CN1830056A - Electrosonic spray ionization method and device for the atmospheric ionization of molecules - Google Patents
Electrosonic spray ionization method and device for the atmospheric ionization of molecules Download PDFInfo
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Abstract
There is described a device and method for generating gaseous ions of a sample material such as molecules in solution at atmospheric pressure. The device includes a conduit for receiving a solution containing the material to be ionized and form a stream. A jet of gas at supersonic velocity is directed at the stream and interacts therewith. Droplets are formed and by the adiabatic expansion of the gas and vigorous evaporation of the solution gaseous ions are generated. In the method a stream of the sample solution is delivered from a conduit with an electric potential. A gas jet at supersonic velocity interacts with the delivered solution and through the action of adiabatic expansion of the gas and evaporation of the solution gaseous ions are formed.
Description
Related application
60/543,096 the priority that the application requires the temporary patent application sequence number of submitting on July 24th, 2,003 60/490,183 and submitted on February 9th, 2004, its open text is hereby incorporated by with their integral body.
Invention field
The present invention relates generally to a kind of be used under atmospheric pressure forms for example molecule of specimen material from liquid, comprise the biomolecule for example equipment and the method for the gas ion of protein, relate more particularly to a kind of equipment and method, the liquid that wherein comprises specimen material or molecule is from terminal emission capillaceous, this capillary is kept current potential to set up electric field at its end, and the supersonic speed annular gas sprays aligning end capillaceous to produce charged ultrafine dust, and this ultrafine dust passes through adiabatic expansion and the violent evaporation formation material of liquid or the gas ion of molecule of gas under atmospheric pressure.
Background of invention
EFI ionization (ESI) mass spectrum
1,2Become the important tool of structure biochemical field rapidly.This technology can make folding protein ionization, sometimes for the evidence that little change is arranged in complete three-dimensional structure.The gained ion can use modern mass spectrum instrument to study in gas phase subsequently.
3-8Not only single protein can use this methodology to study, and multiple proteins and the also intactly ionization of protein-ligand complex sometimes, although thoroughly the quantity of the example of research is wanted much less.Recently, this as whole ribosome
9The ionization of complex structure be proved.In gas phase the protein complex can by cascade or the multistep mass spectrum study.
10-12In this step, initial complex can stand continuous dissociation process, to disclose the molecular weight of each component.Different with most of other technologies, mass spectrum is not limited to detect the molecular complex component of particular types, for example with fluorescent protein labeling or otherwise make for analytical method visible those.
The macromolecular system that protein is relevant with other biological shows a kind of structure of or very few number usually under physiological conditions, this structure is for the essential feature of bearing a kind of univocal biochemical function.It has been generally acknowledged that liquid phase structure is different with structure the most stable in gas phase.
3,4,9,13-15Therefore, be to keep these metastable solution structures for the major requirement of the mass-spectrometric technique of developing success, and this requirement minimize the interior energy of this ion so that keep gas phase to separate Duplication or the rate of dissociation is low as far as possible.This task is usually by avoiding the sex change condition and in the source of instrument with pressure is regulated in the atmosphere interface zone carefully and the lens potential value is realized when preparation is used for mass spectral solution.
10,16The main purpose of these operations is desolvation protein ion and guides them to enter mass spectral high vacuum region and do not influence the noncovalent interaction that keeps the high-sequential structure.This target realizes by applying relatively high pressure and apply low electric potential gradient at whole lens system in atmosphere interface usually
16Hyperbar provides the high collision frequency in first region of no pressure of instrument, and this keeps ion also to promote effectively disassociation simultaneously by the collision cooling at low temperatures.Yet, all be to keep because solution is sealed with ion structure by noncovalent interaction, the condition of the structure that is kept perfectly through being everlasting and those need to obtain compromising between the condition of disassociation fully.In addition, the instrument setting that allows leniently to dissociate is not optimized for the ion transformation efficiency usually, so the sensitivity of instrument may seriously be reduced.
Nanometer spraying (nanospray)
17,18Often be the selected ioning method that is used for obtaining gentle disassociation, and also be provided for the macroion efficient of a small amount of valuable sample simultaneously.Be different from traditional commercially available ESI ion source,
18Nanometer spraying is suitable under the physiological pH water-containing buffering liquid and because the sample consumption of its high this method of Ionization Efficiency has reduced one or two order of magnitude.Macroion efficient and effective disassociation are the characteristics that helps low solution flow rate usually, and known low solution flow rate reduces the size of the electrically charged drop of initial generation.Less initial liquid drop stands less coulomb division and the less solvent of each droplet evaporation, and this causes the low concentration of non-volatile matrix components in the final nano-liquid droplet that produces actual gaseous state protein ion.Less initial liquid drop size also speeding-up ion form and the higher share by this mode drop will be in fact by desolvation fully so that the ion that can use for quality analysis to be provided.It has been generally acknowledged that the nanometer spraying provides the better dissociation efficiency than ESI.Because remarkable lower sample flow rate, this specific character help from than more effectively evaporating solution and the low solvent vapo(u)r load on atmosphere interface the droplet.This intrinsic good dissociation efficiency do not require and apply harsh disassociation condition (high temperature, high awl voltage etc.) on atmosphere interface, itself so that strengthened the survival of the rapid wear biochemical entity that comprises non-covalent complex.Although these advantages are arranged, it is most advanced and sophisticated that nanometer spraying mass spectrum depends on used nanometer spraying strongly; A little less than the spectrogram reappearance from the tip to the tip.In addition, Jian Duan geometry may change owing to arcing or fracture during operation.Another difficult point of nanometer spraying is to lack control in spray process: spraying can not be regulated in practice, and it only can open and close by changing high voltage.
19,20Common demanding flow velocity and extreme pH value.
Under the situation of nanometer spraying and conventional press mobile (electrojet that compressed air is auxiliary), absolute sensitivity not only is subjected to the influence of the width (unit is m/z) at each peak, and is subjected to the shape of whole electronics valence distribution and the influence of width.The shape of electronics valence distribution is through being commonly used for the analysis tool of separating folding degree of determining protein in the ionization process.
21-26Wide electronics valence state substep is accompanied by usually and separates folding structure under high electronics valence state, and narrow distribution is treated to the sign of the natural or similar natural folded ion structure in gas phase under low electronics valence state.The model that is developed recently by people such as Kebarle is based on the maximum charge number of apparent relatively gas phase basicity (GB) the assess proteins ion in the possible electrically charged site on protein molecule
26-29This model description in electron spray, form protein ions by on each electrically charged site, forming the proton constraint complex with buffering molecule and these complex cause cushioning liquid that then dissociate.The branching rate of these complex disassociations depends on the relatively apparent GB of the apparent relatively GB of this buffering molecule (for example being ammonia under the ammonium buffer solution) with respect to protein belt electric charge site.The apparent GB value of specific site can determine that wherein last factor relates to the size of protein ion based on the intrinsic GB value of chemical part, the dielectric constant of protein molecule and the spatial distribution of electric charge on the protein.It is the result of these factors that observed state of charge distributes: the temperature of disassociation and any further electricity price minimizing that occurs in mass spectral atmosphere interface or causing by the reaction of the ion during the ion optics path.
In principle, the charging of spray process and sample can be separated, can pass through different spray techniques by finely divided when beginning with initial charged liquid.This method extensively adopts in commercial ESI source by the compressed air spray method
30, normally for the rough a large amount of fluid samples that disperse from the standard liquid chromatogram.Because d～1/v
g 2, wherein d is the average diameter of drop, v
gBe the linear velocity of spray gas under high gas line speed and high gas/liquid mass velocity ratio, can obtain the drop size that to compare with the nanometer spraying in theory.
31
Although by the complex specimen material of the aqueous solution supply that is buffered to the physiological pH value for example the complete ionization of protein reached the degree that under mass spectral reducing atmosphere, can under atmospheric pressure sample, when material was the protein that is buffered in the aqueous solution of physiological pH value, the gas ion sample was unknown with the one matter basically of the various components of this solution of output before.Scrutinizing of ESI determined that in fact the ionization drop prepares by art methods.After drop was heated and stands repeatedly to collide sometimes, some that causes protein example separated folding, and this causes unfavorable wide CHARGE DISTRIBUTION, and this ionization liquid is sampled and finished evaporation in mass spectrometer.Gas ionization fails to finish before from the specimen material of solution fully outside mass spectrum, although obtained progress on this direction by laser assistant spray ioning method.
32
Goal of the invention and summary of the invention
The object of the present invention is to provide the equipment and the method that are used under atmospheric pressure producing the gas ion of specimen material from the liquid that contains described material.
Another object of the present invention is to be provided at the liquid intermediate ion specimen material ion generator equipment of molecule for example, this equipment comprises and is used at one end receiving liquid and at the sample capillary of the other end with this liquid of liquid stream emission, be used for providing voltage to set up the voltage source of electric field at an end capillaceous, with keep spacing to form the outer tube of annular gap around this capillary and with this capillary, gas-pressurized flows through this annular gap to form the gas blowing around flow of liquid of flowing with supersonic speed, form ultra-fine charged drop, the gas ion that adiabatic expansion and the liquid evaporation of this drop by gas under atmospheric pressure forms material or molecule.This equipment can also comprise that at least one (i) surpasses gas flow rate the device of supersonic speed threshold value with respect to the flow stream velocity of being carried, (ii) regulate the device of current potential intensity, (iii) regulate the first capillary vessel end with respect to the device of the position of the second capillary vessel end and the (iv) device of conditioning equipment operating temperature.
The invention provides a kind of specimen material from solution and produce the method for the gas ion of one matter basically, this method is included under the current potential and this solution is transported in the air-flow that moves with supersonic speed at least with respect to this liquid.
A kind of ion generator equipment is provided, it comprises being used for receiving to have at the liquid of the material sample of solution and at the capillary of other end emission liquid stream, the ring-type injection stream that is used for producing the device of electric field and being used for guiding gas under the speed at 350m/s at least at this other end capillaceous is through first other end capillaceous, with launching fluid is same direction, produce charged ultra-fine drop thus, adiabatic expansion and the violent evaporation of liquid the gas ion that this specimen material be provided of this ultra-fine drop by gas.
Has the quality analysis that the mass-synchrometer of the sampling terminal of sample ions under atmospheric pressure is arranged to receive the gas ion that is formed by ion generator of the present invention and the ionization of sample material is provided.
The accompanying drawing summary
When the description below reading in conjunction with accompanying drawing of the present invention, can more be expressly understood the present invention:
Fig. 1 has schematically shown the quality analysis system that comprises ion generator equipment of the present invention.
Fig. 2 has schematically shown the longitudinal cross-section of an embodiment of ion generator equipment of the present invention.
Fig. 3: bovine protein kinases A catalytic subunit (in 10mM aqueous acetic acid ammonium, be 200nM, (a) ESSI spectrogram pH7.8) and (b) online nanometer spraying spectrogram.
Fig. 4: bovine protein kinases A catalytic subunit (in 10mM aqueous acetic acid ammonium, be 200nM, pH7.8) the ESSI spectrogram when having 100 μ m ATP Mg salt.This enzyme also stands spontaneous phosphorylation in two sites, and this causes the further displacement at observed m/z.
Fig. 5: by ionization 10mM[Fe (bipyridl)
2]
2+And with a piece of paper be exposed under this spraying record as and the spraying tip between the ESSI of function of the distance cross section of spraying.Spray parameters: 1 μ L/min sample flow rate, 3L/min N
2Spray gas, the 2kV current potential of spraying.
Fig. 6:, wherein under the situation of ESSI and nanometer spraying, use the 0.01mg/mL lysozyme that is dissolved in the 10mM ammonium acetate of pH7.8 as (a) signal strength signal intensity and (b) mean charge of the hen albumen lysozyme ion of the function of spraying current potential.
Fig. 7: as (a) of the ox PKAc ion of the function of spray gas flow velocity peak width, (b) overall strength (peak area) in the half-peak eminence as the percentage of theoretical value.
Fig. 8 a-d: the spectrogram of ox cromoci, 0.01mg/ml obtains under different condition in 10mM aqueous acetic acid ammonium.
Fig. 9 a-b: as the mean charge and the peak width of the hen albumen lysozyme ion of the function of the distance between spraying tip and the atmosphere interface.
Figure 10 a-b: as (a) NaCl concentration and (b) intensity of the hen albumen lysozyme ion of the function of glycerol concentration; (c) base peak as the function of NaCl concentration is wide in same system, wherein the tip that experiment is used the most advanced and sophisticated of 5 μ m ID and used 2 μ m ID for nanometer spraying experiment for ESSI.
Figure 11: imidazoles-3-glycerol phosphate synthase (the IGPS)-N-[5 that contains 10mM ammonium acetate (pH7.1) and 6mMPIPES buffer solution,-phosphorus ribulose base]-formimino group]-the ESSI spectrogram of 5-aminooimidazole-4-carboxylic acid amides ribonucleotide (1, special inhibitor) mixture.
Figure 12 (a): lysozyme is (100nM in the 10mM ammonium acetate solution, pH7.8) with the ESSI spectrogram of the spacing spraying of 30cm.(b) similar experiment, spraying make and interact with the piperidines saturated vapor pressure.
Detailed Description Of The Invention
A kind of miniature electron spray that is equipped with variable current potential and high velocity fog gas is provided
33System, and this system and very complete miniature ESI and nanometer spraying ESI technology compared.Because its utilization is similar to the velocity of sound spray technique of Hirabayashi
34,35Supersonic gas spray, this new method is called electricity-sound (electro-sonic) spraying ionization or " ESSI ".This new method produce at low temperatures ultra-fine initial liquid drop (causing) by the adiabatic expansion of spray gas and the violent evaporation of solution and subsequently this method Ionized protein example under physiological condition is obtained narrow peak shape and narrow electronics valence distribution.
Referring to Fig. 1, shown connect according to atmosphere piezoelectricity sound spray ionization device of the present invention (ESSI) 11 with from attachment device for example liquid chromatogram 12 receive the specimen material of liquid forms.The electric sound spray ionization device of describing in detail now under atmospheric pressure forms the gas ion 13 of specimen material and is transported to for example suitable mass-synchrometer 14.Being used for implementing now, the fore-end of the mass-synchrometer 14 of the experiment of description schematically shows at Fig. 1.Illustrated fore-end is from ThermoFinnigan Corporation, the mass spectrometric fore-end that Model LCQ Classic buys.Described ion enters first Room 16 through the capillary terminal transmission of heating, the pressure (about 1 holder) that this chamber keeps the atmosphere than ionization source 11 to force down.Capillary 17 inlet chambers 16 of heating because pressure differential, ion and gas are flowed through.Capillaceous terminal by pipe lens (tube lens) 18 around, should
The pipe lens provide to make and have left the electrostatic field that this ion beam capillaceous focuses on separator apertures 19.Described ion enters mass-synchrometer through second area 21 and by 22 guiding of ion air deflector by second separator 23 subsequently under high vacuum.It will be apparent to one skilled in the art that this ESSI equipment can use together with the mass-synchrometer of any kind, comprise magnetic section, level Four bar, femtosecond, ion trap (2D and 3D), FT-ICR, track trap or their combination in any.In addition, the ion flow spectrometer compatibility of this source and any kind.
Specifically referring to Fig. 2, this figure is the enlarged drawing of electric sound spray ionization device 11 now, and described equipment comprises the T part 24 with end of thread.Sampling capillary 26 is supported by lasso 27 and extends thereon along this part.Second lasso 28 supports second capillary or manages 29, and this capillary or pipe have the internal diameter greater than the external diameter of sampling capillary 26, so that the annular gap between sampling capillary and external capillary or the pipe to be provided.Capillaceous terminal 31 ends that extend beyond external capillary of sampling.The sampling elongation capillaceous that surpasses external capillary can be regulated by move the sampling capillary with respect to external capillary, and vice versa.Regulate described distance in operation to obtain optimum operation condition.Other parts of T part link to each other through high pressure regulator 33 with nitrogen pot or other gas tank 32, the gas pressure that this high pressure regulator adjustments of gas enters the T part and leaves by the annular gap around liquid capillary.Each lasso is screwed into the T part by nut and carries out clamping.
According to the present invention, the size of typical electric sound spray ionization device is as described below:
Sampling capillary-5-100 μ m ID, 0.15mm OD
External capillary-0.025cm ID, 0.40mm OD
Distance-0.1-0.2mm between liquid capillary tip and the external capillary tip
Be applied to voltage on liquid capillary and the liquid-± 0-4kV
Air pressure-approximately 8-25 clings to
Sample flow rate-0.05-50mL/ minute
Material capillaceous is fused silica preferably, although also can use the material of other types, the capillary of preferably sampling is conductive, and voltage can be applied to described tip by capillary thus.External capillary can be pipe or other suitable materials.Yet, have been found that fused silica is suitable.
In operations according to the instant invention, the sampling capillary is applied voltage, set up electric field at this end capillaceous thus.Make specimen material (for example comprising for example molecule of protein of biomolecule) in liquid flow through this capillary and as flow of liquid from this terminal emission capillaceous.Described air pressure provides with greater than 350m/s, preferred 330-1000m/s, more preferably the ring-type of the speed of 400-700m/s is sprayed through regulating the annular gap end that makes between liquid capillary and external capillary, produce charged ultra-fine drop or particulate thus, the violent evaporation that these ultra-fine drops or particulate stand the adiabatic expansion of gas and liquid subsequently is with the gas ion of sampling material under atmospheric pressure.
Described all spectrums are used and to be equipped with the ESSI source (shown in Figure 1) that is similar to electric sound spray ionization device or the Thermo Finnigan LCQ Classic mass spectrum in nanometer spraying source to carry out record.Fluid sample is applied the voltage of 0-4kV by the copper crocodile clip, and this copper crocodile clip is fixed to the stainless steel tip that is used for the injector that sample injects.The temperature that experiment is carried out is a room temperature; Yet described temperature range is the boiling point from the ambient temperature to the solvent, is 20 ℃-100 ℃ for water promptly.Unless otherwise noted, described ion source is calibrated to obtain the highest sensitivity and the narrowest peak width the atmosphere interface of mass spectrometer 14 is careful.Typical instrument parameter is summarized in table 1.
Table 1
The instrument that is used for the LCQ instrument is set
| Parameter | Numerical value |
| Sample flow | 3μL/min |
| The spray gas flow | 3L/min |
| The spraying current potential | 2000V |
| The heated capillary temperature | 150℃ |
| Pipe lens current potential | 120V |
| Spraying distance from heated capillary | 5cm |
| Ends of the earth floating voltage | -1.3V |
| Heated capillary voltage | 30V |
Nanometer spraying spectrum is by using PicoTip
TMInside diameter be that (New Objective Inc., Woburn MA) obtain for the electron spray tip of 1 ± 0.5 μ m or 2 ± 0.5 μ m.Lysozyme, cytochrome c, alcohol dehydrogenase, bovine serum albumin, myoglobins, apomyoglobin and insulin are from Sigma (St Louis, MO) buy, hexokinase, trypsase and chymotrypsin are from Worthington (Lakewood, NJ) obtain, protein kinase, (Madison WI) obtains by Promega in catalytic subunit (PKAc).PKAc is to use Microcon YM-10 centrifugal separation unit, and (Millipore, Billerica is MA) from 350mM KH
2PO
4The buffer solution of initial soln displacement 200mM ammonium acetate solution.Other protein are to be dissolved in aqueous acetic acid ammonium buffer solution simply.The pH value of buffer solution is regulated through adding 1M ammonium hydroxide or acetic acid aqueous solution.
The electronic spraying mass spectrogram of bovine protein kinases A catalytic subunit (PKAc) contrasts the nanometer spraying mass spectrogram of purpose writing down (pH7.8, aqueous acetic acid ammonium buffer solution) under the condition mutually near physiological solution, respectively shown in Fig. 3 a and the 3b with being used to.Between two spectrograms, has remarkable difference aspect observed peak width and the electronics valence distribution.
As at table 2 sum up, also observe similar phenomenon for some other protein.Under the situation of ESSI, for the viewed full width at half maximum of protein ion (FWHM) value of abundant (relative abundance is greater than 10%) 100-150% of the theoretical value that calculates by Isotopic Distribution, and under nanometer was sprayed Ionized situation, typical FWMK value than the high 2-8 of theoretical value doubly.
Table 2
Use the protein spectrogram characteristic of ESSI and nanometer spraying (nS) to compare
| Protein | Peak width (theoretical FWHM%) | Base peak and its contribution to overall strength | ||
| ESSI | nS | ESSI | nS | |
| Lysozyme (egg white) | 105 | 126 | +6(70％) | +8(34％) |
| Cytochromes (horse) | 103 | 155 | +6(98％) | +7(21％) |
| Flesh hemalbumin (ox) | 110 | 260 | +7(85％) | +6(38％) |
| The catalytic subunit of protein kinase (ox) | 102 | 510 | +13(78％) | +12(49％) |
| Hexokinase (yeast) | 117 | 690 | +14(100％)* | +14(24％) |
| Alcohol dehydrogenase (monomer, yeast) | 115 | 340 | +12(72％) | +10(26％) |
| Insulin (pig) | 109 | 250 | +9(76％) | +7(33％) |
| Chymotrypsin (pig) | 105 | 220 | +10(71％) | +8(41％) |
| Concanavalin A (monomer) | 112 | 310 | +11(66％) | +10(18％) |
| Insulin (ox) | 109 | 142 | +4(57％) | +3(45％) |
| BSA | 107 | 760 | +17(100％) | +17(38％) |
* because limiting, the high-quality of instrument do not observe other ions
Second of the comparison of described two kinds of ioning methods is the electronics valence distribution.Depend on the protein of being studied, use the viewed electronics valence distribution of ESSI similar or narrower to the electronics valence distribution of using nanometer vapor recording to arrive.In most of the cases, because other gas ions do not have to surpass 25% relative abundance, single electronics valence state of planting is occupied an leading position on the ESSI spectrogram when ionization.Under the situation of nanometer spraying,, all only in a few eggs white matter, observe similar situation no matter be in our experiment or in data in literature.
With respect to almost completely removing the additive that desolvates under the situation of ESSI, the survival of particular organisms complex is excellent.This result is illustrated by Fig. 4, shows among the figure, and protein kinase A catalytic subunit is converted into its ATP/Mg adduct (spontaneous phosphorylation taking place in two sites) by adding excessive ATP/Mg salt, causes the further migration in viewed m/z value.The gained complex uses ESSI intactly to be transported in the gas phase.The survival rate that it should be noted that this complex is higher than 95%, and high ATP and Mg concentration do not have observable influence on the spectrogram characteristic.Comprise lysozyme-six-N-acetyl group-shell, six sugar (chitohexaose) for other protein ligands complexs, alcohol dehydrogenase-NADH and hexokinase-glucose have been obtained similar result.
The property feature of ESSI and nanometer spraying is shown in table 3.
Table 3
The comparison of the analytical performance of ESSI and nanometer spraying
| The most advanced and sophisticated OD of ESSI | The nanometer most advanced and sophisticated OD2 μ m that sprays | |||
| 100μm | 50μm | 10μm | ||
| | 1 | 4 | 12 | 15 |
| The detectability of PKAc (provides 3: the concentration of 1S/N); Ng/ μ L | 0.44 | 0.11 | 0.05 | 0.03 |
| Dynamic range (progression) | 4-5 | 4-5 | 3-4 | 2-3 |
| Flow (μ L/min) | 0.5- 300 | 0.1-30 | 0.02- 10 | 0.1 |
Although the absolute response factor of nanometer spraying is better, the detectability of these two kinds of technologies is comparability (for identical sample, it is similar that the nanometer spraying obtains higher signal intensity S/N ratio).It is relevant that difference between the response factor and the spraying of ESSI are dispersed, and its data are showed in Fig. 5.Use the thief hatch (standard value of Thermo Finnigan heated capillary) of 0.5mm, the 50-90% of nanometer spray droplet enters instrument under optimal conditions, and the sampling efficiency of ESSI only is 5-25%.This may overcome this defective by the atmosphere interface that use has a different geometries.Under variable concentrations, obtain response factor by the ionization protein solution.The detection limit value that is displayed in Table 3 has reflected that observing signal to noise ratio for the most abundant protein ion is 3: 1 protein concentration.
The dependent form of signal strength signal intensity and spectrogram characteristic is remarkable different (Fig. 6 a and Fig. 6 b) under high voltage (HV) under the situation of ESSI and nanometer spraying.Because spraying forms and drop charge is a process independently, the ESSI ion source imposes a condition at any voltage and produces ion down, is stabilisation and only spray under specific beginning voltage under the situation of nanometer spraying.Can " tuning " voltage be a significant real advantage for ESSI.Under 0V, observe pure velocity of sound spraying spectrogram, and by increasing current potential in low voltage range, intensity among the ESSI and spectrogram characteristic (peak width, average electronics valence state) change sharply.Report not before the appearing at of multiple-charged ion under the situation that does not have electric field.Near the threshold value of nanometer spraying, the ESSI signal stabilization, except faint influence was arranged on intensity, spectrogram did not rely on voltage for typical protein in the 0.8-4kV scope.Because ESSI produces measurable ionic current in whole voltage range, need not Ionized " igniting " in this case.Another advantage of ESSI is not exist arcing, and this may be because the turbulent flow of nitrogen has stoped the formation of corona discharge.
The factor that makes ESSI be different from other electron spray flexible programs the most significantly is gas flow rate.ESSI peak width and total signal strength are shown in Fig. 7 a and 7b for the dependence of spray gas flow velocity.Peak width is along with the rising of spray gas flow velocity sharply descends, and converges on theoretical value, i.e. the width of isotope covering.As can be seen, the rapid variation of peak width occur in about 0.35L/min flow velocity and more than, and the most violent in 0.4L/min place variation.Gas velocity is by calculating volume flow rate divided by the cross-sectional area of annular channel under atmospheric pressure.Usually the data 1L/min that obtains in ESSI equipment represents 943.14 meter per seconds (m/s).Thereby this flow velocity greater than 330m/s is fit to implement the present invention obtains sharp-pointed peak.Have been found that the favor speed scope is 400-700m/s.Overall strength (peak area) raises along with the spray gas flow velocity and descends, although this effect partly is able to balance by the peak shape of improving.The change of spray gas flow velocity makes initial liquid drop form mechanism and moves to pure pneumatic spraying from pure electron spray.The gas flow rate that increases has also changed vapo(u)rizing temperature and has allowed more effective solution evaporation by the adiabatic expansion of gas.Change on the spectrogram characteristic is accompanied by this two factors, and the decline of viewed signal strength signal intensity is because leave the higher line speed of the ion of heated capillary.Back one factor has reduced the sampling efficiency of pipe mirror lens-separator system.
The Ionized another noticeable feature of ESSI is the weak dependence that the spectrogram characteristic is set for different atmosphere interfaces, comprises temperature and electric potential gradient.Under the situation of the nanometer spraying of using the commercial ion source or ESI, desolvation efficient and electronics valence distribution are subjected to the strong influence of these parameters.When using ESI or nanometer to spray to use precipitous electric potential gradient (senior executive's mirror lens or awl voltage) under the Ionized situation, shown in Fig. 8 a and b, mean charge may move to high value.Corresponding ESSI data (Fig. 8 c and d) show more weak influence.
The spectrogram characteristic of ESSI demonstrates the strong dependence (Fig. 9 a and 9b) along the spray position of axle.Be too near at the tip and when entering the awl road, mass spectra peak take place and broaden, and be attended by a large amount of solvents and enter mass spectrometer, cause ion solvation once more in instrument.This explanation is subjected to the support that resolution depends on sample flow rate, and this is presented at the high sample flow rate same variation of flux degree (under the condition of listing＞50 μ L/min) of going down in table 1.More greatly apart from the time, fully desolvation often is accompanied by a small amount of migration of average electronics valence state, the electric charge that means ion descends and occurs in the barometric pressure range.Multi-charge protein ion stands when the high-pressure area of instrument and solvent and buffer solution interaction of molecules that hydrogen-bonded adduct forms and disassociation.Because from the ionic dissociation neutral flux molecule in specific charge site is that reversible process electric charge reduction then is not, although these electric charge sites have the GB value that is higher than any other kind, it will stand electric charge reduction slowly
24,26Although exist this electric charge to reduce process, protein solution can use ESSI from the distance spraying greater than 3m (rice), still obtains having the signal of S/N～30 under normal conditions.This observation has been opened up and has been used for the new possibility of the ion-molecule reaction of postgraduate's compounds under atmospheric pressure.
The sample flow rate of the sample flow rate of ESSI and nanometer spraying is overlapping; Yet the latter's average sample consumption is lower usually, and this is convenient to the off-line experiment.(use 10 μ m ID spray capillary and 1 μ L syringe, the dead volume of ESSI still is 2-3 μ L, and nanometer spraying spectrum can rise sample volume from sub-micro easily and carries out record.) as shown in table 3, the lower limit of sample flow rate depends on the cross section of spray capillary.This phenomenon shows that the principal element that stops flow velocity constantly to reduce is from the capillary tip evaporating solvent in ESSI.Because many interested analytes (protein and other biological macromolecule) are assumed that it is necessary that droplet-shaped is paired in its ionization by the ionization of electric charge residual (CR) technology.Evaporation can be suppressed at the exposed area of capillary tip by reducing liquid.The upper limit of sample flow rate in the scope of conventional H PLC eluent flow rate, this means that described ion source can use in the LC-MS interface in ESSI.
The aqueous solution that use contains different concentrations of sodium chloride and glycerol is tested the susceptibility of matrix effect the ESSI technology.Data are shown in Figure 10 a and 10b.Signal strength signal intensity shows that to NaCl concentration the sensitiveness that ESSI sprays to the sensitiveness and the nanometer of inorganic salts is similar.Yet than nanometer spraying or little spraying ESI, ESSI is more insensitive for high glycerol concentration.As if 20% glycerol concentration is incompatible with the nanometer spraying, and this may be the high viscosity because of sample, ESSI obtains stable signal under up to 70% glycerol content.In some cases, such as lysozyme, be successful by the ESSI ionization from pure cushioning liquid based on glycerol.ESSI also can tolerate the 2-amino-2-(methylol)-1 of high concentration (0.1-0.5M) well, ammediol (trishydroxymethylaminomethane (Tris base)).This feature is relevant with the rapid evaporation of drop.Because it is also therefore especially irreversible that initial liquid drop size and liquid/gas than all little, occur on the high-ratio surface.Under these conditions, even have all potential evaporations of material of low-steam pressure.
Three major advantages of ESSI are: eliminate the peak effectively and broaden (Fig. 3), narrow, normally unimodal electronics valence distribution under the situation of multi-charge unfolded protein ion, the ability (seeing below) of effective ion protein complex.Yet it is the phenomenon of well-known less relatively research that the peak that writes down the protein ion in electrospray mass spectrometer broadens.This is not enough or assemble on the electric charge site of protein ion owing to buffer salt owing to the desolvation of ion in atmosphere interface usually.(do not consider for example influence of slaine or carbohydrate of non-volatile component at this, can easily eliminate by buffer solution displacement or dialysis because these disturb usually.) covalency or ion cluster are present in some site of protein ion in both cases.In order to eliminate these extra materials, can change the composition of solution phase or the average interior energy of system.Yet if the main target of experiment is to study from the protein in physiology source or the folded structure of protein complex, all there is critical limitations in two kinds of replacement schemes.Protein unfolding or precipitation in the solution are brought out in the change of solvent or solvent pH value, and high-potential gracient in the front end vacuum area of atmosphere interface or high-intensity ion source temperature are brought out similar process in the electron spray nano-liquid droplet.Further exciting of incomplete desolvated gaseous state protein ion also may relate to the folding or disassociation of separating of structure of interest.Therefore, this class research of great majority is forced to implement under the low-res condition.Fig. 3 and 11 and table 2 in the result show that clearly ESSI has avoided making the requirement of this compromise.
Figure 11 shows, ESSI produces ion and shows its characteristic by the prevailing extremely narrow peak of single electron valence state thus from the protein complex efficiently.Should note another advantage of occurring in the figure.Under certain conditions, example is condition as used in this, and some protein fragments are sex change; These protein molecules can not form complex with the part combination and their occur as a series of peaks that broaden in some different electronics valence states, use the asterisk mark.This feature common in the ESI spectrogram also can be observed in the ESSI spectrogram.Remaining protein ion can and form complex really and they are as single rich complex peak.The ability of distinguishing the protein of natural and sex change is another advantage of ESSI.
The weak dependence that the electronics valence distribution is set atmosphere interface in ESSI shows that strongly the main distinction of ESSI and ESI (perhaps nanometer spraying) is that gas ion forms the position that takes place.Under the situation of traditional electrical spray technique, the formation of detected macroion occurs in the atmosphere interface-ion guides district of instrument.As if in ESSI, this process occurs in the atmospheric pressure region of instrument.For other evidences of this hypothesis are provided, use ESSI spraying lysozyme (100fm/ μ L), and described spraying is exposed to the highly basic piperidines.Shown in Figure 12 a and 12b, average electronics valence state moves to 6 from 7, and observes a large amount of adducts and form.Piperidines (the pK that in liquid phase, only exists with 1mM concentration
a=11.8) successfully suppressed the ionization of lysozyme.These results clearly show: gaseous state protein ion is atmospheric pressure region Already in.
Because under atmospheric pressure ESSI produces complete desolvated macroion, this feature provides under high pressure ability to these ion modifications to the user.These modifications comprise different separation and other technology based on mobility, ion reactivity, the cracked property of collision.The major advantage of atmospheric pressure operation ion is the macroscopic property of these processes.
ESSI demonstrates two phenomenons, and this makes it be different from other electro-spray ionization method, i.e. the high desolvation efficient of folded protein plastidome and observe prevailing a kind of electronics valence state.Good desolvation efficient may be relevant with little initial liquid drop size, and the latter causes by the supersonic speed spray gas with from the quick solvent evaporation of the high-ratio surface of droplet.Because high spray gas flow velocity and this make that solvation speed is low again, occurs in the low environment of solvent partial pressure.This helps to explain the following fact: be dissolved at protein under the situation of water-containing buffering liquid of physiological pH scope, observe single electronics valence state in the ESSI spectrogram.The low temperature of the spraying that the adiabatic expansion of spray gas and the violent evaporation of solvent cause helps to keep the prototype structure of these molecules.Folding protein structure contains the embedding electric charge (buried charge) of accurate qualification quantity, and it can carry the electric charge of specific quantity on its surface.Back one numeral is determined with respect to the gas phase basicity (GB) of solvents/buffer by apparent gas phase basicity (GB) value of lip-deep basic site.Because desolvation takes place under high pressure, this system can be assumed that and be in the thermodynamical equilibrium form, makes that these GB values are confirmable amount, and described confirmable amount strictness defines the surface charge capacity of protein molecule.Be understood that the charge capacity that will be higher than protein molecule in the quantity that comprises electric charge on the final drop of a single protein molecule easily.Therefore, during complete desolvation, buffer solution that some electric charge is dissociated or lyate ion or be pulled away as charged bunch.The result is that desolvated protein ion is recharged up to its capacity, and further the electric charge reduction is insignificant, because the dividing potential drop of solvent or buffer solution molecule is enough low.
Electron spray and use the supersonic speed spray gas combination results a kind of new electron spray flexible program-electric sound spray ionization-have this method is different from other specific characteristics based on the method for electron spray or velocity of sound spraying.The result has some unique new methods of analyzing advantage and some shortcomings.This analysis of technology performance comprises sample consumption or sensitivity, can compare with traditional E SI, not equal to can compare with the nanometer spraying ionization techniques of extensive use.In addition, ESSI demonstrates the ratio nano better reappearance and more stable of spraying.Opposite with the nanometer spraying, the major parameter of ESSI (sample flow, spraying air-flow, high voltage) can change arbitrarily, and this provides the more controls on the spectrogram characteristic.
The notable attribute of ESSI is desolvated degree and observed extremely narrow electronics valence distribution.These features are particular importances, because they mean the ionization of unfolded protein structure.These phenomenons are assumed that the position that forms with ion is relevant to the atmospheric pressure region domain migration of instrument.This makes ESSI become the method likely that allows the complete desolvation of protein molecule and do not lose tertiary structure and allow to keep specific non-covalent structure.Similarly, the continuous electric charge of multi-charge protein ion reduces generation gradually; Each electric charge decline step distributes according to the viewed narrow electric charge of different proton affinities (PA) the separated generation of the value site in each electric charge site.Because these features, the present invention can successfully allow even complicated and fragile more structure is transported to gas phase from solution, makes and can carry out biochemical system research more completely by mass spectrometer.
List of references
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(28)Experimental-Measurement?of?Coulomb?Energy?and?Intrinsic?DielectricPolarizability?of?a?Multiply?Protonated?Peptide?Ion?Using?Electrospray-Ionization?Fourier-Transform?Mass-Spectrometry.Gross，D.S.；Williams，E.R.Journal?of?the?American?Chemical?Society?1995，117，883-890.
(29)Dissociation?of?heme-globin?complexes?by?blackbody?infrared?radiativedissociation：Molecular?specificity?in?the?gas?phase？Gross，D.S.；Zhao，Y.X.；Williams，E.R.Journal?of?the?American?Society?for?Mass?Spectrometry?1997，8，519-524.
(30)Ion?Spray?Interface?for?Combined?Liquid?Chromatography/AtmosphericPressure?Ionization?Mass-Spectrometry.Bruins，A.P.；Covey，T.R.；Henion，J.D.Analytical?Chemistry?1987，59，2642-2646.
(31)Energy?Considerations?in?Twin-Fluid?Atomization.Lefebvre，A.H.Journal?ofEngineering?for?Gas?Turbines?and?Power-Transactions?of?the?Asme?1992，114，89-96.
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Claims (34)
1. method at liquid intermediate ion specimen material comprises:
Provide to have the end that be fit to receive described liquid and from the capillary of the other end with this liquid of stream emission,
Make this other end capillaceous remain essentially in atmospheric pressure,
The other end capillaceous produce electric field and
Make the gas ring-type spray the direction that flows through described described other end directional liquid capillaceous with the velocity flow that is at least 330m/s, produce charged ultra-fine drop thus, the adiabatic expansion of this drop by gas and the gas ion of the violent evaporation sampling material of liquid.
2. the process of claim 1 wherein that it is by making flow through described capillary and form around the annular gap between this second pipe capillaceous of gas-pressurized that described ring-type is sprayed, described second pipe has the internal diameter greater than the external diameter capillaceous of flow of liquid warp.
3. the process of claim 1 wherein that the speed that ring-type is sprayed is about 330m/s-1000m/s.
4. the process of claim 1 wherein that the speed that ring-type is sprayed is 400-700m/s.
5. the process of claim 1 wherein that the speed of gas expands by control gaseous and liquid evaporation is regulated.
6. the process of claim 1 wherein that gas is selected from dry air, argon gas, neon, oxygen and nitrogen.
7. the process of claim 1 wherein that gas temperature is 20 ℃-100 ℃.
8. the process of claim 1 wherein that gas temperature is through regulating to obtain the desirable desolvation degree of ultra-fine drop.
9. electron spray ion generator that is used at liquid intermediate ion specimen material comprises:
Be used at one end receiving described liquid and launch the capillary of flow of liquid from the other end,
The described other end capillaceous with the direction of the flow of liquid of being launched produce electric field device and
Be used to make the gas ring-type to spray and point to the device that institute is launched the equidirectional that flows through the other end capillaceous with the velocity flow of 330m/s at least, produce charged ultra-fine drop thus, the adiabatic expansion of this drop by gas and the gas ion of the violent evaporation sampling material of liquid.
10. the electron spray ion generator of claim 9 comprises that its internal diameter flows through annular gap capillary and second pipe between to form injected in gaseous state around this pipe capillaceous and gas-pressurized by this pipe greater than the capillary external diameter.
11. be used for the instrument of specimen material quality analysis, comprise:
Mass-synchrometer with the sampling terminal that can under atmospheric pressure sample,
Be used at one end receiving the specimen material of described liquid and launch the capillary of flow of liquid from the other end, spaced apart between this other end capillaceous and the sampling terminal,
Be used for described end capillaceous by apply between the terminal in this end capillaceous and sampling voltage set up electric field device and
Be used to make the gas ring-type to spray and point to the device that institute is launched the equidirectional that flows through the other end capillaceous with the velocity flow of 330m/s at least, produce charged ultra-fine drop thus, the adiabatic expansion of this drop by gas and the gas ion of the violent evaporation sampling material of liquid, this gas ion is introduced analytical equipment through this terminal.
12. the instrument of claim 11 wherein is used to guide the flow through device of end capillaceous of ring-type gas blowing to comprise around described capillary with the pipe that forms annular gap be used to make gas-pressurized to flow through described annular gap to form the device of ring-type gas blowing.
13. the instrument of claim 11 comprises the device that changes the distance between capillary end and the sampling terminal.
14. the instrument of claim 11 comprises the device of regulating described pipe end and intercapillary distance.
15. under atmospheric pressure to form the method for gas ion, comprising at the effects of ion specimen material:
Under a current potential, solution is conveyed into gas blowing as stream from sample line, this gas blowing is moved with supersonic speed with respect to the speed of solution, form ultrafine dust thus, the adiabatic expansion of this ultrafine dust by gas and the gas ion of the violent evaporation sampling material of solution.
16. the method for claim 15, wherein said gas blowing are to spray around the ring-type of flow of liquid.
17. the method for claim 16, the gas-pressurized that wherein said ring-type is sprayed by the annular channel of flowing through forms, and this annular channel is formed by the conduit around described sample line.
18. the method for claim 15, wherein said specimen material is a molecule.
19. the method for claim 18, wherein said molecule is a biomolecule.
20. the method for claim 19, wherein said molecule is a protein molecule.
21. the method for claim 16, wherein the speed of gas is greater than 330 meter per seconds.
22. the method for claim 16, wherein the speed of gas is 400-700m/s.
23. one kind is used at the effects of ion specimen material comprising under atmospheric pressure to form the system of gas ion:
Be used at one end receiving sample and carry the conduit of sample flow at the other end,
Be used for to described sample flow apply voltage device and
Be used under supersonic speed, air-flow being directed to the device of sample flow direction of the other end of conduit, make itself and sample flow interact, thereby produce charged drop that this drop provides gas ion by the adiabatic expansion of gas and the evaporation of solution.
24. the system of claim 23, the device that wherein is used for guiding gas stream comprises around first conduit with second conduit that forms annular channel be used for providing gas to form the pressurized-gas source around the ring-type gas stream of sample flow to described annular channel.
25. the system of claim 23, the terminal end of another conduit relatively of wherein said conduit is adjustable mutually.
26. be used under atmospheric pressure producing from the solution that contains described material the equipment of the gas ion of material of interest, this equipment comprises:
A., first capillary vessel of solution is provided by it;
B. second concentric with the first capillary vessel basically capillary vessel, this second capillary vessel are applicable to that to carry with respect to provided solution for ultrasonic speed with respect to solution speed be the gas stream of ring-type; The terminal common nozzle that limits of first and second capillary vessels that solution and gas therefrom flow through;
C. be used for applying the power supply of current potential to described solution; With
D. at least one (i) is used to regulate air-flow with respect to the device of the solution of being carried greater than the supersonic speed threshold value, (ii) be used to regulate the device of current potential intensity, the end that (iii) is used to regulate first capillary vessel is with respect to the device of the position of the end of second capillary vessel and (iv) be used for the device of conditioning equipment operating temperature;
Produce charged ultra-fine drop thus, this drop produces gas ion by the adiabatic expansion of gas and the evaporation of solution.
27. the equipment of claim 26, wherein the end of first capillary vessel is taper, to minimize its internal diameter and its external diameter difference at the end of delivered solution.
28. the equipment of claim 26 further comprises mass spectrometer with the inlet that is used for the atmospheric pressure sampling and the device that is used to change the distance between this inlet and the nozzle, this inlet is set to receive at least a portion gas ion.
29. the information that provides at least about the mass-charge ratio of gas ion is provided for the equipment of claim 28, wherein said mass spectrometer.
30. the equipment of claim 29, wherein can operate at least one device that is used for the adjustments of gas flow velocity, be used to regulate the first capillary vessel end with respect to the device of the position of the second capillary vessel end, be used to regulate current potential intensity device, be used for the device of conditioning equipment temperature and be used to regulate the device of the distance of inlet and nozzle, thereby change the relative abundance of the gas ion that produces by equipment.
31. be used under atmospheric pressure producing from the solution that contains described material the method for the gas ion of material, this method comprises:
A. in the equipment of claim 24, guiding enters the gas stream that the end at second capillary vessel provides from the solution of the first capillary vessel end, and this gas stream moves with supersonic speed at least with respect to solution.
32. the method for claim 31, wherein said material are the protein that is buffered in the aqueous solution of physiological pH value, most of gas ion produces the single chemical substance corresponding to each component of solution.
33. the method for claim 31, wherein said material are biomolecule or molecular complex in the aqueous solution that is buffered to the physiological pH value, and the gas ion that is produced is the single substantially material corresponding to each component of solution.
34. the method for claim 31, wherein the gas ion of specimen material stands the operation of gas phase atmospheric pressure.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US49018303P | 2003-07-24 | 2003-07-24 | |
| US60/490,183 | 2003-07-24 | ||
| US60/543,096 | 2004-02-09 | ||
| US10/888,869 | 2004-07-09 |
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| CN1830056A true CN1830056A (en) | 2006-09-06 |
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| CN 200480021444 Pending CN1830056A (en) | 2003-07-24 | 2004-07-23 | Electrosonic spray ionization method and device for the atmospheric ionization of molecules |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102142352A (en) * | 2011-02-12 | 2011-08-03 | 浙江好创生物技术有限公司 | Electro-spray ion generator |
| CN102142352B (en) * | 2011-02-12 | 2013-05-08 | 浙江好创生物技术有限公司 | Electro-spray ion generator |
| CN103048378A (en) * | 2012-12-20 | 2013-04-17 | 上海华质生物技术有限公司 | Mass spectrum sampling and ionization device for direct extraction and ionization of sample, and method of device |
| CN103545166A (en) * | 2013-11-04 | 2014-01-29 | 清华大学深圳研究生院 | Portable electrospray ion source device and mass spectrometer |
| CN103545166B (en) * | 2013-11-04 | 2016-01-27 | 清华大学深圳研究生院 | Portable electrospray ion source device and mass spectrometer |
| CN104714567A (en) * | 2013-12-15 | 2015-06-17 | 中国科学院大连化学物理研究所 | Air pressure regulator for vacuum chamber |
| CN108697333A (en) * | 2016-02-18 | 2018-10-23 | 国际香料和香精公司 | Electron spray ionisation smell counter device, system and application method |
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