WO2001078880A1

WO2001078880A1 - Method of reducing ion fragmentation in mass spectrometry

Info

Publication number: WO2001078880A1
Application number: PCT/US2001/006625
Authority: WO
Inventors: Alec M. Wodtke
Original assignee: The Regents Of The University Of California
Priority date: 2000-04-12
Filing date: 2001-02-28
Publication date: 2001-10-25
Also published as: AU2001241907A1

Abstract

A method for the reducing ion fragmentation in mass spectrometry analysis is described. A sample and a carrier gas are expanded to form a molecular beam of sample molecules having the carrier gas adsorbed, or condensed, thereon. Ionization of the sample is accompanied by rapid evaporation of the adsorbed carrier gas, which cools the ions and effictively reduces ion fragmentation. An apparatus for mass spectrometry analysis according to the subject methods is provided. By using the method of the invention and reducing ion fragmentation it is possible to carry out mass spectrometry on molecules such as DNA sequences which could not ordinarily be so analyzed.

Description

METHOD OF REDUCING ION FRAGMENTATION IN MASS SPECTROMETRY

STATEMENT OF FEDERALLY SPONSORED RESEARCH This invention was made with Government support under Grant No.

9633002, awarded by the National Science Foundation. The Government has certain rights in this invention.

FIELD OF THE INVENTION This invention relates generally to the mass analysis of molecules.

More particularly, the invention relates to a method of reducing ion fragmentation in mass spectrometry, particularly when analyzing very low vapor pressure compounds.

BACKGROUND OF THE INVENTION

Mass spectrometry is a commonly used method for chemical analysis in many areas of industry. It has had particular importance in recent years in the analysis of low vapor pressure compounds, e.g. organic, inorganic polymers and biopolymers. Applications of this technology permeate the chemical, materials and biotechnology economy. As an example, consider one of the most technologically important biopolymers, DNA. Approximately 4,000 human disorders are currently attributed to genetic causes. Hundreds of genes responsible for various disorders have been mapped, and sequence information is being accumulated rapidly. A principal goal of the federal government's Human Genome Project is to find all genes associated with each disorder. The definitive diagnostic test for any specific genetic disease (or predisposition to disease) will be the identification of epigenetic modifications, mutations and polymorphic variations in DNA sequence in affected cells that result in alterations of gene function. Sequence information will also allow the development of gene-based diagnostics and therapies. The ultimate goal of the Project is the determination of the complete sequence of the nearly 3 billion base pairs that make up the human genome. Conventional genetic sequence analysis is based on gel electrophoretic separation of labeled DNA fragments generated by chemical (Maxam and Gilbert (1977) Proc. Natl. Acad. Sci. USA 74:560-564) or more widely favored enzymatic (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463-5467) sequencing methods. High resolution denaturing gels are available, and capillary techniques using DNA arrays, chips or microplates have been developed that have advanced sequencing technology. However, gel-based separations suffer from limited throughput (the time required to perform gel separations can be hours) and frequent errors due to band compression and/or the presence of non-specific sequencing products. These factors, and the expense of the reagents needed to purify DNA, pour gels and perform sequencing reactions make gel-based separations expensive and time-consuming.

The development of soft ionization techniques such as electrospray ionization (ESI) (J. B. Fenn et al. (1989) Science 246:64-71) and matrix- assisted laser desorption/ionization (MALDI) (Karas et al. (1988) Anal. Chem. 60:2299-2301 ; Tanaika et al. (1988) Rapid Commun. Mass Spectrom. 2:15 1- 153 have made mass spectrometry a rapid and accurate alternative method for the sequencing and analysis of DNA and other large biomolecules (Fitzgerald et al. (1995) Annu. Rev. Biophys. Biomol. Struct. 24:117-140; Murray (1996) J. Mass Spectrom. 31 :1203-1215). Using mass spectrometry, sequencing products, or PCR products, are ionized and the resulting ions separated and detected on the basis of mass.

For a mixture of Sanger sequencing products, four mass spectra are obtained (each in about 1 minute), one for each of the four bases, and overlaid such that the order of the peaks correspond to the DNA strand sequence. Mass spectrometry of Maxani and Gilbert or ladder sequencing products can also be performed. Because separation is accomplished on the basis of absolute molecular mass (i.e., by size), and not relative gel mobility (i.e., by structure), the accuracy of the sequence data is high.

The resolution and mass range needed for accurate mass determinations of large biomolecules by mass spectrometry is frequently compromised by ion fragmentation. Fragmentation occurs when an initially formed, excited molecular ion breaks up or fragments to form more stable ions in order to dissipate or release internal energy. Fragmentation mechanisms for DNA include base loss and backbone cleavage. Fragmentation decreases the intensity of the signal detected from the intact molecular ions because the fragmentation reduces the number of intact molecules. Fragmentation also masks the signal of the intact molecule within signals from fragments. Fragmentation is commonly observed in the mass spectrometric analysis of samples that are difficult to vaporize or ionize without degradation, e.g., hydrogen bonded molecules and molecules having a low vapor pressure. U.S. Patent No. 5,742,050 discloses a method and apparatus for introducing a sample into a mass spectrometer for analysis using laser desorption of a sample and supersonic molecular beam expansion of a mixture of the sample and a carrier gas for analysis by mass spectrometry. Samples analyzed in accordance with the method include anthracene, lidocaine, pyrene, 9,10-dichloroanthracene, methylparathion and caffeine. The patent does not teach or suggest the reduction or suppression of ion fragmentation or the analysis of low vapor pressure molecules.

U.S. Patent No. 5,760,393 discloses methods for improving resolution in MALDI-TOF mass spectrometry using delayed ion extraction. The patent does not teach or suggest the reduction or suppression of ion fragmentation.

There is a clear need in the art for a method of reducing or suppressing ion fragmentation in the mass spectrometric analysis of molecules which are difficult to vaporize or ionize, e.g., large, fragile polymeric molecules. Such a method would facilitate the accurate mass spectrometric analysis of biomolecules such as DNA (but certainly not limited to DNA) with high resolution and detection limits.

SUMMARY OF THE INVENTION

The present invention provides a novel method for reducing ion fragmentation in mass spectrometry. The method comprises forming a molecular beam by expanding a composition comprising a sample and a carrier gas in a manner which condenses said carrier gas on said sample, ionizing said sample of said molecular beam to produce ions, analyzing the mass of said ions, and detecting said ions. The methods of the invention effectively reduces ion fragmentation which occurs during the analysis of unstable molecules, and as such is applicable to the analysis and sequencing of a range of molecules with very low vapor pressure such as biomolecules, including proteins and nucleotide sequences, e.g., DNA and RNA.

An apparatus for mass spectrometry is also disclosed, comprising a means for forming a molecular beam by expanding a composition comprising a sample and a carrier gas in a manner which condenses said carrier gas on said sample, a means for ionizing said sample of said molecular beam to produce ions, a means for analyzing the mass of said ions, and a means for detecting said ions.

An object of the invention is to provide a method of reducing ion fragmentation in mass spectrometry analysis particularly with samples with very low or substantially no vapor pressure. A specific aspect of the invention is the formation of a molecular beam by expanding a composition comprising a sample and a carrier gas in a manner which condenses the carrier gas with the sample.

An important feature of the invention is reducing ion fragmentation when carrying out mass spectrometry analysis of a sample which has substantially no vapor pressure at STP.

An advantage of the invention is that ion fragmentation can be reduced to the extent that analyzable signals can be obtained using mass spectrometry on samples which have substantially no vapor pressure.

A feature of the invention is molecular beam expansion of the composition comprising a sample and a carrier gas.

Another feature of the invention is the condensation of a carrier gas on a sample.

Yet another feature of the invention is the ionization of a sample having a carrier gas condensed thereon in a molecular beam. Yet another feature of the invention is the rapid evaporation of the condensed carrier gas effectively cooling the ion, removing energy from said ion.

An advantage of the invention is that ion fragmentation is effectively reduced. Another advantage of the invention is that simplified mass spectra of fragile molecules which have substantially no vapor pressure at STP can be obtained.

Still another advantage of the invention is that parent ion signals from fragile molecules can be observed with improved limits of detection.

Yet another advantage of the invention is that it is amenable to high throughput analysis.

It is an object of this invention to provide a method for reducing ion fragmentation in mass spectrometry. These and other aspects, objects, advantages, and features of the invention will become apparent to those skilled in the art upon reading this disclosure.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a schematic overview of a molecular beam photoionization reflection TOF mass spectrometer useful in the invention.

Figure 2A is a mass spectrum graph recorded using a weak H20/Ar expansion.

Figure 2B is a portion of the graph of Figure 2 starting at the p31. Figure 2C is a mass spectrum graph recorded under strong expansion conditions.

Figure 3 is a graph showing the relative abundance of unprotected parent ions (solid line) and daughter ions produced by loss of OD (long- dashed line) or by loss of OD followed by loss of D2O from the same parents (short-dashed line).

Figure 4A is a graph which shows fragmentation in mass spectrometry of Octane with the analysis carried out on a gaseous sample at room temperature.

Figure 4B is a graph which shows the suppression of fragmentation (compared to 4A) in mass spectrometry of Octane with the analysis carried out under rapid evaporation cooling conditions.

Figure 5A is a graph which shows a mass spectrographic analysis using N2 as a carrier gas showing fragmentation suppression relative to 5B. Figure 5B is a graph which shows a mass spectrographic analysis using Neon as a carrier gas.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Before the devices, formulations, and methodology of the present invention are described, it is to be understood that this invention is not limited to the particular device, components, formulations and methodology described, as such may, of course, vary. It is also to be understood that the terminology used herein is with the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a formulation" includes mixtures of different formulations and reference to "the method of treatment" includes reference to equivalent steps and methods known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to describe and disclose specific information for which the reference was cited in connection with.

All publications mentioned herein are incorporated herein by reference to described and disclose specific information for which the reference was cited in connection with. The publications discussed herein are provided solely for their stated disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such publications by virtue of prior invention. Further, the actual publication date may be different from that stated on the publication and as such may require independent verification of the actual publication dates.

DEFINITIONS The term "substantially no vapor pressure at STP" is used here to mean substances which exhibit very little detectable vapor pressure at STP. More specifically, substances which preferably have a vapor pressure of about 1/10 the vapor pressure of water at STP. Water has a vapor pressure of about 20 torr at STP. Thus, the invention is particularly useful with substances which have a vapor pressure of 2 or less at STP, preferably 1 torr less, still more preferably about 1 x 10-3 torr or less, and still more preferably substances with a vapor pressure of about 1 x 10-7 Torr or less at STP.

The term "mass spectrometry" is used herein to refer to an analytical technique for determining the mass and structural information of any particles, e.g., neutral atoms, molecules, clusters (i.e., aggregates of atoms or molecules) or polymers. Traditionally, an analyte in the vapor phase is bombarded by a high-energy beam of electrons, atoms or molecules causing the formation of ions. The resulting ions are accelerated through a magnetic, electrostatic or electrodynamic field which confines and directs the ions into a mass analyzer, where they are separated on the basis of their mass-to-charge ratio (m/z). An ion detector measures the relative abundance of each ion and produces a spectrum of signal intensity v. m/z for each ion detected.

A mass spectrometer combines ion formation or ionization, mass analysis and ion detection. Exemplary ionization methods include chemical ionization (Cl), plasma discharge ionization, glow discharge ionization, electron impact ionization (El), electrospray ionization (ESI), fast-atom bombardment ionization (FAB), field ionization, laser desorption/ionization, matrix-assisted laser desorption/ionization (MALDI), laser multi-photon ionization, laser single-photon ionization, electron capture ionization, Penning ionization, plasma desorption/ionization, resonance ionization, secondary ionization and spark ionization or thermal ionization. Exemplary mass analyzer designs include time-of-flight (TOF), magnetic sector, electric sector, combined magnetic/electric sector instruments, Fourier-transform ion cyclotron resonance, quadrupole, and quadrupole trap; see Brunee (1987) Int. J. Mass Spectrom. 76:125-237 and any combination of these or other mass spectrometries in the many so-called "hyphenated mass spectrometries". Exemplary ion detectors include electron multiplier, multichannel plate microchannel plates, microsphere plates, ceratrons, and cryogenic detectors. The mass spectrometer type is denoted by the mass analyzer design. For example, a mass spectrometer equipped with a time-of-flight mass analyzer is herein referred to as a time-of-flight mass spectrometer.

The term "analyte" is used herein to refer to any particle or group of particles, e.g., neutral atom, molecule, cluster (i.e., aggregate of atoms or molecules) or polymer to be analyzed by mass spectrometry. Analytes of particular interest in the present invention are compounds which have substantially no vapor pressure at STP and includes nucleic acids (DNA and RNA), polynucleotides, peptides, proteins, peptide nucleic acids, carbohydrates, glycoconjugates, glycoproteins or synthetic variants thereof, organic polymers, inorganic polymers and any other compound where mass spectrometry has been applied.

The term "sample" is used herein to denote a portion of an analyte that is subjected to mass spectrometric analysis according to the subject methods. A sample may be volatile (i.e., have a high vapor pressure at STP) or nonvolatile (i.e., have a low or no detectable vapor pressure at STP). In the latter case, vaporization of the sample is accomplished prior to or accompanying ionization.

A sample may be embedded in a solid or liquid matrix comprising a small, light absorbing compound, adsorbed on a porous or non-porous solid surface (e.g., glass, powder, stainless steel, polymeric film or bead; conductive metal foil, wire or rod) or a liquid surface (e.g., water, blood), or attached to an insoluble support (e.g., microtiter plate, petri dish, microspheres or particles) such that the sample is cleavable by chemical, enzymatic or photolytic methods prior to analysis.

The term "carrier gas" is used herein to refer to a gas that is condensed or adsorbed onto a sample for mass spectrometric analysis according to the subject methods. Properties of a carrier gas useful in the present invention include being substantially inert to the analytical conditions. Exemplary carrier gases include inert gases, such as argon and krypton, oxygen, nitrogen, carbon dioxide and carbon monoxide. Experiments show that many molecules can be used and exhibit suitable properties as the carrier gas in this disclosure. Therefore, the present list of possible carrier gasses is by no means comprehensive. They are given only as examples of typical gases that can be used. The disclosure intends to include any new carrier gases that may be used exhibiting the property of rapidly cooling the newly formed ions in the mass spectrometric application.

The terms "ion," is used herein to refer to a charged species produced by ionization of a sample, wherein a sample molecule either loses or gains a charged particle, such as an electron, a proton or any other charge carrier.

The terms "parent ion" and "molecular ion" and the like are used interchangeably herein to refer to a charged species produced by ionization of a sample, and the resulting ions which possess a mass-to-charge ratio equal to the mass of the neutral (for singly charged ions) or some integral fraction thereof (m/(z n), where n is an integer) for multiply charged species.

The term "ion fragmentation" is used herein to mean the formation of lighter ions from initially formed parent ions having a range of different masses. Ion fragmentation may occur during ionization, ion acceleration or ion flight from the mass analyzer to the ion detector.

The term "expansion" is used herein to refer to an increase in the volume of a composition in the vapor phase, where the composition may contain one or a more components. Molecular beam expansion is carried out by admitting a composition at high pressure (typically 0.1 10 atmospheres) into a vacuum system through a very small inlet or aperture, e.g., a pinhole nozzle, slit nozzle, pulsed valve and the like. The intensity of an expansion is characterized as strong, moderate or weak, depending on conditions, including the backing pressure, and the driving voltage and diameter of the aperture, of the expansion. For a strong expansion, adiabatic cooling accompanying the expansion typically lowers the sample temperature to about 10K and densities are sufficient to form clusters of rare gas atoms, e.g. Argon. For weak expansions, cooling is not as effective and densities are not as high, so that condensation of hydrogen bonded species (e.g. water) is still readily effected, but Argon containing clusters are not formed. GENERAL ASPECTS OF THE INVENTION

The invention provides a method whereby molecules which could not previously be analyzed by mass spectrometry, due to low efficiency for parent ion formation, can be analyzed. In particular many larger molecules which have substantially no detectable vapor pressure at STP can be analyzed via the methodology of the present invention. The method comprises forming a molecular beam by expanding a composition of a sample and a carrier gas so that the carrier gas condenses on the sample. The sample is then ionized and the ions produced are detected and analyzed. The rapid evaporation of the condensed carrier gas reduces ion fragmentation by rapidly removing energy from the newly formed parent ion, making possible a simpler and often much more meaningful analysis (in terms of the resolution of useful understandable data) of the ions of the sample. The performance of a mass spectrometer is defined by factors which include, but are not limited to, mass accuracy, mass resolution and mass range. Mass accuracy is the measure of error in designating a mass to an ion signal. Mass resolution is the measure of the ability of a mass spectrometer to produce separate signals from ions of similar mass. Mass range, or the range of masses that can be accurately detected, is often limited by the ionization method. The current mass range for MALDI-TOF mass spectrometry is from 1 to 1000 kD (Nelson et a1. (1995) Rapid Commun. Mass Spectrom. 9:625).

For many applications of mass spectrometry, e.g. MALDI, both mass accuracy and resolution degrade significantly as the mass of the analyte increase. For example, this is significant above approximately 30 kD for MALDI of oligonucleotides. One important cause of reduced mass resolution and range is ion fragmentation. The methods of the present invention have broad application to the mass spectrometric analysis of high mass analytes that are prone to fragment upon ionization using currently available ionization techniques.

The relative importance of the various factors defining overall performance in mass spectrometry depends primarily on the type of sample, but generally several parameters must be specified and simultaneously optimized to obtain satisfactory performance for a particular application. These parameters may be varied for optimal resolution in the method of the invention, which would be obvious to one skilled in the art upon reading the present disclosure. The invention can be better understood by reference to drawings where Figure 1 is a schematic overview of a molecular beam photoionization reflection TOF mass spectrometer useful in the present invention. A pulsed source expands a mixture of sample and carrier gas into the source chamber. After the molecular beam is skimmed, it enters the ion source of the mass spectrometer. Sample is ionized using XUV radiation, produced by frequency tripling the second harmonic of a pulsed dye laser in a pulsed jet. The XUV light thus obtained, is captured by a quartz capillary and guided to the ion source. The resulting ions are extracted by the applied electric fields into the drift tube. A mass gate mounted in this tube can select part of the spectrum. Parent and daughter ions are separated in time in the reflection (respective trajectories indicated by the dashed and by the dotted line) and are detected on a MCP detector after passing the second field free region.

The results of using such a system are shown in Figure 2A which is a mass spectrum as recorded using a weak H2O/Ar expansion. In Figure 2B a small part of the same spectrum as displayed in Figure 2A, starting at the p31 shows a clear progression of "triplets", separated by 18 amu. The first peak of each group is the parent pn ion for n=31-36. The second and third peak are daughter ion peaks, produced by loss of one and two water monomers from the protonated parent ion, respectively. Figure 2C shows the mass spectrum recorded under strong expansion conditions. An extra peak with a mass that is one amu less than that of the pn parent is observed. For comparison, a vertical dashed line indicates the position of the p31. The low mass member of each quartet is assigned as the "unprotonated" water cluster. The structure of these ions is best described as a solvated H3O+ ion with an OH radical caged somewhere in the cluster, i.e. H3O+ (H2O)kCOH (k=29-34). The small peaks between the quartets are the same unprotonated ions with varying numbers of Ar atoms attached to them.

Figure 3 is a graph showing the relative abundance of unprotonated parent ions (solid line) and daughter ions produced by loss of OD from these parents (long-dashed line) or by loss of OD followed by loss of D20 from the same parents (short-dashed line). The horizontal axis refers to the cluster size of the parent ion.

Figures 4A and 4B are graphs which show the suppression of fragmentation in mass spectrometry carried out on octane. Figure 4A shows the photoionization mass spectrum of normal octane which is obtained from a conventional room temperature-sample. Figure 4B shows the mass spectrum obtained under rapid evaporative cooling conditions. By comparing Figure 4A with Figure 4B it is clear that there is a substantial increase in parent ion signal. Thus, a comparison of Figures 4A and 4B demonstrated how the rapid evaporative cooling process for the present invention (shown in 4B) decreases ion fragmentation and provides a clear signal for the user to read.

Figures 5A and 5B also demonstrate the improved results which can be obtained from the present invention. In Figure 5A a graph is shown of a mass spectrum wherein rapid evaporative cooling is carried out using N2 as a carrier gas. In Figure 5B molecular beam ionization is carried out using Neon as a carrier gas. A clearer signal with less ion fragmentation shown in Figure 5A as compared to Figure 5B.

MOLECULAR BEAM EXPANSION

In the present invention, a sample is mixed with a carrier gas to form a composition that is expanded into a vacuum chamber to form a free jet, or beam, of sample molecules having the carrier gas adsorbed, or condensed, thereon as a result of the cooling of sample molecules during the expansion process. The sample molecules having the carrier gas condensed thereon are carried along, or entrained, in the molecular beam in a manner such that fragment-producing collisions are minimized or excluded.

Molecular beam expansion may be accomplished at ambient or high pressure. Preferred in the methods of the invention is a supersonic molecular beam expansion at high pressure through a pulsed valve. The molecular beam may be passed through one or more additional, smaller apertures, or skimmers, prior to sample ionization to narrow the velocity distribution of the molecules in the beam. Preferred for this invention is the expansion of gas through a 1 mm diameter orifice with backing pressures above one atmosphere. Application of the principles of the present invention may be important over a broader range of orifice sizes as small as 0.001 inch in diameter or as large as 5 mm. Pressure ranges from as low as 0.1 atmosphere to as high as 50 atmospheres may also serve to implement the principles of the present invention.

Figure 1 is a schematic overview of a molecular beam photoionization reflection TOF mass spectrometer 1 useful in the present invention. A pulsed source 2 expands a mixture of sample and carrier gas into the source chamber. After the molecular beam is skimmed in a skimmer 3, it enters the ion source 4 of the mass spectrometer. Sample is ionized using XUV radiation, produced by frequency tripling out of the tripling valve 5 the second harmonic of a pulsed dye laser 6 in a pulsed jet. The XUV light this obtained, is captured by a quartz capillary 7 and guided to the ion source 4. The resulting ions are extracted by the applied electric fields into the drift tube. A mass gate 8 mounted in this tube can select part of the spectrum. Parent and daughter ions are separated in time in the reflectron 9 (respective trajectories indicated by the dashed and by the dotted line) and are detected on a MCP detector 10 after passing the second field free region.

SAMPLE IONIZATION lonization of a sample in a molecular beam, said sample having a carrier gas adsorbed, or condensed, thereon, is accompanied by rapid evaporation of the adsorbed carrier gas. Evaporation of the carrier gas removes energy from the ions formed, thereby cooling the ions and reducing or suppressing ion fragmentation, lonization techniques useful in the present invention include, but are not limited to, laser desorption/ionization and matrix- assisted laser desorption/ionization (MALDI), multiphoton laser ionization, electron capture ionization, single photon ionization. Laser desorption/ionization is used to ionize nonvolatile samples. A pulse of laser radiation desorbs the sample directly by means of vaporization or microscopic ablation of the sample molecules and produces a gaseous plume of intact ions. Lasers suitable for use in the laser desorption/ionization include pulsed UV and IR lasers, e.g., nitrogen lasers, excimer lasers, frequency-doubled excimer-pumped dye lasers, and Q-switched frequency- tripled and quadrupled Nd.YAG lasers.

Direct laser desorption typically employs sufficiently short pulses (frequently less than 10 nanoseconds) to minimize temporal uncertainty. However, in some cases, ion generation may continue for some time after the laser pulse terminates causing loss of resolution due to temporal uncertainty. Also, in some cases, the laser pulse generating the ions is much longer than the desired width of mass spectral peaks (for example, several IR lasers). The longer pulse length can limit mass resolution. The performance of laser desorption/ionization may be substantially improved by the addition of a small organic matrix molecule to the sample material, that is highly absorbing, at the wavelength of the laser. The matrix facilitates desorption and ionization of the sample. In matrix-assisted laser desorption/ionization (MALDI), the sample is embedded in either a solid or liquid matrix comprising a small, highly absorbing compound, adsorbed on a surface or covalently attached to a support, as known in the art. MALDI uses a beam of laser radiation tuned to an absorption band of the matrix to desorb a nonvolatile sample indirectly. The matrix compound absorbs energy at the wavelength of the laser and used the energy to eject and ionize the embedded sample molecules. Representative matrix compounds include sinapinic acid, 2,5-hydroxybenzoic acid, D-cyano-4-hydroxycinnamic acid, gentisic acid, dithranol, 2-amino-4- methyl-5-nitropyridine, 2-amino-5-nitropyridine, 6-aza-2-thiothymine, caffeic acid, 3-hydroxypicolinic acid, nicotinic acid, 2,4,6-trihydroxyacetophenone and 3-hydroxy-4-methoxybenzaldehyde. MALDI is of particular interest in the mass analysis of large, nonvolatile analytes such as nucleic acids.

In a preferred embodiment, the sample is ionized by single photon ionizaiton where the laser radiation is vacuum ultraviolet radiation (VUV) or by multiphton ionization or by election capture ionization. MASS ANALYSIS

The ions generated by ionization of a sample according to the methods of the invention are accelerated in a field and separated according to their mass-to-charge ratio (m/z) in a mass analyzer. Mass analysis techniques useful in the present invention include time-of-flight (TOF), magnetic sector, Fourier-transform ion cyclotron resonance, quadrupole mass filter, and quadrupole ion trap electric sector, combined magnetic/electric sector instruments and any combination of these or other mass spectrometries in the many so-called "hyphenated mass spectrometries." One embodiment of the present invention employs TOF mass analysis, in which ions are separated by measuring their time of flight from the ion source to an ion detector. The ions travel through an electric field-free region in a vacuum with velocities corresponding to their respective mass-to charge ratios (m/z). Accordingly, smaller m/z ions will travel through the vacuum region faster than the larger m/z ions, thereby causing a separation.

TOF mass analysis takes only microseconds, and its mass range is limited only by the ionization technique employed and the detector efficiency. Exemplary TOF mass spectrometers useful in the present invention are disclosed in U.S. Patent Nos. 5,045,694; 5,160,840 and 5,627,369 and incorporated herein by reference.

The flight path of an ion in a TOF mass analyzer may be linear or reflective (Mamyrin et al. (1973) Sov. Phys. JETP) 37:45-48). In a reflection TOF mass analyzer, ions with the same m/z are separated by their difference in kinetic energy. An ion reflector (or reflection or ion mirror) is positioned at the end of the free-flight region and consists of one (single-stage) or more (e.g., dual stage) homogeneous, retarding electrostatic fields. As the ions penetrate the reflector, they are decelerated until the velocity component in the direction of the field becomes zero. Then, the ions reverse direction and are accelerated back through the reflector. The ions exit the reflector with energies identical to their incoming energy but with velocities in the opposite direction. Ions with larger energies penetrate the reflector more deeply and consequently will remain in the ion reflector for a longer time. In a properly designed reflector, the potentials are selected to modify the flight paths of the ions such that ions of like mass and charge arrive at the detector at the same time regardless of their initial energy. Furthermore, lighter ions produced by metastable decay of parent ions in the field free region can be separated in time. In linear TOF mass spectrometry, ions are extracted by an electric field gradient and move quickly into a long field free region followed by an ion detector. All ions possess nominally identical kinetic energies and their velocities (and therefore their arrival times at a detector) are determined by their mass to charge ratio. Linear TOF mass analysis is preferred in the present invention.

MASS DETECTION

The separated ions collide with a detector that generates a signal as each set of ions of a particular mass-to-charge ratio strikes the detector. In a preferred embodiment of the invention, the ion detector is a multichannel plate

(MCP) detector. An exemplary MCP detector suitable for use in the practice of the present invention is a double-staged MCP detector.

APPLICATIONS The methods of the invention are useful for a variety of applications, including genomics, proteomics, sequencing, identification, combinatorial chemistry, and the determination of natural and synthetic polymers. Nucleic acids, polynucleotides, proteins peptides, peptide nucleic acids carbohydrates, glycoconjugates, glycoproteins or synthetic variants thereof, and inorganic polymers or nanoparticles may be analyzed according to the methods disclosed herein. Aspects, advantages and features of these methods are demonstrated by the following examples.

EXAMPLES The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what, the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Example 1 : Suppression of the Fragmentation of Water Cluster Ions

Molecular beam expansion. A mixture of room-temperature water vapor and carrier gas (total backing pressure between 2 and 6 bar) is expanded through a pulsed (200 Ds duration) solenoid valve (General Valve). The standard orifice of the valve is replaced with a slowly diverging conical nozzle (smallest diameter: 0.8 mm) to enhance water cluster formation. The gas mixture is expanded into the source chamber (pumped by an 1100 L/s turbomolecular pump). Under these conditions the cooling during the expansion allows efficient production of clusters containing up to ~80 water monomers. After passing a 0.5 mm diameter skimmer, the molecular beam enters the ionization region (pumped by a 400 L/s turbomolecular pUmp) of a reflection TOF mass spectrometer (R.M. Jordan Co.). Sample ionization. Single-photon ionization of the water clusters is performed with vacuum-ultraviolet (VUV), radiation produced as follows: the output of a Nd:YAG laser (Continuum, PL7010). pumped dye laser (Spectra Physics, PDL-1) running on Fluorescein 548 is frequency doubled in a KDP crystal, providing radiation around 277 nm with an energy of ~15 mJ/pulse in a 0.7 cm-1 bandwidth. The second harmonic is separated from the fundamental of the dye laser and guided into a tripling chamber. The laser is focused close to the orifice of a pulsed beam of pure N2 (General Valve, backing pressure 10 bar) by a lens with a 25 cm focal length. This results in frequency tripling of the 277 nm radiation, which is enhanced by tuning the laser to a well-known two-photon resonance in molecular nitrogen (Page, R.H. et al (1987) Rev. Sci. Instrum. 58:1616). The VUV radiation (92.5 nm/13.4 eV) is captured by a 37.5 cm long quartz capillary with an inner diameter of 2 mm that guides the VUV radiation (and a large fraction of the remaining 277 nm radiation) to the ion source of the mass spectrometers (Tonkyn, R.G. et al. (1989) Rev. Sci. Instrum. 60:1245). A slotted hole is placed about halfway down the capillary so that most of the tripling gas can be pumped out of the capillary into a differentially pumped (70 L/s turbomolecular pump) bridging chamber, thus reducing the gas load of the tripling chamber to the ion source of the mass spectrometer. The capillary provides efficient transfer of the VUV light to the ionization region as well as a > 106 pressure reduction between the tripling chamber and the ionization region. Thus, under typical operating conditions, the tripling chamber exhibits a pressure of -10-2 Torr (pumped by a 400 Us turbo drag pump) while the ionization chamber exhibits a pressure of less than 10-8 Torr. The water clusters are ionized between the first two extraction plates (10 mm apart) of the double electric field ion source (Wiley, W.C. et al. (1955) Rev. Sci. Instrum. 26:1150) of the mass spectrometer.

Ion mass analysis. Ionized water clusters are deflected into the drift tube of the mass spectrometer. The distance from the ion source to the first grid of the reflection is 1.1 m. The distance from this same grid to the detector, defining the second field-free region, is approximately 0.6 m. A mass gate is mounted in the first field-free region. A voltage difference of 400 V can be applied between the plates of the mass gate, rejecting (i.e., deflecting) the majority of the masses. Only a narrow part of the spectrum is transmitted when the applied voltage is pulsed to ground during a certain time interval. Unless stated otherwise, this mass gate is not used (i.e., grounded).

Ion detection. Different masses are detected according to their arrival time on a double-staged 40 mm diameter microchannel plate (MCP) detector. The ion signal detected by the MCP detector is fed into a digitizing oscilloscope with a 10 bit vertical resolution and a 100 MHZ sampling rate (LeCroy 9430). The signal is summed over a couple of thousands of laser shots in the 16 bit memory of the oscilloscope and subsequently read out by a PC via a GPIB interface. Triggering of the laser, tripling valve, pulsed source, and oscilloscope is regulated by a delay generator (Stanford Research Systems, DG535).

Suppression of ion fragmentation. Figure 2A shows a typical mass spectrum as recorded under modest expansion conditions using Ar as a carrier gas (backing pressure: 2 bar). A long progression of mass features, up to ~1350 amu, resulting from neutral clusters containing up to about 75 water molecules, is readily observed. A small part of this same spectrum (greatly expanded) is shown in Figure 2B. The spectrum appears as a series of "triplets". The protonated parent ion p31 (i.e., H+(H2O)31 produced by intracluster proton transfer and subsequent loss of the OH) is labeled. The low-mass members of each triplet are the pn ions (n = 31-36). The most intense member of each triplet corresponds to protonated ions that have evaporated one water molecule during PSD (pn D pn-1 + H2O, where n = 32- 36). The high-mass member of each triplet corresponds to evaporation of two water molecules during PSD (pn D pn-2 + 2H2O, where n = 33-37). The rate constant for loss of each additional water monomer decreases rapidly as, for larger water clusters, each evaporated water molecule removes between 0.27 and 0.44 eV from the cluster (Shi, Z. et al. (1993) J. Chem Phys. 99:8009; Magnera, FT. et al. (1991) J. Chem. Phys. Lett. 182:363). The low pressure in the field-free drift tube (under these conditions below 3 x 10-8 Torr) almost certainly excludes collisions of cluster ions with background gas, and the observed metastable decay is due to unimolecular decomposition of the energized water cluster ions. Figure 2C also shows evidence of fragmentation during the time where the ion is accelerated in the ion source, referred to as "in source decay" (ISD). In source decay (ISD) appears as an asymmetric peak shape with a tail to longer flight times (higher masses when converted to the mass scale). The tail terminates on the metastable peak to which it correlates in PSD (Kuhlewind, H. et al. (1986) J. Chem Phys. 85:4427). Such asymmetric peak shapes are clearly evident for the pn, family of mass features. Although observable, ISD is only a minor contribution to the total fragmentation.

It should be noted that the mass scale that is mdicated on the horizontal axis is only valid for the parent ions. Their flight times are proportional to Dm. Because the mass of the daughter ions changes in the drift tube, this relation is no longer valid, and the horizontal scale is not correct for them. The time delay between parent and daughter ions, however, can be exactly calculated since both geometry of and voltages applied to the reflection are known.

When backing pressure and driving voltage of the pulsed source are increased (thereby increasing the intensity of the gas pulse), additional families of features are observed in the mass spectrum (Figure 4). Unlike Figure 3, this mass spectrum is composed of a series of strong quartets with additional weak mass features between the quartets. The low-mass members of the quartets appear 1 amu lower than the pn family. (For comparison, a vertical dashed line is indicated in FIGS. 2b and 2c.) This family of mass features nominally has the formula (H2O)n+ and is formally designated un ("unprotonated" cluster ions with n H2O molecules).

Figure 3 shows the results of fragmentation analysis for deuterated water clusters. Analysis of the data from the Reflection experiments for per- Deuterated water clusters allowed the determination of fragmentation fractions and decay mechanisms of the "unprotonated" water cluster ions. The results of these experiments are shown in Figure 3. The "unprotonated" water cluster ions which have been subjected to rapid evaporative cooling decay exactly as newly ionized water clusters, i.e. loss of hydroxyl radical, in this case loss of deuterated hydroxyl radical. However, the fraction of decay shown here, indicates that, for example, only about 20% of the parent ion of the n=15 cluster ion has fragmented after the flight time through the mass spectrometer. This flight time (~50 Ds) is about 10,000 longer than the decay time of unprotonated" water cluster ions that have not been subjected to rapid evaporative cooling. These experiments give empirical evidence that the fragmentation rates have been reduced by about a factor of 10,000. Example 2: Suppression of the Fragmentation of normal-Octane Ions

This Example demonstrates the application of rapid evaporative cooling mass spectrometry to the suppression of fragmentation in n-octane (C8H18). This compound fragments dramatically under many ionization conditions. For example, Figure 4A shows the photoionization (D=92.5 nm) mass spectrum measured by a linear time-of-flight machine. The parent ion makes up only about 8% of the total ions (Figure 4A) in the mass spectrum. The numbers above the mass features give the mass-to-charge ratio in atomic mass units (equivalently Dalton) corresponding to that peak. The parent mass is labeled 114. The many other mass features correspond to breaking the long chain hydrocarbon at one of the seven C-C bonds. Under rapid evaporative cooling conditions more than 70% (Figure 4B) of the ions present in the spectrum are parent ions. Figures 5A and 5B show a comparison between photoionization mass spectra carried out with N2 (Figure 5A) carrier gas (where rapid evaporative cooling is possible) and Neon (Figure 5B) carrier gas (where rapid evaporative cooling is impossible). With N2, it is possible to pre-adsorb the carrier gas to the. octane molecules under these conditions, whereas with Neon it is impossible. This is simply due to the fact that Neon is much more weakly bound to Octane than is N2. The enhancement in the parent ion signal with Rapid evaporative cooling is clearly seen.

The instant invention is shown and described herein in a manner, which is considered to be the most practical and preferred embodiments. It is recognized, however, that departures may be made there from which are within the scope of the invention and that obvious modifications will occur to one skilled in the art upon reading this disclosure.

Claims

What is claimed is:

1. A method of reducing ion fragmentation in mass spectrometiy analysis, comprising: forming a molecular beam by expanding a composition comprising a sample and a carrier gas in a manner which condenses said carrier gas with said sample; ionizing said sample of said molecular beam to produce ions; analyzing the mass of said ions; and detecting said ions; wherein the fragmentation of said ions is effectively reduced and wherein the sample has substantially no detectable vapor pressure at STP.

2. The method of claim 1 , wherein the sample is comprised of naturally occurring biological polymer or fragment thereof.

3. The method of claim 1 , wherein the sample is selected from the group consisting of a nucleic acid, methylated nucleic acid, methylated polynucleotide, polynucleotide, oligonucleotide, methylated-oligonucleotide, protein, peptide, peptide nucleic acid, carbohydrate, glycoconjugate, glycoprotein, a synthetic variant thereof, an inorganic polymer, an organic polymer, and a nanoparticle.

4. The method of claim 2, wherein the biological polymer is a nucleotide sequence.

5. The method of claim 1 , wherein the sample is embedded in a matrix.

6. The method of claim 5, wherein the matrix is a dye compound.

7. The method of claim 5, wherein the matrix is a dye compound diluted in a solvent.

8. The method of claim 5, wherein the matrix is a dye compound diluted in a frozen solvent.

9. The method of claim 5, wherein the matrix is a frozen solvent.

10. The method of claim 5, wherein the matrix is selected from the group consisting of sinapinic acid, 2,5-hydroxybenzoic acid, D-cyano-4- hydroxycinnamic acid, gentisic acid, dithranol, 2-amino-4-methyl-5- mtropyridine, 2-amino-5-nitiopyridine, 6-aza-2-thiothymine, caffeie acid, 3- hydroxypicolinic acid, nicotinic acid, 2,4,6-thhydroxyacetophenone and 3- hydroxy-4-methoxybenzaldehyde.

11. The method of claim 1 , wherein the sample is adsorbed on a surface.

12. The method of claim 1 , wherein the mass analysis is carried out using a technique selected from the group consisting of time-of-flight (TOF), magnetic sector, electric sector, ion cyclotron resonance, quadrupole mass filter, and quadrupole ion trap.

13. The method of claim 7, wherein the mass analysis is carried out by time-of-flight analysis.

14. The method of claim 1 , wherein the mass of said ions is analyzed by a reflection time-of-flight mass analyzer.

15. The method of claim 1 , wherein the carrier gas is a gas with vapor pressure greater than 1 Torr at STP.

16. The method of claim 1 , wherein the carrier gas is selected from the group consisting of argon, krypton, oxygen, nitrogen, carbon dioxide and carbon monoxide, NO, HCI, HBr, SO2, SF6, H2O, CH4, C2H6, and C3H8.

17. The method of claim 1 , wherein the carrier gas is argon.

18. The method of claim 1 , wherein the molecular beam is formed by expanding said composition at high pressure.

19. The method of claim 1 , wherein the molecular beam is a supersonic molecular beam.

20. The method of claim 1 , wherein the sample is vaporized by laser desorption.

21. The method of claim 1 , wherein the sample is vaporized by matrix-assisted laser desorption.

22. The method of claim 1 , wherein the composition is ionized using ultraviolet radiation.

23. The method of claim 1 , wherein the sample is ionized by laser desorption/ionization.

24. The method of claim 1 , wherein the sample is ionized by matrix- assisted laser desorption/ionization.

25. The method of claim 1 , wherein the sample is ionized using multi-photon laser ionization.

26. The method of claim 1 , wherein the sample is ionized using electron capture.

27. The method of claim 25, wherein election capture is performed with an electron gun.

28. The method of claim 25, wherein electron capture is performed with laser generated elections.

29. The method of claim 1 , wherein the ions are detected by an electron multiplier

30. The method of claim 1 , wherein the ions are detected by an election multiplier with coversion dynode.

31. The method of claim 1 , wherein the ions are detected by a microchannel plate detector.

32. The method of claim 1 , wherein the ions are detected by a microsphere plate detector.

33. An apparatus for mass spectrometiy analysis, comprising: a means for forming a molecular beam by expanding a composition comprising a sample and a carrier gas in a manner which condenses said carrier gas on the sample; a means for ionizing said sample of said molecular beam to produce ions; a means for analyzing the mass of said ions; and a means for detecting said ions.

34. The apparatus of claim 33, wherein the molecular beam forming means is pulsed or continuous.

35. The apparatus of claim 33, wherein the molecular beam forming means is a pulsed source.

36. The apparatus of claim 33, wherein the molecular beam forming means is a pulsed solenoid valve.

37. The apparatus of claim 33, wherein the molecular beam forming means is a pulsed piezoelectric valve.

38. The apparatus of claim 33, wherein the ionizing means is a laser radiation.

39. The apparatus of claim 33, wherein the ionizing means is a pulsed laser.

40. The apparatus of claim 33, wherein the ionizing means is a continuous laser.

41. The apparatus of claim 33, wherein the ionizing means uses ultraviolet radiation.

42. The apparatus of claim 33, wherein the ionizing means uses visible or infrared radiation.

43. The apparatus of claim 33, wherein the ionizing means uses electron capture.

44. The apparatus of claim 33, wherein the ionizing means uses electron capture with an election gun.

45. The apparatus of claim 33, wherein the ionizing means uses electron capture with laser generated electrons.

46. The apparatus of claim 33, wherein one or more skimmers are positioned between the molecular beam forming means and the ionizing means.

47. The apparatus of claim 33, including a delay generator.

48. The apparatus of claim 33, wherein the analyzing means is a time-of-flight mass analyzer, a magnetic sector mass analyzer, an ion cyclotron resonance mass analyzer, a quadrupole mass filter mass analyzer or a quadrupole ion trap mass analyzer.

49. The apparatus of claim 33, wherein the analyzing means is a time-of-flight mass analyzer.

50. The apparatus of claim 33, wherein the analyzing means is a reflection time-of-flight mass analyzer.

51. The apparatus of claim 33, wherein the detecting means is an election multiplier.

52. The apparatus of claim 33, wherein the detecting means is an electron multiplier with a conversion dynode.

53. The apparatus of claim 33, wherein the detecting means is a microchannel plate detector.

54. The apparatus of claim 33, wherein the detecting means is a microsphere plate detector.