented just with some or all structures or process of the invention.In order to explain
Definition for, specific number, configuration and sequence are elaborated, however, it will be apparent that these specific details the case where
Under the present invention also can be implemented.It in other cases, will no longer for some well-known features in order not to obscure the present invention
It is described in detail.
A kind of laser desorption ionisation Mass Spectrometry detection method is proposed according to the present invention, is included the following steps：
Step (1) prepares efficient light absorption material, as load sample substrate；
Sample to be tested is added dropwise to the load sample substrate by step (2), and keeps the sample to be tested dry；
Step (3) carries out laser desorption ionisation mass spectral analysis.
In above-mentioned steps (1), the efficient light absorption material used is made on the alloys such as the metals such as copper sheet or stainless steel.Production
Method such as patent application CN103627883.
In above-mentioned steps (2), before sample to be tested is added drop-wise to load sample substrate, organic solvent can be used, such as methanol or second
Alcohol cleans load sample substrate, and cleaning method is ultrasonic cleaning 5 minutes.
In above-mentioned steps (2), using water, methanol or ethyl alcohol equal solvent, the sample preparation being analysed to is 1mM's at concentration
Solution is simultaneously diluted, and the sample of a certain concentration of certain volume is taken to be added dropwise to efficient light absorption material surface, without adding matrix,
After being spontaneously dried in air, puts it into MALDI-TOF mass spectrograph and analyzed.Sample to be tested is added drop-wise to efficient light absorption material
Upper not formed apparent crystalline solid.Detect obtained LDI mass spectrogram does not have strong background peaks to interfere at low quality end, clean background.
Molecular weight analyte range used in above-mentioned application in 100~2000Da, including carbohydrate, amino acid, drug molecule,
Metabolin etc..
Sample to be analyzed further includes the biologies such as complex samples, such as onions extracting liquid and yeast cells in above-mentioned application
Sample.
In addition, method of the invention can also include step (4)：It is cleaned by ultrasonic the load sample base using organic solvent and water
Bottom, and make the load sample substrate drying, remain next use.Organic solvent includes methanol, ethyl alcohol.
Efficient light absorption material used in embodiment uses femtosecond pulse processing method, in metals such as copper or stainless
It is made on the alloys such as steel (304).Absorptance of the light absorbent at 355nm in embodiment 1 is 80%, in other embodiments
Absorptance of the light absorbent at 355nm is 95%.
All mass spectral analyses use the autoflex speed type MALDI-TOF mass spectrum of Bruker company in embodiment
Instrument, the laser of outfit are the Nd that wavelength is 355nm:YAG laser.Acceleration voltage is set as 20kV, and working method is reflection mould
Formula is detected in the positive-ion mode, and every mass spectrogram 500 laser scanning results that are averaged obtain.The control used
Software is flex Control 3.3, and Data Analysis Software is flex Analysis 3.3.
Embodiment 1 does the LDI mass spectral analysis of substrate using the light absorbent of absorptance 80%
Analyze the preparation of sample：Configuration concentration is the reserpine solution of 1mM, and solvent is methanol/water 3/7, is placed in 4 DEG C of refrigerators
In it is spare.
LDI mass spectral analysis：
(1) the light absorbing metal material that absorptance is 80% is prepared on copper.
(2) organic solvent cleaning material is used, is placed in air after thoroughly drying, secures it on target plate and as load
Sample substrate takes 1 μ L reserpine solution to be added dropwise to material surface and be dried.
(3) target plate is placed in progress laser desorption ionisation mass spectral analysis in MALDI-TOF mass spectrograph.
(4) light absorbent after testing is cleaned by ultrasonic and is dried using organic solvent and water, remains next use.
Obtained efficient light absorption material is processed to be fixed on target plate as shown in Figure 1.Testing result shows that reserpine molecule obtains
Effective desorption and ionization have been arrived, there is good signal-to-noise ratio, has been generated without molecular fragment.
Embodiment 2 uses LDI mass spectrum routine MALDI (matrix used is DHB) and do substrate using efficient light absorption material
The comparison of analysis
The preparation of matrix solution：Configuration concentration is the DHB solution of 20mg/mL, and solvent is acetonitrile/water 3/7.
Analyze the preparation of sample：The deionized water solution that concentration is 1mM is configured by glucose, is placed in standby in 4 DEG C of refrigerators
With.
The conventional MALDI mass spectral analysis of sample：1 μ L matrix DHB solution point is taken on the stainless steel target plate of commercialization, to it
After drying forms matrix thin layer, the glucose solution of same volume is taken to drip above thin layer.It is put into after it is spontaneously dried
It is analyzed by mass spectrometry in MALDI-TOF mass spectrograph.It carries out 5 repetitions to test, calculates its leading ion peak ([M+Na]+) be averaged
Signal-to-noise ratio.
The LDI mass spectral analysis of load sample substrate is done using efficient light absorption copper product：
(1) efficient light absorption metal material, absorptance 95% are prepared on copper.
(2) organic solvent cleaning material is used, is placed in air after thoroughly drying, secures it on target plate and as load
Sample substrate takes 1 μ L glucose solution to be added dropwise to material surface and be dried.
(3) target plate is placed in progress laser desorption ionisation mass spectral analysis in MALDI-TOF mass spectrograph.Carry out 5 weights
Multiple experiment, calculates its leading ion peak ([M+Na]+) average signal-to-noise ratio.
(4) light absorbent after testing is cleaned by ultrasonic and is dried using organic solvent and water, remains next use.
The microstructure of obtained efficient absorption copper product is as shown in Figure 2.In MALDI analysis, sample and matrix meeting
Co-crystal thereof is generated on stainless steel target plate.In LDI analysis, sample solution is added dropwise to the photo of efficient light absorption material surface such as
Shown in Fig. 3, not formed crystalline solid after drying.Testing result as shown in figure 4, glucose generate leading ion peak be [M+Na]+.
Wherein, when carrying out MALDI analysis using matrix, strong matrix peak is had at low quality end, so that sample signal is pressed down
System.When using efficient light absorption copper product to carry out LDI analysis as substrate, interfered at low quality end without background peaks, the electricity of sample
Higher from efficiency, the signal-to-noise ratio at sample ions peak greatly improves.Should the result shows that, using efficient light absorption copper product as laser solution
The method of absorption ionization substrate is fine to the analytical effect of small-molecule substance.
Embodiment 3 does load sample substrate to the laser desorption ionisation of neutral carbohydrate admixture using efficient light absorption copper product
Mass spectral analysis
Analyze the preparation of sample：Lactose, maltotriose, maltopentaose, beta-cyclodextrin and gamma-cyclodextrin are respectively configured as
Concentration is the deionized water solution of 1mM, takes five kinds of isometric solution to mix, obtains mixture solution, is placed in standby in 4 DEG C of refrigerators
With.
The LDI mass spectral analysis of load sample substrate is done using efficient light absorption copper product：
(1) efficient light absorption metal material, absorptance 95% are prepared on copper.
(2) organic solvent cleaning material is used, is placed in air after thoroughly drying, secures it on target plate and as load
Sample substrate takes 2 μ L carbohydrate admixture solution to be added dropwise to material surface and be dried.
(3) target plate is placed in progress laser desorption ionisation mass spectral analysis in MALDI-TOF mass spectrograph.
(4) light absorbent after testing is cleaned by ultrasonic and is dried using organic solvent and water, remains next use.
Testing result is main as shown in figure 5, five kinds of neutral saccharide compounds have all obtained effective desorption and ionization
Molecular ion peak is [M+Na]+Peak, generates without molecular fragment, and every kind of substance all has good signal-to-noise ratio.It is inhaled using efficient
Light copper product carries out laser desorption ionization mass spectral analysis to neutral saccharide compound as substrate, without carrying out chemistry to carbohydrate
Derivatization, which can be realized, quickly, accurately and efficiently to be detected.
Embodiment 4 does load sample substrate using efficient light absorption copper product and carries out laser desorption ionisation mass spectral analysis lactose
Detection limit
Analyze the preparation of sample：Configuration concentration is the lactose deionized water solution of 1mM, and constantly diluted concentration is to 100nM
And 20nM, it is placed in spare in 4 DEG C of refrigerators.
It is limited using the detection that efficient light absorption copper product makees load sample substrate analysis lactose：
(1) efficient light absorption metal material, absorptance 95% are prepared on copper.
(2) organic solvent cleaning material is used, is placed in air after thoroughly drying, secures it on target plate and as load
Sample substrate takes the lactose solution that 1 μ L concentration is 1mM, 100nM and 20nM to drip in material surface and be dried respectively.
(3) target plate is placed in progress laser desorption ionisation mass spectral analysis in MALDI-TOF mass spectrograph.
(4) light absorbent after testing is cleaned by ultrasonic and is dried using organic solvent and water, remains next use.
Testing result is as shown in fig. 6, molecular ion peak 365.12 and 381.09 is [M+Na] respectively+[M+K]+.Three kinds dense
The milk sugar specimen of degree can detect, and when concentration is higher, main molecular ion peak is [M+Na]+, when concentration reduces, lactose
Mainly with [M+K]+Form ionization, signal peak still has preferable signal-to-noise ratio.Testing result explanation uses efficient light absorption copper material
When material carries out laser desorption ionization analysis to lactose as load sample substrate, detection limit can achieve fmol magnitude, have preferable
Sensitivity.
Embodiment 5 does load sample substrate to the attached ion of the laser desorption of ispol using efficient light absorption stainless steel material
Change mass spectral analysis
Analyze the preparation of sample：Respectively by glutamic acid, histidine, arginine, phenylalanine, proline, tryptophan and junket
Propylhomoserin is configured to the deionized water solution that concentration is 1mM, takes seven kinds of isometric solution to mix, it is molten to obtain ispol
Liquid is placed in spare in 4 DEG C of refrigerators.
The LDI mass spectral analysis of substrate is done using efficient light absorption stainless steel material：
(1) efficient light absorption material, absorptance 95% are prepared on stainless steel (304).
(2) organic solvent cleaning material is used, is placed in air after thoroughly drying, secures it on target plate and as load
Sample substrate takes 2 μ L amino acid mixture solutions drop in material surface and is dried.
(3) target plate is placed in progress laser desorption ionisation mass spectral analysis in MALDI-TOF mass spectrograph.
(4) light absorbent after testing is cleaned by ultrasonic and is dried using organic solvent and water, remains next use.
For testing result as shown in fig. 7, the seven kinds of amino acid surveyed can be all detected, the molecular ion peak of formation is [M+
H]+,[M+Na]+Or [M+K]+Peak.In the mass range surveyed, background interference is smaller, and resulting mass spectrogram is simple and is easy to area
Point, signal resolution is preferable.Analysis the result shows that, the stainless steel material of efficient light absorption is as laser desorption ionization mass spectral analysis
Load sample substrate when, also can get good desorption efficiency.
Embodiment 6 does load sample substrate to the laser desorption ionisation mass spectrum of onions extracting liquid using efficient light absorption copper product
Analysis
Onions extracting liquid is produced：Fresh onion is removed into outer epidermal layer, then shreds onion epidermis, grinds 5 points
Clock is simultaneously freeze-dried.Later, the onion sample of freeze-drying deionized water is extracted 1 hour, and uses centrifuge by solution
With 10,000rpm centrifugation 20 minutes, takes supernatant to be analyzed, do not need any other purifying and concentration processes.
The laser desorption ionisation mass spectral analysis of load sample substrate is done using efficient light absorption copper product：
(1) efficient light absorption metal material, absorptance 95% are prepared on copper.
(2) organic solvent cleaning material is used, is placed in air after thoroughly drying, secures it on target plate and as load
Sample substrate takes 1 μ L onions extracting liquid directly to drip in material surface and be dried.
(3) target plate is placed in progress laser desorption ionisation mass spectral analysis in MALDI-TOF mass spectrograph.
(4) light absorbent after testing is cleaned by ultrasonic and is dried using organic solvent and water, remains next use.
Testing result is as shown in figure 8, can detecte monosaccharide and the degree of polymerization as 2~6 oligosaccharide.Due to containing in onion
The K of high concentration+, the major molecular ion peak that each oligosaccharide is formed is [M+K]+.Mass spectrogram is completely simple, within the scope of low quality
Almost without background peaks interference, there is good signal-to-noise ratio.Use efficient light absorption copper product can be directly right as load sample substrate
Onions extracting liquid carries out laser desorption ionisation mass spectral analysis, without complicated purifying and concentration processes, may be implemented quickly,
Accurately, sensitive oligomeric sugar detection.
Embodiment 7 does load sample substrate to the laser desorption ionisation of yeast cells metabolin using efficient light absorption copper product
Mass spectral analysis
Yeast cells sample is produced：Saccharomyces cerevisiae is cultivated in yeast extract peptone glucose (YPD) culture medium.
Cell culture is transferred in centrifuge tube, and is centrifuged 10 minutes with 800 × g using centrifuge to separate cell and culture medium.
Cell precipitation is stored in spare (sample 1) at -80 DEG C.Cell is dispersed in the methanol/water solution (50/50v/v) of pre-cooling
And with ultrasonic cell disruptor by clasmatosis, 10 minutes then are centrifuged to obtain clasmatosis with 15000 × g at 4 DEG C
Product (sample 2), is stored in spare at -80 DEG C.Cell metabolism extract is obtained by following procedure.Cell is released rapidly
It is put into the methanol/water solution (60/40v/v) of pre-cooling to stop its metabolic activity.Acquired solution is cooled down on ice, then
With 10000 × g centrifugation 5 minutes at -9 DEG C, supernatant is removed.Then two kinds of cooling in advance extraction solution are added to respectively
In cell precipitation, cell is suspended in solution by upper and lower imbibition, is held the mixture in 15 minutes on ice.Finally mixture is existed
With 15000 × g centrifugation 10 minutes with sedimentation cell fragment at 4 DEG C, take supernatant be stored in -80 DEG C it is spare.Extract solution 1：First
Alcohol/acetonitrile/water 40/40/20 (sample 3)；Extract solution 2：Methanol/water 80/20 (sample 4).
The laser desorption ionisation mass spectral analysis of load sample substrate is done using efficient light absorption copper product：
(1) efficient light absorption material, absorptance 95% are prepared on copper.
(2) organic solvent cleaning material is used, is placed in air after thoroughly drying, secures it on target plate and as load
Sample substrate takes 1 μ L sample 1,2,3,4 directly drop in material surface respectively and is dried.
(3) target plate is placed in progress laser desorption ionisation mass spectral analysis in MALDI-TOF mass spectrograph.
(4) light absorbent after testing is cleaned by ultrasonic and is dried using organic solvent and water, remains next use.
It is as shown in Figure 9 to the testing result of four kinds of samples.Load sample substrate is done to yeast cells sample using efficient light absorption material
When this progress laser desorption ionization mass spectra analysis, it can detecte multiple molecular ion peaks, the sample being treated variously for
In the ion that detects there is overlapping and differences.The present invention pays close attention to 100~400Da of mass range, the identification master of ion
Will be by following two approach, one is be based on mass accuracy Δ m/z≤0.1, the ion that will test and multiple metabolins
Metabolin in database (including KEGG, MetaboLights and METLIN) is matched；Yeast cells sample also makes simultaneously
It is analyzed with electric spray ion source mass spectrograph, obtains its second level mass spectrogram further to be identified.Four kinds of samples measure
Quasi-molecular ions altogether can identify 13 kinds of metabolins, mainly include amino acid, amino acid derivativges/precursor, nucleoside base and
Carbohydrate etc..Specifically include γ-aminobutyric acid (4-Aminobutyric acid), uracil (Uracil), proline
(L-Proline), phenylethylamine (Phenylethylamine), 4- amino -5- methylol -2- methylpyrimidine (4-Amino-5-
Hydroxymethyl-2-methylpyrimidine), glutamine (L-Glutamine), histidine (L-Histidine),
Aminoadipic acid (Aminoadipic acid), arginine (L-Arginine), divides phenylalanine (L-phenylalanine)
Branch acid (Chorismic acid), monosaccharide and disaccharides etc..Wherein, the ionic species that sample 1 can detecte is at most (9 kinds).Inspection
It surveys the result shows that efficient light absorption material can be used for the laser desorption ionisation mass spectral analysis of metabolin in cell, the present invention is
Cell metabolite analysis provides a kind of quick, simple method.
Claims (14)
1. a kind of laser desorption ionisation Mass Spectrometry detection method, it is characterised in that：Include the following steps：
Step (1) prepares the efficient light absorption material as load sample substrate；
Sample to be tested is added dropwise to the load sample substrate by step (2), and keeps the sample to be tested dry；
Step (3) carries out laser desorption ionisation mass spectral analysis.
2. according to the method described in claim 1, it is characterized in that：The efficient light absorption material is in ultraviolet, visible light and infrared
The absorptivity of wave band (250~2500nm)>70%.
3. according to the method described in claim 1, it is characterized in that：The efficient light absorption material is processed by ultrafast pulsed laser
It arrives, contains micro-/nano compound structure with high light absorptive.
4. according to the method described in claim 1, it is characterized in that：The efficient light absorption material is efficient light absorption metal material
Material, by including but is not limited to that copper, titanium, aluminium, magnesium or alloy are made.
5. according to the method described in claim 1, it is characterized in that：It is molten using water, methanol or ethyl alcohol in the step (2)
Agent and the sample to be tested are made into sample solution, and sample solution is taken to drop to the efficient light absorption material surface, and in air certainly
It is so dry.
6. according to the method described in claim 1, it is characterized in that：The sample to be tested include but is not limited to amino acid, carbohydrate,
Drug molecule, metabolin, molecular weight ranges are 100~2000Da.
7. according to the method described in claim 1, it is characterized in that：The sample to be tested is complex sample or biological sample, institute
Stating complex sample includes mixture, and the biological sample includes biological cell sample.
8. according to the method described in claim 7, it is characterized in that：The biological cell sample can directly be added dropwise in the height
It imitates and carries out laser desorption ionisation mass spectral analysis on light absorbent.
9. according to the method described in claim 7, it is characterized in that：The biological sample is using extractant to biological cell
The cell extract of extraction.
10. according to the method described in claim 9, it is characterized in that：The extractant is the mixing of water, methanol and acetonitrile
Object.
11. according to the method described in claim 7, it is characterized in that：The biological sample is using Ultrasonic Cell Disruptor to biology
Cell carries out the clasmatosis product generated after being crushed.
12. according to the method described in claim 1, it is characterized in that：In the step (3), mass spectrometric analysis method is according to mark
Quasi- laser desorption ionisation Mass Spectrometer Method program.
13. according to the method described in claim 1, it is characterized in that：Further include step (4), uses organic solvent and water ultrasound
The load sample substrate is cleaned, and makes the load sample substrate drying, remains next use.
14. according to the method for claim 13, it is characterised in that：The organic solvent includes methanol, ethyl alcohol.
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