GB2310950A - Method for the ionization of heavy molecules at atmospheric pressure - Google Patents

Description

method for the ionization of heavy molecules at atmospheric pressure The invention relates to the ionization of heavy, nonvolatile analyte molecules at atmospheric pressure, especially for the analysis by mass spectrometry.

Interest in the mass-spectrometric analysis of macromolecules, primarily large biomolecules or polymer molecules, has grown extensively during recent years. The analysis has become possible through a series of partially new ionization methods for these molecules. In the technical and scientific literature, the following abbreviations can be found for these ionization methods: SIMS (secondary ion mass spectrometry), PD (plasma desorption), MALDI (matrix-assisted laser desorption and ionization), FAB (fast atom bombardment), LSIMS (liquid SIMS), ESA (electrospray ionization). The specialist in the field is well acquainted with these types of ionization.

With the exception of electrospray ionization, all of these methods share a relatively low yield of ions. Out of 10,000 substance molecules, only about one ion is produced. The success of these methods is nonetheless very great, since 60 ions can still be produced from one attomol of substance (i.e. from 600,000 molecules). In principle, these can generate a spectrum which, in suitable mass spectrometers, may be sufficient to determine molecular weight. In practice, one cannot yet attain this sensitivity, since at least 10 to 100 attomol of substance is still necessary for this determination in good time of-flight spectrometers.

As always, it remains a disadvantage for these methods (except ESI) that the sample located on sample supports must be troublesomely introduced into the vacuum via vacuum locks. In biochemical laboratories, such treatment of samples is unusual and unpopular, since it is much easier and more common to leave the sample supports outside the vacuum. With sample supports which must be introduced into the vacuum, the coupling of mass spectrometry with chromatographic and electrophoretic separation methods is also made more difficult.

The great success of electrospray ion sources partially lies in the fact that ionization takes place outside the mass spectrometer. They are generally used at normal atmospheric pressure. The ions which are now generated in this way can be introduced into the vacuum and fed to the mass spectrometer relatively effectively and without excessive losses. Depending upon the technical embodiment, transfer yields of between 0.1 to 1% can thereby be achieved. Since vacuum-external ionization approaches close to 100% yield, the spray methods have become very effective in the meantime and are superior to ionization methods in the vacuum by one to two orders of magnitude. However they have only been applicable up to now with the highest sensitivity for storage mass spectrometers (RF quadrupole ion traps or ICR mass spectrometers).

/ evertheless, not all substances can be ionized using the electrospray method, and the wmand for more sensitive methods of ionization from a solid state continues to remain as great as ever. For example, efforts are being made to analyze the proteins from a single cell in situ. In particular however, substances separated two-dimensionally in gel layers can be ionized down better from surfaces rather than extracting them individually in complicated steps from the gel or from blot membranes in a solution which would then be sprayable.

The invention seeks to find a method which transfers large, nonvolatile analyte molecules located on a solid sample support from the solid state into the state of ionized individual molecules with great yield and make them accessible to mass spectrometric analysis. The overall ion yield should be increased in comparison to methods standard today and the handling of the sample support should be simplified.

The objective of this invention can be best achieved if the generation of ions from macromolecular analyte substances, which previously were ionized by processes like MALDI in vacuum with rather mediocre yield, is performed outside the vacuum. The ionization process, which had previously been part of the desorption process, can thereby be separated from it. Using the known methods of ionization at atmospheric pressure (API), especially chemical ionization at atmospheric pressure (APCI), however also charge exchange (CE) or electron capture (EC), a much higher ionization yield approaching 100% can be attained, so that a much higher ion yield from the analyte substance can be attained for the analysis despite the transfer losses in the vacuum.

This external ionization has become possible because methods have been revealed recently by which ions generated in a gas at atmospheric pressure can be introduced very effectively and economically to mass spectrometric analysis in a vacuum. The ions in the gas can be introduced into the vacuum through appropriate capillaries, whereby high transfer rates for the ions can be achieved in practice. Two-stage turbomolecular pumps, new on the market, with additional drag stages for differential pumping of such ion inlet systems, have made ion introduction relatively economical. Furthermore, the effective capture and guidance of ions through different pumping stages to the mass spectrometer in extended multipole arrangements has increased the transfer rate of ions. Nowadays vacuum-externally generated ions can be transferred to the mass spectrometer with high yields of up to 1%.

The problem is thereby reduced to the nondestructive transfer of nonvolatile analyte molecules from the sample support surface into the ambient gas. The transfer must be very fast since otherwise the molecules decompose through acquisition of energy. The analyte molecules must be transported far into the cool gas in front of the sample support in order to avoid recondensation. In addition, this must not lead to cluster formation with matrix material or to condensation of the analyte molecules, which occur quite easily in ambient gas.

It is therefore a basic idea of the invention to assist the desorption process moving the analyte molecules into the ambient gas by a photolytic or thermolytic self-decomposing matrix substance. Decomposition must take place extremely fast and must preferredly produce light gases. The gases must blow the heavy molecules into the ambient gas.

For this objective, organic explosive substances such as cellulose trinitrate, TNT, picric acid or xylite, or even metallo-organic substances such as silver azide or lead azide can be favorably used. Also material which is not normally used as explosives, such as cellulose dinitrate (used as a basis for nitrocellulose lacquer and detonates when heated), can be used. Organic explosives decompose into water, carbon monoxide, carbon dioxide and nitrogen vapors, with no residue remaining. They can be mixed with metallo-organic detonating agents (lead azide) in order to reduce the detonating temperature. The addition of other organic materials, which decompose easily by thermolytic processes but thereby acquire energy, such as simple sugars, can be used for reduction of gas temperature.

The decomposition processes of the matrix molecules are initiated by irradiation with laser light, as known from the vacuum MALDI process. In this case, a pulse laser is preferable to a continuous wave laser since decomposition then leads to an explosion-like expansion of a small cloud of decomposition vapor and the macromolecules are gas-dynamically entrained before they can reconnect themselves to the background by adsorption.

However a continuous wave irradiation is also possible if there is good focusing and the sample support and laser focus can be moved relative to one another in such a way that continuously fresh matrix material can be decomposed photolytically or thermolytically.

These molecule-decomposing processes must not affect the analyte molecules themselves. For this reason it is especially favorable not to imbed the analysis molecules in the matrix, but rather to deposit them on the surface. If the matrix substances are selected in such a way that their decomposition products are gaseous in their normal state, the large analyte molecules adsorbed on the surface are gently catapulted into the gas phase during rapid decomposition of the underlying matrix layer.

Most organic explosives are not soluble in water, but in some organic solvents like acetone. They can therefore (such as for nitrocellulose lacquer) be very simply applied to the sample support as a thin lacquer layer. Most of these substances, e. g. nitrocellulose (i. e. cellulosis dinitrate), are also very adsorptive so that large analyte molecules which are applied in an aqueous solution bind themselves to the surface adsorptively. It is even possible to wash away salts and other buffering agents again which were added to the solutions without great losses to the analyte molecules.

The lacquer layers can be made sufficiently thin that explosive decomposition remains limited to the section of the layer irradiated by laser light. Explosives can be easily derivatized in general without losing their decomposability. They can thus be modified relatively easily so that they absorb the light from the laser at the wavelength used.

By contrast with MALDI, the decomposed matrix molecules being released need not perform the task of ionizing the large analysis molecules during desorption at atmospheric pressure. The selection of matrix molecules is therefore only directed toward their ability to desorptively liberate the macromolecules. In contrast to this, with MALDI a compromise had to be made between the absorptive energy acquisition of the matrix by the photons, volatility, and ionizing power, which has meant that until now, no optimal matrix substance for all proteins, other biomolecules and polymers could be found. There are many different matrix substances in use and often the optimal matrix substance must be determined case by case in time-consuming steps.

It is therefore a further basic idea of the invention to add to the gas stream into which the large analyte molecules are catapulted, an excess of ions from moderately large reactant gas molecules in order to positively or negatively ionize the analyte molecules, in a manner basically known as API (= atmospheric pressure ionization).

The chemically ionizing reactant gas ions are selected according to the ionization energies of the biomolecules and their proton affinities, both relative to those of the reactant ions. In ambient gas, reactant gases must form stable ions which can easily ionize other substances by release of protons. Thus their ionization energy must be above and their proton affinity must be below that of the macromolecules to be ionized, otherwise there is no limit at all to this selection.

The ionization of reactant gases can occur in a known manner, for example using a gas cell with a beta emitter or a corona discharge. It has thereby proven useful to first ionize only slightly moist air or slightly moist nitrogen with the beta emitter or corona discharge.

First the excess available nitrogen is ionized, however water ions are formed very quickly (in milliseconds) by charge exchange. The reactant gas is mixed into the stream of this mixture of gas molecules and water ions in a concentration of a few percent, upon which the water ions react very quickly with the reactant gas molecules by the formation of reactant gas ions, which then exclusively remain for energetic reasons.

In contrast to normal chemical ionization, for which methane, ethane or isobutane are preferably used, heavy reactant gases can be used here by preference. Xylene has particularly proven itself for this purpose since it ionizes the large biomolecules without causing fragmentation. The difference in ionization energies between xylene and the large biomolecules is so limited that no excess energy is available for fragmentation. Conversely xylene's ionization energy lies below the ionization energies of possible contaminations of the ambient gas so that xylene can be regarded as a relatively universal reactant gas.

There is however a large number of substances which are just as favorable as xylene.

Ashe ionization yield of large analyte molecules can be particularly increased by moving the small reactant gas ions through an axially arranged electrical field relative to the flowing gas, similar as in an ion mobility spectrometer. The number of collisions of smaller reactant gas ions with as many flowing analyte molecules as possible is increased by the fact that the reactant gas ions literally plow through the gas.

It is a further basic idea of the invention to filter out again the rest of these medium large reactant gas ions before reaching the mass spectrometer. This filtering out can take place in a simple manner in the vacuum using the ion guiding multipole arrangements which have a lower mass cut-off limit for the capture and further guidance of the ions.

It is however also possible to filter out the excess reactant gas ions already in the input capillary to the mass spectrometer. Along the input capillary, an electrical longitudinal field is normally applied. The ions are then transported by viscous friction by the gas stream against this field and raised to a higher potential. Inside the flowing gas, the ions move against the gas stream due to their ion mobility, drawn by the elctrich field. Since light ions move more quickly than heavy ones, there is a lower transport limit in gas flow direction for the ions. Lighter ions can move more quickly than is appropriate for the gas velocity in the capillary, and they are therefore not transported into the direction of the mass spectrometer. By installing a filtering section along which the electrical field is so great that the reactant gas ions cannot be transported, the ions can be filtered out. This method has the advantage that the space charge density in the capillary becomes smaller and the heavy analysis molecules show a better transport yield. Additionally, there is an especially high yield for analyte ions at the beginning of this filter section.

With molecular ions from smaller reactant gas molecules, multiple ionization of heavy molecules can also be achieved.

Instead of using positively charged reactant ions, it also possible to add negative ions or thermal electrons to the gas stream in order to generate negative ions from the large biomolecules. This type of ion generation is especially significant for nucleotides.

Further Advantages of the Invention The addition of photolytically non-explosively decomposable matrix molecules can cause cooling of the matrix product gases after explosion, and therefore achieve better stability for the analyte molecules. This type of cooling has become known, for example, through the admixture of photolytically decomposable sugars as a so-called "co-matrix" in previous MALDI methods.

For certain types of mass spectrometers, it is especially advantageous that the ions in the input capillary of the mass spectrometer can be transported against a potential difference.

in this way they can be raised to the acceleration potential of the mass spectrometer. This timing against a potential difference is automatically associated with a movement of all ions relative to the neutral molecules of the gas which then, in turn favorably influences the ionization yield for macromolecules. It cannot be ruled out that even large molecule ions are focused in this way into the middle of the gas jet in the capillary, which increases the transfer yield.

Especially advantageous however is the easy handling of the sample support outside the vacuum. The sample support need not first to be troublesomely introduced into the vacuum system via a vacuum lock. In an especially favorable embodiment, the sample support can simply be placed upon a small moving device and the mass spectrometer is then immediately prepared for the scanning of spectra.

Also favorable is the possibility of two-dimensional movement of the sample support at atmospheric pressure. This is, in contrast to movement within the vacuum, extraordinarily easy and economical to produce. Movement within the vacuum is complicated and expensive in comparison, since the drives must remain outside the vacuum and the movements must be transferred via bellows or other transfer elements. In addition, the use of lubricants in the vacuum is not possible so that very expensive self-lubricating or sliding materials must be used.

Brief Description of the Drawings Figure 1 shows a diagram of a preferred device according to this invention.

(1) Suction port for moist air, (2) High voltage feeder and needle for the corona discharge, (3) ionization chamber for air, (4) Feeder for the reactant gas, (5) Work plate with hole for placement of the sample supports (in a movable frame, not shown) (6) Window for the admission of focused laser light, (7) Sample support with analysis substance on the underside, with a movement device, adjustable in two dimensions, not shown here, (8) Focusing lens for the laser light, (9) Channel for feeding the mixture of gas and ions into the input capillary, (10) Wall of the vacuum system for the mass spectrometer, (11) Input capillary through which the mixture is introduced into the differential pump system, (12) First chamber of the differential pump system, 3) Gas skimmer with through-hole for the ions, in the wall to the next chamber of the wfferential pump arrangement, (14) Wall between the first and second chambers of the differential pump system, (15) Second chamber of the differential pump system, (16) lon guide consisting of an extended multipole field with rod-shaped poles, (17) Opening in the wall of the second chamber to the main vacuum chamber of the mass spectrometer, (18) Main vacuum chamber of the mass spectrometer, (19) End cap of a mass spectrometer based on a quadrupole RF ion trap, (20) Ring electrode of the ion trap, (21) Laser for the desorption of analysis substance, (22) Pump nozzle in the first chamber of the differential pump system, (23) Pump nozzle in the second chamber, (24) Pump nozzle in the main vacuum chamber of the mass spectrometer.

Figure 2 shows a hexapole arrangement as an ion guide. The pole rods are covered with an RF voltage, whereby the phase changes respectively between adjacent rods.

Figure 3 shows a slightly changed arrangement relative to Figure 1 with a mixing chamber (25) in which the gas stream with the sample molecules is enveloped by a gas stream with the reactant gas ions. The arrangement to a large extent prevents wall collisions of the sample molecules. The other numbers have the same meaning as in Figure 1.

Description of Preferred Embodiments Figure 1 shows a diagram of a preferred device according to this invention. Through an opening (1), moist air is suctioned into an ionization chamber (3) in which a needle (2) at high voltage develops a corona discharge. The corona discharge can also be replaced by a beta emitter on the wall of the ionization chamber (3), for example by Ni63. When using Ni63, the ionization chamber (3) should have a diameter of about 10 millimeters, since the Ni63 electrons pass along a path of about 6 millimeters before they lose their kinetic energy in air at atmospheric pressure and are stopped. They form most of the ions at the end of their path.

In the ionization chamber (3), nitrogen ions are preferredly formed at first, which however quickly react with H2O water molecules and become the water ions OH+ and Or2+. The water ions react further with water molecules and a large share of OH3+ ions are formed.

These ions are especially capable of chemical ionization by the release of a proton.

small percentage of reactant gas, for example xylene, is mixed into the flowing gas trough the feeder (4). Rapidly, the xylene molecules are transformed through protonation by the water ions into energetically more favorable protonized xylene ions, whereby the water ions are used up. When the gas stream reaches the hole in the work plate (5), almost only xylene ions are still present.

The sample support (7) lies on the work plate (5), the latter having a hole. The analyte substance forms a very thin layer (less than monomolecular) on the underside of the carrier plate (7) on top of a layer of matrix molecules.

The matrix substance layer consists preferredly of cellulose dinitrate, the layer is about 5 micrometers thick. A nicely pure lacquer of nitrocellulose can be obtained by solving nitrocellulose from commercial blot membranes in acetone. This nitrocellulose has a molecular size of some hundred monomer units only. The matrix can be mixed with other substances increasing the absorption of the laser light, e. g., with low concentrations of a cyano4-hydroxy-cinnamic acid ( < 5 %). The increased absorption decreases the need for high laser power.

The sample support is fastened in a frame, not shown, of an x-y movement device, also not shown, which maintains a precise distance between the sample support and work plate. In this way the analysis substance is protected from contact with the work plate.

Through the precision gap, some ambient air (or nitrogen) is drawn into the gas channel intentionally - as a second gas stream. From an inexpensive nitrogen pulse laser (21), flashes of light with 337 nanometer wavelength (about 10 microjoule each) are emitted which, focused through the lens (8), pass through the window (6) and the hole in the work plate (5) onto the sample support in a focus of about 150 micrometer diameter and vaporize the matrix molecules in explosion-like detonations. By the detonations, the analyte molecules are desorbed into the gas stream. They are entrained by the additional second stream which penetrates through the precision gap between the support plate and work plate, and mix with the gas and reactant ion mixture.

The sample support plate can also be produced from a transparent material, such as glass or plastic for example. It is then possible to arrange the laser above the plate and introduce the laser light through the sample support plate. This arrangement leads to a simpler design.

The mixture of air molecules, reactant gas ions, matrix molecules and molecules from the analysis substance is now fed through the channel (9) of the input capillary (11) which extends through the wall (10) of the mass spectrometer into the vacuum. The input capillary, with an inside diameter of 0.5 millimeter and a length of 10 to 15 centimeters thereby draws one to two liters of air per minute into the vacuum. This suction stream maintains the gas stream through the suction port (1) in the ionization chamber, the second stream through the gap between the work and carrier plates, and the stream ,trough the channel (9), without requiring any additional pump or evacuation. In the -annel (9) with a diameter of about 1.5 millimeters, a rather steady stream with a central velocity of about 20 meters per second is thereby attained. The channel (9) is designed conically for reasons of practicality, in order to offer a good and non-turbulent transfer into the input capillary (11).

The analyte molecules are ionized by chemical ionization at atmospheric pressure (APCI) essentially in this channel (9). This ionization is generally very effective and reaches 100% ion yield if the concentration of reactant gas ions is sufficiently high. Due to wall collisions in the channel (9) and in the input capillary (11), about 90% of the ions are lost however, but the yield is still quite high. The channel (9) should therefore be kept as short as possible.

At low concentrations of reactant gas ions, it is also possible to increase the ionization yield by designing a part of the channel (9) as an ion drift region by the application of an axially arranged electrical field. If the input capillary (11) is used to prime the ions against an electrical potential, an increase in the yield of heavy ions is thereby automatically achieved.

In the first chamber (12) of the differential pump unit, which is evacuated via the nozzle (22) by a prevacuum pump, the ions are accelerated by adiabatic expansion of the gas at the end of the input capillary and are simultaneously cooled. They form a cone-shaped effluent jet of about 20 aperture angle. Using an electrical drawing field (not shown) directed to the gas skimmer (13), a considerable portion of the ions can be transferred into the second chamber (15) of the differential pump system through the opening of the gas skimmer (13), which has a diameter of about 1.2 millimeters. In the second chamber (15), the ions are almost completely accepted by the ion guide (16) which is made of extended pole rods and generates an electrical multipole field. The capture of ions by the ion guide is thereby supported to a large extent by the gas-dynamic processes in the gas skimmer.

The ion guide leads the ions through the chamber (15), a wall opening (17), and the main vacuum chamber (18) to the mass spectrometer which is designed here as an RF quadrupole ion trap with end cap (19) and ring electrode (20).

The ion guide preferably takes the form of a hexapole arrangement and consists of six pole rods, each about 15 centimeters long and only one millimeter in diameter (see Figure 2), which are attached to one another by ceramic holders, not shown. The thin pole rods are arranged around the circumference of a cylinder and enclose an empty inner cylinder of only 2 millimeters diameter. With an RF voltage of about 600 volts at 3.5 megahertz, this multipole has a lower cut-off limit of about 150 atomic mass units for singly charged ions. For this reason the xylene ions, which when protonized are only 107 atomic mass units in weight, have no stable trajectories inside the ion guide and are eliminated. Ions from the remains of matrix molecules can also be eliminated in this way if their molecular height is correspondingly small. Only the heavy ions of the analysis substance can reach mass spectrometer as required.

Figure 3 shows a slightly changed arrangement in contrast to Figure 1. The substance desorbed explosively by the light from the laser (21) is first entrained here by the second stream which penetrates through the gap between the work plate (5) and sample support plate (7). The second gas envelops the stream of analyte sample molecules and prevents wall collisions by the analyte molecules. The gas stream with the analyte sample molecules is first enveloped with the gas stream in a mixing chamber (25) which contains the reactant gas ions. These penetrate by diffusion into the central gas stream and cause chemical ionization of the analyte molecules.

Here too, a transparent version of the sample support plate can simplify the design of an appropriate instrument. The dead space between the hole in the work plate (5) and the window (6) is then unnecessary for the laser beam.

It is not absolutely necessary to generate the reactant gas ions before mixing with the gas stream which contains the sample molecules. The analyte molecules may be ionized in a mixture of air, water vapor, reactant gas, matrix decomposing products and analyte molecules inside channel (9), for example by a wall coating with Ni63.

The sample carrier or support plate (7) can be adjusted in its movement device (not shown) in two directions on the work plate (5). Adjustment is controlled by a computer which allows the carrier plate to be coated with substances two-dimensionally. In this way plates with substances separated by two-dimensional electrophoresis can be scanned and analyzed for the distribution of proteins or other analysis substances. In particular, blot membranes which are made of cellulose dinitrate (gun cotton) can be applied directly to the sample support plate after the usual loading with two dimensionally separated substances and used for this method.

The ions can be primed against a high voltage in the input capillary (11), whereby however a lower cut-off threshold exists for light ions which can be used for filtering out reactant gas ions. For example, nitrogen has a velocity of 128 meters per second in a 16 centimeter long capillary with an inside diameter of 0.4 millimeters. If one applies an opposing electrical field of 2,000 volts per centimeter to a section of the capillary, ions with a mass of 110 atomic mass units then also move at 128 meters per second in the opposite direction through the nitrogen, and are therefore no longer transported forwards by the gas. The xylene ions are therefore not introduced into the vacuum of the mass spectrometer. Ion electron capture best takes place after mixing with the sample molecules since the electrons dissipate very quickly.

However, through the electrons, negative reactant gas ions can also be generated in the known manner if a reactant gas of higher electron affinity is used. For ionization with negative ions at atmospheric pressure, the abbreviation APNCI is sometimes used. This type of ionization is especially important for nucleotides.

Claims (12)

Claims

1. A method for the ionization of heavy analyte molecules on a solid sample support in a gaseous environment at atmospheric pressure, wherein a decomposable matrix substance is present on the solid sample support along with the analyte molecules, and wherein the matrix substance is decomposed by the light from a laser, whereby the products of decomposition transport the analyte molecules into the gaseous environment, and the analyte molecules are ionized by ionization at atmospheric pressure.

2. A method as claimed in Claim 1, wherein the matrix comprises an explosive substance.

3. A method as claimed in Claim 2, wherein the matrix also comprises an non-explosive substance.

4. A method as claimed in one of the preceding Claims, wherein the matrix material is applied as a thin lacquer layer on the solid sample support.

5. A method as claimed in one of the preceding Claims, wherein the analyte molecules are mixed homogeneously with the matrix material.

6. Method as claimed in Claim 4, wherein the analyte molecules are applied to the surface of the lacquer layer made of matrix material.

7. A method as claimed in any one of the preceding Claims, wherein the gas flows in front of the sample support and transports the analysis molecules.

8. A method as claimed in Claim 7, wherein the reactant gas ions for the ionization at atmospheric pressure are in the flowing gas before it reaches the sample support.

9. A method as claimed in Claim 7, wherein the reactant gas ions are in a second gas stream which is mixed with the gas stream coming from the sample support.

10. A method as claimed in Claims 8 or Claim 9, wherein the analyte ions which form in the gas stream are fed to a mass spectrometer.

11. A method as claimed in Claim 10, wherein the reactant gas ions are filtered out before reaching the mass spectrometer.

12. A method as claimed in one of the preceding Claims, wherein the yield of large ions from the analysis substance is increased, in that a part of the gas flow to the mass spectrometer moves through an axial electrical field in the form of an ion drift region.