External shutter for electrospray ionization mass spectrometry Number:6,828,550 from the United States Patent and Trademark Office (PTO) owispatent Novel methods and apparatuses for mass spectrometry are disclosed wherein a time slice of ions are selectively accumulated in an ion reservoir of a mass spectrometer and subsequently are allowed to undergo an ion-molecule reaction with a reactive species or are dissociated with coherent radiation prior to mass analysis. These methods and apparatuses are amenable to mass spectrometric analysis of singly or multiply charaged ions of peptides, proteins, carbohydrates, oligonucleotides, nucleic acids and small molecules as prepared by combinatorial or classical medicinal chemistry.<H spectrometry, electrospray, External, shutte, 6828550.html, Patent, pto, 6828550

Title: External shutter for electrospray ionization mass spectrometry

Abstract: Novel methods and apparatuses for mass spectrometry are disclosed wherein a time slice of ions are selectively accumulated in an ion reservoir of a mass spectrometer and subsequently are allowed to undergo an ion-molecule reaction with a reactive species or are dissociated with coherent radiation prior to mass analysis. These methods and apparatuses are amenable to mass spectrometric analysis of singly or multiply charaged ions of peptides, proteins, carbohydrates, oligonucleotides, nucleic acids and small molecules as prepared by combinatorial or classical medicinal chemistry.

Patent Number: 6,828,550 Issued on 12/07/2004 to Griffey, et al.

Inventors: Griffey; Richard (Vista, CA); Hofstadler; Steven (Oceanside, CA)
Assignee: ISIS Pharmaceuticals, Inc. (Carlsbad, CA)

Appl. No.: 459791

Filed: June 12, 2003

Current U.S. Class: 250/281 ; 250/287; 250/288; 250/292

Field of Search: 250/281,287-292

References Cited [Referenced By]

U.S. Patent Documents


4238678	December 1980	Castleman et al.
5436446	July 1995	Jarrell et al.
5545304	August 1996	Smith et al.
5616918	April 1997	Oishi et al.
5811800	September 1998	Franzen et al.
6124592	September 2000	Spangler

Foreign Patent Documents


3226803	Jul., 1982	DE

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Primary Examiner: Wells; Nikita
Assistant Examiner: Hashmi; Zia R.
Attorney, Agent or Firm: Cutler; Wilmer Pickering Hale and Dorr LLP

Government Interests

This invention was made with United States Government support under NIST Contract 97-0025. The United States Government has certain rights in the invention.

Parent Case Text

CROSS REFERENCED TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No. 09/332,685, filed Jun. 14, 1999, pending, the entirety of which is incorporated herein by reference.

Claims

What is claimed:

1. A system for processing ions, comprising: an ion source for generating ions within a first space; a vacuum chamber which forms a second space, said vacuum chamber comprising a gate electrode having an outlet opening and a wall having an inlet opening; said vacuum chamber being maintained at a lower uressure than said first space so that gas contained within said first space flows from said first space into said vacuum chamber when said inlet opening is unobstructed; an ion reservoir disposed within said vacuum chamber, wherein said ion reservoir is capable of maintaining ions within said vacuum chamber; an inlet shutter, wherein said inlet shutter can block said inlet opening to prevent said ions and said gas from entering said vacuum chamber; and an outlet shutter, wherein said outlet shutter can block said outlet opening to prevent ions from exiting said vacuum chamber.

2. The system of claim 1, wherein said ion reservoir comprises at least one of the following: a rf-multipole ion reservoir, an electrostatic lens ion reservoir and a jet expansion ion reservoir.

3. The system of claim 1, wherein said ion reservoir comprises at least one of the following: a Paul ion trap and a Penning ion trap.

4. The system of claim 1, further comprising: an electrode having an orifice, wherein said electrode is disposed within said vacuum chamber between said inlet opening and said ion reservoir.

5. The system of claim 1, wherein said ion source comprises at least one of the following: an electron impact (EI) ionization source, an electrospray ionization (ESI) source, a chemical ionization (CI) source and a matrix-assisted laser desorption ionization (MALDI) source.

6. The system of claim 1, further comprising: a gas source having a reactant gas, wherein said gas source is in fluid communicate with said vacuum chamber and wherein, upon the introduction of said reactant gas into said vacuum chamber, at least a portion of said ions maintained within said vacuum chamber react with said reactant gas to form product ions.

7. The system of claim 6, wherein said reactant gas comprises at least one of the following: gaseous molecules and gaseous ions.

8. The system of claim 1, further comprising: a laser source in operative association with said vacuum chamber, wherein said laser source dissociates at least a portion of said ions maintained within said vacuum chamber to form fragment ions.

9. The system of claim 8, wherein said laser source comprises an infrared laser source.

10. The system of claim 1, further comprising: an inlet tube having a capillary disposed therethrough, said inlet tube being disposed within said inlet opening of said vacuum chamber.

11. The system of claim 1, wherein said ion reservoir acts as an ion desolvating chamber.

12. The system of claim 1, wherein said ion reservoir acts as an ion collision chamber.

13. The system of claim 1, wherein said inlet shutter includes a seal, and wherein said seal of said inlet shutter can form a fluid-tight seal around said inlet opening between said wall and said inlet shutter.

14. The system of claim 13, wherein said outlet shutter includes a seal, and wherein said seal of said outlet shutter can form a fluid-tight seal around said outlet opening between said gate electrode and said outlet shutter.

15. The system of claim 1, further comprising a mass analyzer for analyzing ions, wherein said mass analyzer is located downstream of said vacuum chamber.

16. The system of claim 15, wherein said mass analyzer comprises at least one of the following: a magnetic sector mass analyzer, a Fourier transform mass spectrometry mass analyzer, a time-of-flight mass analyzer, a multipole mass analyzer or an ion trap mass analyzer.

17. The system of claim 1, further comprising: a first actuator coupled to said inlet shutter, wherein said first actuator is capable of positioning said inlet shutter so as to block said inlet opening; and a second actuator coupled to said outlet shutter, wherein said second actuator is capable of positioning said outlet shutter so as to block said outlet opening.

18. A mass spectrometry system, comprising: an ion source for generating ions within a first space; a first vacuum chamber which forms a second space, said first vacuum chamber comprising a gate electrode having an outlet opening and a wall having an inlet opening, wherein said ions can be directed from said first space into said first vacuum chamber via said inlet opening; said vacuum chamber being maintained at a lower pressure than said first space so that buffer gas contained within said first space flows from said first space into said vacuum chamber when said inlet opening is unobstructed; an ion reservoir disposed within said first vacuum chamber, wherein said ion reservoir is capable of maintaining ions within said first vacuum chamber; an inlet shutter, wherein said inlet shutter can block said inlet opening to prevent said ions and said buffer gas from entering said first vacuum chamber; an outlet shutter, wherein said outlet shutter can block said outlet opening to prevent ions from exiting said first vacuum chamber; a gas source having a reactant gas, wherein said gas source is in fluid communication with said first vacuum chamber and wherein, upon the introduction of said reactant gas into said first vacuum chamber, at least a portion of said ions maintained within said first vacuum chamber react with said reactant gas to form product ions; and a mass analyzer, wherein said mass analyzer is disposed within a second vacuum chamber.

19. The mass spectrometry system of claim 18, wherein said reactant gas comprises at least one of the following: gaseous molecules and gaseous ions.

20. The mass spectrometry system of claim 18, wherein said ion reservoir comprises at least one of the following: a rf-multipole ion reservoir, an electrostatic lens ion reservoir and a jet expansion ion reservoir.

21. The mass spectrometry system of claim 18, wherein said mass analyzer comprises at least one of the following: a magnetic sector mass analyzer, a Fourier transform mass spectrometry mass analyzer, a time-of-flight mass analyzer, a multipole mass analyzer or an ion trap mass analyzer.

22. A mass spectrometry system, comprising: an ion source for generating ions within a first space; a first vacuum chamber which forms a second space, said first vacuum chamber comprising a gate electrode having an outlet opening and a wall having an inlet opening, wherein said ions generated by said ion source can be directed from said first space into said first vacuum chamber via said inlet opening; said vacuum chamber being maintained at a lower pressure than said first space so that gas contained within said first space flows from said first space into said vacuum chamber when said inlet opening is unobstructed; an ion reservoir disposed within said first vacuum chamber, wherein said ion reservoir is capable of maintaining ions within said first vacuum chamber; an inlet shutter, wherein said inlet shutter can block said inlet opening to prevent said ions and said gas from entering said first vacuum chamber; an outlet shutter, wherein said outlet shutter can block said outlet opening to prevent ions from exiting said first vacuum chamber; a laser source in operative association with said first vacuum chamber; and a mass analyzer, wherein said mass analyzer is disposed within a second vacuum chamber.

23. The mass spectrometry system of claim 22, wherein said laser source comprises an infrared laser source.

24. The mass spectrometry system of claim 22, wherein said laser source dissociates at least a portion of said ions maintained within said first vacuum chamber to form fragment ions.

25. The mass spectrometry system of claim 22, wherein said laser source excites solvent in said first vacuum chamber to vaporize said solvent.

26. A method of processing ions, comprising: providing a vacuum chamber comprising a gate electrode having an outlet opening and a wall having an inlet opening; providing an ion reservoir within said vacuum chamber, wherein said ion reservoir is capable of maintaining ions within said vacuum chamber; providing an inlet shutter, wherein said inlet shutter can block said inlet opening to prevent ions and non-ionized gas from entering said vacuum chamber; providing an outlet shutter, wherein said outlet shutter can block said outlet opening to prevent ions from exiting said vacuum chamber; generating ions within a first space; opening said inlet shutter to allow ions to be directed from said first space into said vacuum chamber; closing said inlet shutter to isolate said ions directed into said vacuum chamber from said first space; maintaining ions in said vacuum chamber for a period of time; and opening said outlet shutter and altering an electrical potential of said gate electrode to release ions from said vacuum chamber.

27. The method of claim 26, wherein ions are continuously generated within said first space.

28. The method of claim 26, further comprising: introducing a reactive moiety into said vacuum chamber for a time sufficient for at least some of said reactive moiety to react with at least some of said ions maintained within said vacuum chamber to form product ions; and releasing said product ions from said vacuum chamber.

29. The method of claim 28, wherein said reactive moiety comprises at least one of the following: gaseous molecules, gaseous ions and plasma.

30. The method of claim 28, wherein said reaction comprises an ion-molecule reaction and said reactive moiety comprises a gas phase deuterated solvent, gas phase acid, a gas phase base or reactive electrophile.

31. The method of claim 28, wherein said reactive moiety comprises a deuterated solvent selected from D.sub.2 O, ND.sub.3 or CH.sub.3 OD.

32. The method of claim 28, wherein said reactive moiety comprises an acid selected from acetic acid, trifluoroacetic acid or hydroiodic acid.

33. The method of claim 28, wherein said reactive moiety comprises a base selected from ammonia, dimethylamine, trimethylamine, N,N,N',N'-tetramethyl-1,8-naphthalenediamine, tetramethyldiamine, imidazole, triethylamine and tripropylamin.

34. The method of claim 28, wherein said reaction comprises an ion-ion reaction and said reactive moiety comprises perfluoro-1,3-dimethylcyclohexane.

35. The method of claim 28, wherein said reactive moiety comprises at least one chemical isotope that is absent from the isotopic species that form the elemental building blocks of said generated ions.

36. The method of claim 35, wherein said chemical isotope is deuterium.

37. The method of claim 26, further comprising: irradiating at least a portion of said ions maintained within said vacuum chamber to form fragment ions; and releasing said fragment ions from said vacuum chamber.

38. The method of claim 26, further comprising: directing said ions released from said vacuum chamber to a mass analyzer.

39. The method of claim 38, wherein said mass analyzer comprises at least one of the following: a magnetic sector mass analyzer, a Fourier transform mass spectrometry mass analyzer, a time-of-flight mass analyzer, a multipole mass analyzer or an ion trap mass analyzer.

40. The method of claim 26, wherein said ions are generated by at least one of the following: an electron impact (EI) ionization source, an electrospray ionization (ESI) source, a chemical ionization (CI) source and a matrix-assisted laser desorption ionization (MALDI) source.

41. The method of claim 26, further comprising: desolvating said ions maintained in said vacuum chamber.

42. The method of claim 26, wherein said generated ions are comprised of protein ions, peptide ions, oligonucleotide ions, nucleic acid ions, or carbohydrate ions.

43. The method of claim 26, wherein said generated ions are comprised of protein ions, peptide ions, oligonucleotide ions, nucleic acid ions, or carbohydrate ions and complexes of said protein ions, peptide ions, oligonucleotide ions, nucleic acid ions, or carbohydrate ions with other molecules that bind to said protein ions, peptide ions, oligonucleotide ions, nucleic acid ions, or carbohydrate ions.

44. The method of claim 26, wherein said ion reservoir comprises at least one of the following: a rf-multipole ion reservoir, an electrostatic lens ion reservoir and a jet expansion ion reservoir.

45. The method of claim 26, wherein said generated ions are generated from a compound obtained via an analytical separation technique.

46. The method of claim 45, wherein said analytical separation technique comprises high pressure liquid chromatography.

47. The method of claim 45, wherein said analytical separation technique comprises capillary electrophoresis chromatography.

48. The method of claim 45, wherein said analytical separation technique comprises capillary electrophoresis.

Description

FIELD OF THE INVENTION

The present invention relates to improved methods and apparatus for mass spectrometry. In particular the invention provides methods and apparatus that allows for an accumulation of a time slice of ions to be stored in an external ion reservoir of a mass spectrometer for subsequent ion-molecule, ion-ion or dissociation reactions. The methods and apparatus of the invention can be used in the analysis of ions of macromolecules including peptides, proteins, carbohydrates, oligonucleotides and nucleic acids as well as small molecules as prepared by combinatorial or classical medicinal chemistry.

BACKGROUND OF THE INVENTION

Mass spectrometry (MS) is a powerful analytical tool for the study of molecular structure and interaction between small and large molecules. The current state of the art in MS is such that sub-femtomole quantities of material can be readily analyzed to afford information about the molecular contents of the sample. An accurate assessment of the molecular weight of the material may be quickly obtained, irrespective of whether the sample's molecular weight is several hundred, or in excess of a hundred thousand, atomic mass units or Daltons (Da). It has now been found that mass spectrometry can elucidate significant aspects of important biological molecules. One reason for the utility of MS as an analytical tool is the availability of a variety of different MS methods, instruments, and techniques which can provide different pieces of information about the samples.

A mass spectrometer analyzes charged molecular ions and fragment ions from sample molecules. These ions and fragment ions are then sorted based on their mass to charge ratio (m/z). A mass spectrum is produced from the abundance of these ions and fragment ions that is characteristic of every compound. In the field of biotechnology, mass spectrometry can be used to determine the structure of a biomolecule. Of particular interest is the ability of mass spectrometry to be used in determining the sequence of oligonucleotides, peptides, and oligosaccharides. Particular mass spectrometric techniques have been used to deduce the sequence of an oligonucleotide (Murray, J. Mass Spec., 1996, 31, 1203-1215). Mass spectrometry is also commonly used for the sequencing of peptides and proteins (Biemann, Annu. Rev. Biochem., 1992, 61, 977-1010).

In principle, mass spectrometers consist of at least four parts: (1) an inlet system; (2) an ion source; (3) a mass analyzer; and (4) a mass detector/ion-collection system (Skoog, D. A. and West, D. M., Principles of Instrumental Analysis, Saunders College, Philadelphia, Pa., 1980, 477-485). The inlet system permits the sample to be introduced into the ion source. Within the ion source, molecules of the sample are converted into gaseous ions. The most common methods for ionization are electron impact (EI), electrospray ionization (ESI), chemical ionization (CI) and matrix-assisted laser desorption ionization (MALDI). A mass analyzer resolves the ions based on mass-to-charge ratios. Mass analyzers can be based on magnetic means (sector), time-of-flight, quadrupole and Fourier transform mass spectrometry (FTMS). A mass detector collects the ions as they pass through the detector and records the signal. Each ion source can potentially be combined with each type of mass analyzer to generate a wide variety of mass spectrometers.

Mass spectrometry ion sources are well-known in the art. Two commonly used ionization methods are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) (Smith et al., Anal. Chem., 1990, 62, 882-899; Snyder, in Biochemical and Biotechnological Applications of Electrospray Ionization Mass Spectrometry, American Chemical Society, Washington, D.C., 1996; and Cole, in Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation, Wiley, N.Y., 1997).

ESI is a gentle ionization method that results in no significant molecular fragmentation and preserves even weakly bound complexes between biopolymers and other molecules so that they are detected intact with mass spectrometry. ESI produces highly charged droplets of the sample being studied by gently nebulizing a solution of the sample in a neutral solvent in the presence of a very strong electrostatic field. This results in the generation of highly charged droplets that shrink due to evaporation of the neutral solvent and ultimately lead to a "coulombic explosion" that affords multiply charged ions of the sample material, typically via proton addition or abstraction, under mild conditions. Electrospray ionization mass spectrometry (ESI-MS) is particularly useful for very high molecular weight biopolymers such as proteins and nucleic acids greater than 10 kDa in mass, for it affords a distribution of multiply-charged molecules of the sample biopolymer without causing any significant amount of fragmentation. The fact that several peaks are observed from one sample, due to the formation of ions with different charges, contributes to the accuracy of ESI-MS when determining the molecular weight of the biopolymer because each observed peak provides an independent means for calculation of the molecular weight of the sample. Averaging the multiple readings of molecular weight so obtained from a single ESI-mass spectrum affords an estimate of molecular weight that is much more precise than would be obtained if a single molecular ion peak were to be provided by the mass spectrometer. Further adding to the flexibility of ESI-MS is the capability of obtaining measurements in either the positive or negative ionization modes.

ESI-MS has been used to study biochemical interactions of biopolymers such as enzymes, proteins and macromolecules such as oligonucleotides and nucleic acids and carbohydrates and their interactions with their ligands, receptors, substrates or inhibitors (Bowers et al., Journal of Physical Chemistry, 1996, 100, 12897-12910; Burlingame et al., J. Anal. Chem., 1998, 70, 647R-716R; Biemann, Ann. Rev. Biochem., 1992, 61, 977-1010; and Crain et al., Curr. Opin. Biotechnol., 1998, 9, 25-34). While interactions that lead to covalent modification of biopolymers have been studied for some time, one of the most significant developments in the field has been the observation, under appropriate solution conditions and analyte concentrations, of specific non-covalently associated macromolecular complexes that have been promoted into the gas-phase intact (Loo, Mass Spectrometry Reviews, 1997, 16, 1-23; Smith et al., Chemical Society Reviews, 1997, 26, 191-202; Ens et al., Standing and Chernushevich, Eds., New Methods for the Study of Biomolecular Complexes, Proceedings of the NATO Advanced Research Workshop, held Jun. 16-20 1996, in Alberta, Canada, in NATO ASI Ser., Ser. C, 1998, 510, Kluwer, Dordrecht, Netherlands).

A variety of non-covalent complexes of biomolecules have been studied using ESI-MS and reported in the literature (Loo, Bioconjugate Chemistry, 1995, 6, 644-665; Smith et al., J. Biol. Mass Spectrom. 1993, 22, 493-501; Li et al., J. Am. Chem. Soc., 1993, 115, 8409-8413). These include the peptide-protein complexes (Busman et al., Rapid Commun. Mass Spectrom., 1994, 8, 211-216; Loo et al., Biol. Mass Spectrom., 1994, 23, 6-12; Anderegg and Wagner, J. Am. Chem. Soc., 1995, 117, 1374-1377; Baczynskyj et al., Rapid Commun. Mass Spectrom., 1994, 8, 280-286), interactions of polypeptides and metals (Loo et al., J. Am. Soc. Mass Spectrom., 1994, 5, 959-965; Hu and Loo, J. Mass Spectrom., 1995, 30, 1076-1079; Witkowska et al., J. Am. Chem. Soc., 1995, 117, 3319-3324; Lane et al., J. Cell Biol., 1994, 125, 929-943), and protein-small molecule complexes (Ganem and Henion, ChemTracts-Org. Chem., 1993, 6, 1-22; Henion et al., Ther. Drug Monit., 1993, 15, 563-569; Ganguly et al., Tetrahedron, 1993, 49, 7985-7996, Baca and Kent, J. Am. Chem. Soc., 1992, 114, 3992-3993). Further, the study of the quaternary structure of multimeric proteins (Baca and Kent, J. Am. Chem. Soc., 1992, 114, 3992-3993; Light-Wahl et al., J. Am. Chem. Soc., 1994, 116, 5271-5278; Loo, J. Mass Spectrom., 1995, 30, 180-183, Fitzgerald et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 6851-6856), and of nucleic acid complexes (Light-Wahl et al., J. Am. Chem. Soc., 1993, 115, 803-804; Gale et al., J. Am. Chem. Soc., 1994, 116, 6027-6028; Goodlett et al., Biol. Mass Spectrom., 1993, 22, 181-183; Ganem et al., Tet. Lett., 1993, 34, 1445-1448; Doctycz et al., Anal. Chem., 1994, 66, 3416-3422; Bayer et al., Anal. Chem., 1994, 66, 3858-3863; Greig et al., J. Am. Chem. Soc., 1995, 117, 10765-766), protein-DNA complexes (Cheng et al., Proc. Natl. Acad. Sci. U.S.A., 1996, 93, 7022-7027), multimeric DNA complexes (Griffey et al., Proc. SPIE-Int. Soc. Opt. Eng., 1997, 2985, 82-86), and DNA-drug complexes (Gale et al., JACS, 1994, 116, 6027-6028) are known in the literature.

ESI-MS has also been effectively used for the determination of binding constants of noncovalent macromolecular complexes such as those between proteins and ligands, enzymes and inhibitors, and proteins and nucleic acids. The use of ESI-MS to determine the dissociation constants (K.sub.D) for oligonucleotide-bovine serum albumin (BSA) complexes have been reported (Greig et al., J. Am. Chem. Soc., 1995, 117, 10765-10766). The K.sub.D values determined by ESI-MS were reported to match solution K.sub.D values obtained using capillary electrophoresis.

ESI-MS measurements of enzyme-ligand mixtures under competitive binding conditions in solution afforded gas-phase ion abundances that correlated with measured solution-phase dissociation constants (K.sub.D) (Cheng et al., JACS, 1995, 117, 8859-8860). The binding affinities of a 256-member library of modified benzenesulfonamide inhibitors to carbonic anhydrase were ranked. The levels of free and bound ligands and substrates were quantified directly from their relative abundances as measured by ESI-MS and these measurements were used to quantitatively determine molecular dissociation constants that agree with solution measurements. The relative ion abundance of non-covalent complexes formed between D- and L-tripeptides and vancomycin group antibiotics were also used to measure solution binding constants (Jorgensen et al., Anal. Chem., 1998, 70, 4427-4432).

Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) is another ion source method that can be used for studying biomolecules (Hillenkamp et al., Anal. Chem., 1991, 63, 1193A-1203A). This technique ionizes high molecular weight biopolymers with minimal concomitant fragmentation of the sample material. This is typically accomplished via the incorporation of the sample to be analyzed into a matrix that absorbs radiation from an incident UV or IR laser. This energy is then transferred from the matrix to the sample resulting in desorption of the sample into the gas phase with subsequent ionization and minimal fragmentation. One of the differences of MALDI-MS versus ESI-MS is the simplicity of the spectra obtained, as MALDI spectra are generally dominated by singly charged species. Typically, the detection of the gaseous ions generated by MALDI techniques, are detected and analyzed by determining the time-of-flight (TOF) of these ions. While MALDI-TOF MS is not a high resolution technique, resolution can be improved by making modifications to such systems, by the use of tandem MS techniques, or by the use of other types of analyzers, such as Fourier transform (FT) and quadrupole ion traps.

ESI and MALDI techniques have found application for the rapid and straightforward determination of the molecular weight of certain biomolecules (Feng and Konishi, Anal. Chem., 1992, 64, 2090-2095; Nelson et al., Rapid Commun. Mass Spectrom., 1994, 8, 627-631). These techniques have been used to confirm the identity and integrity of certain biomolecules such as peptides, proteins, oligonucleotides, nucleic acids, glycoproteins, oligosaccharides and carbohydrates. Further, these MS techniques have found biochemical applications in the detection and identification of post-translational modifications on proteins. Verification of DNA and RNA sequences that are less than 100 bases in length has also been accomplished using ESI with FTMS to measure the molecular weight of the nucleic acids (Little et al, Proc. Natl. Acad. Sci. USA, 1995, 92, 2318-2322).

While data generated and conclusions reached from ESI-MS studies for weak non-covalent interactions generally reflect, to some extent, the nature of the interaction found in the solution-phase, it has been pointed out in the literature that control experiments are necessary to rule out the possibility of ubiquitous non-specific interactions (Smith and Light-Wahl, Biol. Mass Spectrom., 1993, 22, 493-501). The use of ESI-MS and MALDI-MS has been applied to study multimeric proteins because the gentleness of the electrospray/desorption process allows weakly-bound complexes, held together by hydrogen bonding, hydrophobic and/or ionic interactions, to remain intact upon transfer to the gas phase. The literature shows that not only do ESI-MS data from gas-phase studies reflect the non-covalent interactions found in solution, but that the strength of such interactions may also be determined. The binding constants for the interaction of various peptide inhibitors to src SH2 domain protein, as determined by ESI-MS, were found to be consistent with their measured solution phase binding constants (Loo et al., Proc. 43.sup.rd ASMS Conf. on Mass Spectrom and Allied Topics, 1995). ESI-MS has also been used to generate Scatchard plots for measuring the binding constants of vancomycin antibiotics with tripeptide ligands (Lim et al., J. Mass Spectrom., 1995, 30, 708-714).

Similar experiments have been performed to study non-covalent interactions of nucleic acids. Both ESI-MS and MALDI-MS have been applied to study the non-covalent interactions of nucleic acids and proteins. While MALDI does not typically allow for survival of an intact non-covalent complex, the use of crosslinking methods to generate covalent bonds between the components of the complex allows for its use in such studies. Stoichiometry of interaction and the sites of interaction have been ascertained for nucleic acid-protein interactions (Jensen et al., Rapid Commun. Mass Spectrom., 1993, 7, 496-501; Jensen et al., 42.sup.nd ASMS Conf. on Mass Spectrom. and Allied Topics, 1994, 923). The sites of interaction are typically determined by proteolysis of either the non-covalent or covalently crosslinked complex (Jensen et al., Rapid Commun. Mass Spectrom., 1993, 7, 496-501; Jensen et al., 42.sup.nd ASMS Conf. on Mass Spectrom. and Allied Topics, 1994, 923; Cohen et al., Protein Sci., 1995, 4, 1088-1099). Comparison of the mass spectra with those generated from proteolysis of the protein alone provides information about cleavage site accessibility or protection in the nucleic acid-protein complex and, therefore, information about the portions of these biopolymers that interact in the complex.

So-called "hyphenated" techniques can be used for structure elucidation because they provide the dual features of separation and mass detection. Such techniques have been used for the separation and identification of certain components of mixtures of compounds such as those isolated from natural products, synthetic reactions, or combinatorial chemistry. Hyphenated techniques typically use a separation method as the first step: liquid chromatography methods such as HPLC, microbore LC, microcapillary LC, or capillary electrophoresis are typical separation methods used to separate the components of such mixtures. Many of these separation methods are rapid and offer high resolution of components while also operating at low flow rates that are compatible with MS detection. In those cases where flow rates are higher, the use of `megaflow` ESI sources and sample splitting techniques have facilitated their implementation with on-line mass spectrometry. The second stage of these hyphenated analytical techniques involves the injection of separated components directly into a mass spectrometer, so that the spectrometer serves as a detector that provides information about the mass and composition of the materials separated in the first stage. While these techniques are valuable from the standpoint of gaining an understanding of the masses of the various components of multi component samples, they are incapable of providing structural detail. Some structural detail, however, may be ascertained through the use of tandem mass spectrometry, e.g., hydrogen/deuterium exchange or collision induced disassociation (CID).

Tandem mass spectrometry (MSN) involves the coupled use of two or more stages of mass analysis where both the separation and detection steps are based on mass spectrometry. The first stage is used to select an ion or component of a sample from which further structural information is to be obtained. This selected ion is then fragmented by (CID) or photo dissociation. The second stage of mass analysis is then used to detect and measure the mass of the resulting fragments or product ions. The advent of Fourier Transform ion cyclotron resonance mass spectrometry (FT-ICR MS) has made a significant impact on the utility of tandem, MS.sup.N procedures because of the ability of FTICR to select and trap specific ions of interest and its high resolution and sensitivity when detecting fragment ions. Such ion selection followed by fragmentation routines can be performed multiple times so as to essentially completely dissect the molecular structure of a sample. A two-stage tandem MS experiment would be called a MS-MS experiment while an n-stage tandem MS experiment would be referred to as a MS.sup.N experiment. Depending on the complexity of the sample and the level of structural detail desired, MS.sup.N experiments at values of n greater than 2 may be performed.

While tandem ESI mass spectra of oligonucleotides are often complex, several groups have successfully applied ESI tandem MS to the sequencing of large oligonucleotides (McLuckey et al., J. Am. Soc. Mass Spectrom., 1992, 3, 60-70; McLuckey and Habibigoudarzi, J. Am. Chem. Soc., 1993, 115, 12085-12095; Little et al., J. Am. Chem. Soc., 1994, 116, 4893-4897). General rules for the principal dissociation pathways of oligonucleotides, as formulated by McLuckey (McLuckey et al., J. Am. Soc. Mass Spectrom., 1992, 3, 60-70; Mcluckey and Habibigoudarzi, J. Am. Chem. Soc., 1993, 115, 12085-12095) have assisted interpretation of mass spectra of oligonucleotides, and include observations of fragmentation such as, for example, the stepwise loss of a base followed by cleavage of the 3'--C--O bond of the relevant sugar. Besides the use of ESI with tandem MS for oligonucleotide sequencing, two other mass spectrometric methods are also available: mass analysis of products of enzymatic cleavage of oligonucleotides (Pieles et al., Nucleic Acids Res., 1993, 21, 3191-3196; Shaler et al., Rapid Commun. Mass Spectrom., 1995, 9, 942-947; Glover et al., Rapid Commun. Mass Spectrom., 1995, 9, 897-901), and the mass analysis of fragment ions arising from the initial ionization/desorption event, without the use of mass selection techniques (Little et al., Anal. Chem., 1994, 66, 2809-2815; Nordhoff et al., J. Mass Spectrom., 1995, 30, 99-112; Little et al., J. Am. Chem. Soc., 1994, 116, 4893-4897; Little and McLafferty, J. Am. Chem. Soc., 1995, 117, 6783-6784). While determining the sequence of deoxyribonucleic acids (DNA) is possible using ESI-MS and CID techniques (McLuckey et al., J. Am. Soc. Mass Spectrom., 1992, 3, 60-70; McLuckey and Habibigoudarzi, J. Am. Chem. Soc., 1993, 115, 12085-12095), the determination of RNA sequence is much more difficult. Thus while small RNA, such as 6-mers, have been sequenced (McCloskey et al., J. Am. Chem. Soc., 1993, 115, 12085-1095), larger RNA have been difficult to sequence using mass spectrometry. Tandem ESI-MS methods can also be used to determine the binding sites for small molecules that bind to RNA targets (Griffey et al., Journal of the American Society for Mass Spectrometry, 1995, 6, 1154-1164).

Ion trap-based mass spectrometers are particularly well suited for such tandem experiments because the dissociation and measurement steps are temporarily rather than spatially separated. For example, a common platform on which tandem mass spectrometry is performed is a triple quadrupole mass spectrometer. The first and third quadrupoles serve as mass filters while the second quadrupole serves as a collision cell for collisionally activated dissociation (CAD), also known as collision induced dissociation (CID). In a trap-based mass spectrometer, parent ion selection and dissociation take place in the same part of the vacuum chamber and are effected by control of the radio frequency wavelengths applied to the trapping elements and the collision gas pressure. Hence, while a triple quadrupole mass analyzer is limited to two stages of mass spectrometry (i.e. MS/MS), ion trap-based mass spectrometers can perform MS.sup.n analysis in which the parent ion is isolated, dissociated, mass analyzed and a fragment ion of interest is isolated, further dissociated, and mass analyzed and so on. A number of MS.sup.4 procedures and higher have appeared in the literature in recent years and can be used here. See, Cheng et al., Techniques in Protein Chemistry, VII, pp. 13-21.

ESI tandem MS has been used for the study of high molecular weight proteins, for peptide and protein sequencing, identification of post-translational modifications such as phosphorylation, sulfation or glycosylation, and for the study of enzyme mechanisms (Rossomando et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 5779-578; Knight et al., Biochemistry, 1993, 32, 2031-2035). Covalent enzyme-intermediate or enzyme-inhibitor complexes have been detected using ESI and analyzed by ESI-MS to ascertain the site(s) of modification on the enzyme. The literature has shown examples of protein sequencing where the multiply charged ions of the intact protein are subjected to collisionally activated dissociation to afford sequence informative fragment ions (Light-Wahl et al., Biol. Mass Spectrom., 1993, 22, 112-120). ESI tandem MS has also been applied to the study of oligonucleotides and nucleic acids (Ni et al., Anal. Chem., 1996, 68, 1989-1999; Little et al., Proc. Natl. Acad. Sci., 1995, 92, 2318-2322).

Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) is an especially useful analytical technique because of its ability to resolve very small mass differences to make mass measurements with a combination of accuracy and resolution that is superior to other MS detection techniques, in connection with ESI or MALDI ionization (Amster, J. Mass Spectrom., 1996, 31, 1325-1337, Marshall et al., Mass Spectrom. Rev., 1998, 17, 1-35). FT-ICR MS may be used to obtain high resolution mass spectra of ions generated by any of the other ionization techniques. The basis for FT-ICR MS is ion cyclotron motion, which is the result of the interaction of an ion with a unidirectional magnetic field. The mass-to-charge ratio of an ion (m/q or m/z) is determined by a FT-ICR MS instrument by measuring the cyclotron frequency of the ion. The insensitivity of the cyclotron frequency to the kinetic energy of an ion is one of the fundamental reasons for the very high resolution achievable with FT-ICR MS. Each small molecule with a unique elemental composition carries an intrinsic mass label corresponding to its exact molecular mass, identifying closely related library members bound to a macromolecular target requires only a measurement of exact molecular mass. The target and potential ligands do not require radio labeling, fluorescent tagging, or deconvolution via single compound re-synthesis. Furthermore, adjustment of the concentration of ligand and target allows ESI-MS assays to be run in a parallel format under competitive or non-competitive binding conditions. Signals can be detected from complexes with dissociation constants ranging from <10 nM to .about.100 mM. FT-ICR MS is an excellent detector in conventional or tandem mass spectrometry, for the analysis of ions generated by a variety of different ionization methods including ESI and MALDI, or product ions resulting from CAD.

FTICR-MS, like ion trap and quadrupole mass analyzers, allows selection of an ion that may actually be a weak non-covalent complex of a large biomolecule with another molecule (Marshall and Grosshans, Anal. Chem., 1991, 63, A215-A229; Beu et al., J. Am. Soc. Mass Spectrom., 1993, 4, 566-577; Winger et al., J. Am. Soc. Mass Spectrom., 1993, 4, 566-577; Huang and Henion, Anal. Chem., 1991, 63, 732-739), or hyphenated techniques such as LC-MS (Bruins et al., Anal. Chem., 1987, 59, 2642-2646; Huang and Henion, J. Am. Soc. Mass Spectrom., 1990, 1, 158-65; Huang and Henion, Anal. Chem., 1991, 63, 732-739) and CE-MS experiments (Cai and Henion, J. Chromatogr., 1995, 703, 667-692). FTICR-MS has also been applied to the study of ion-molecule reaction pathways and kinetics.

The use of ESI-FTICR mass spectrometry as a method to determine the structure and relative binding constants for a mixture of competitive inhibitors of the enzyme carbonic anhydrase has been reported (Cheng et al., J. Am. Chem. Soc., 1995, 117, 8859-8860). Using a single ESI-FTICR-MS experiment these researchers were able to ascertain the relative binding constants for the noncovalent interactions between inhibitors and the enzyme by measuring the relative abundances of the ions of these noncovalent complexes. Further, the K.sub.D S so determined for these compounds paralleled their known binding constants in solution. The method was also capable of identifying the structures of tight binding ligands from small mixtures of inhibitors based on the high resolution capabilities and multistep dissociation mass spectrometry afforded by the FTICR technique. A related study (Gao et al., J. Med. Chem., 1996, 39, 1949-55) reports the use of ESI-FTICR-MS to screen libraries of soluble peptides in a search for tight binding inhibitors of carbonic anhydrase II. Simultaneous identification of the structure of a tight binding peptide inhibitor and determination of its binding constant was performed. The binding affinities determined from mass spectral ion abundance were found to correlate well with those determined in solution experiments. Further, the applicability of this technique to drug discovery efforts is limited by the lack of information generated with regards to sites and mode of such noncovalent interactions between a protein and ligands.

Improvements in mass spectrometric instrumentation and methodologies are needed to address increasingly challenging applications in a number of research arenas including the physical, biological, and medical sciences. In many implementations of mass spectrometers based on Penning and Paul traps, ion formation, isolation, and detection take place in the same region of a vacuum chamber and are temporally rather than spatially separated. In a typical pulse, sequence ions are alternatively formed and detected; the ionization duty cycle is defined as the fraction of time ions are formed compared to the overall experiment time. In high resolution measurements, which may take several seconds to perform yet require ionization intervals of only a few milliseconds, the overall ionization duty cycle is only a few percent. A number of approaches have been explored to improve the ionization duty cycle including schemes in which ions are formed and continuously accumulated in an external ion reservoir and periodically gated into the mass analyzer. For example, a Penning trap in the fringing magnetic field of an Fourier transform ion cyclotron resonance (FTICR) mass spectrometer was used to accumulate ions formed by EI during high resolution measurements in the FTICR cell. See, Hofstadler and Laude, Jr., Anal. Chem., 1991, 63, 2001-2007. An external ion reservoir formed by an rf-only multipole bounded by two electrostatic elements can efficiently accumulate ions generated by electrospray ionization and the ion ensemble can be periodically pulsed into the FTICR cell for mass analysis has also been demonstrated (Senko et al., J. Amer. Soc. Mass Spectrom., 1997, 8, 970-976).

Another means of improving mass spectra is the use of dissociation to fragment the molecular ions. Dissociation strategies for tandem ESI-MS can be separated into two general categories: those which take place in the ESI source prior to mass analysis, and those which take place after the ESI source and often rely on some form of m/z dependent ion manipulation. For example, it has been demonstrated that large multiply-charged proteins could be effectively dissociated by employing a relatively large voltage difference between the exit of the desolvating capillary and the skimmer cone (Loo et al., Anal. Chim. Acta, 1990, 241, 167-173). It has also been demonstrated that ions could be thermally dissociated in the ESI source by heating the desolvation capillary to extreme temperatures (Rockwood et al., Rapid Comm. Mass Spectrom., 1991, 5, 582-585). Both of these "in-source" dissociation schemes produce mass spectra which are rich in fragment ions and can provide sequence information for peptides, proteins, or oligonucleotides. Alternatively, a number of post-source dissociation schemes have been presented which are now widely employed. In general, scanning MS/MS instruments such as triple quadrupoles and magnetic sector instruments employ collisionally activated dissociation (CAD) to effect the dissociation of an m/z selected parent ion (Dagostino et al., J. Chrom., 1997, 767, 77-85). In addition to employing various forms of CAD (Gauthier et al., Chim. Acta, 1991, 246, 211-225; and Senko et al., Anal. Chem., 1994, 66, 2801-2808), FTICR instruments have successfully demonstrated the use of UV-photodissociation (Williams et al., J. Amer. Soc. Mass Spectrom., 1990, 1, 288-294), infrared multiphoton dissociation (IRMPD) (Little et al., Anal. Chem., 1994, 66, 2809-2815), surface induced dissociation (SID) (I james and Wilkins, C. L., Anal. Chem., 1990, 62, 1295-1299; and Williams et al., J. Amer. Soc. Mass Spectrom., 1990, 1, 413-416), blackbody infrared radiative dissociation (BIRD) (Price et al., Anal. Chem., 1996, 68, 859-866), and more recently, electron capture dissociation (ECD) (Zubarev et al., J. Am. Chem. Soc., 1998, 120, 3265-3266) to fragment precursor ions.

Collisionally activated dissociation (CAD), also known as collision induced dissociation (CID), is a method by which analyte ions are dissociated by energetic collisions with neutral or charged species, resulting in fragment ions which can be subsequently mass analyzed. Mass analysis of fragment ions from a selected parent ion can provide certain sequence or other structural information relating to the parent ion. Such methods are generally referred to as tandem mass spectrometry (MS or MS/MS) methods and are the basis of the some of MS based biomolecular sequencing schemes being employed today.

Infrared