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.
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 xe2x80x9ccoulombic explosionxe2x80x9d 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 (KD) for oligonucleotide-bovine serum albumin (BSA) complexes have been reported (Greig et al., J. Am. Chem. Soc., 1995, 117, 10765-10766). The KD values determined by ESI-MS were reported to match solution KD 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 (KD) (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. 43rd 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., 42nd 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., 42nd 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 xe2x80x9chyphenatedxe2x80x9d 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 xe2x80x98megaflowxe2x80x99 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, MSN 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 MSN experiment. Depending on the complexity of the sample and the level of structural detail desired, MSN 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 3xe2x80x2xe2x80x94Cxe2x80x94O 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 MSn 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 MS4 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  less than 10 nM to xcx9c100 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 KDS 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 xe2x80x9cin-sourcexe2x80x9d 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 multi-photon dissociation (IRMPD) uses photodissociation generally in combination with FTICR or quadrupole ion trap mass analyzers. In this method, ions are collected in the FTICR analyzer cell and the laser interacts with ions within the cell. In IRMPD, the laser dissociates ions into fragment ions, as opposed to an ionization method involving lasers, e.g. MALDI. The most common method of ionization used in IRMPD methods is electrospray ionization as this provides more highly charged ions that are more easily dissociated, as compared to MALDI. Little et al., Anal. Chem., 1994, 66, 2809-2815. IRMPD has been used for protein and nucleotide sequencing (Little et al., Anal. Chem., 1994, 66, 2809-2815). IRMPD has also been used with quadrupole ion trap mass spectrometers (Colorado et al., Anal. Chem., 1996, 68, 4033-4043).
Studies have demonstrated that oligonucleotides and nucleic acids obey certain fragmentation patterns during collisionally induced dissociation (CID), and that these fragments and patterns can be used to determine the sequence of the nucleic acid (McLuckey et al., J.Am.Soc. Mass Spectrom., 1992, 3, 60-70; McLuckey and Haaabibigoudarzi, J.Am.Chem. Soc., 1993, 115, 12085-12095). Electrospray ionization produces several multiply charged ions of the parent nucleic acid, without any significant fragmentation of the nucleic acid. Typically, a single charge state of the nucleic acid is isolated using a triple quadrupole ion trap, or ion cyclotron resonance (ICR) device. This ion is then excited and allowed to collide with a neutral gas such as helium, argon, or nitrogen so as to afford cleavage of certain bonds in the nucleic acid ion, or excited and fragmented with a laser pulse. Typically, two series of fragment ions are found to be formed: the a-Base series (a-B) and the w series.
The series of a-Base fragments originates from initial cleavage of the glycosidic bond by simultaneous abstraction of a C-2xe2x80x2 proton, followed by the elimination of the 3xe2x80x2-phosphate group and the C-4xe2x80x2 proton. This fragmentation scheme results in a residual fragment attached to the 3xe2x80x2-phosphate and affords a series of a-Base fragments whose masses increase sequentially from the 5xe2x80x2-terminus of the nucleic acid. Measurement of the masses of these collisionally induced fragments therefore affords the determination of the sequence of the nucleic acid in the 5xe2x80x2 to 3xe2x80x2 direction. The w series of fragments is generated via cleavage of the nucleic acid in a manner that leaves a phosphate residue on each fragment. Similarily, y fragments are based on cleavage of the nucleic acid in a manner that cleaves a phosphate residue. Thus monitoring the masses of w-series and y-series fragments allows determination of the sequence of the nucleic acid in the 3xe2x80x2 to 5xe2x80x2 direction. Using the sequence information generated from the series of fragments the sequence of deoxyribonucleic acids (DNA) may be ascertained. Obtaining similar mass spectrometric information for ribonucleic acids (RNA), is a much more difficult task. Collisionally induced dissociation (CID) of RNA is much less energetically favored than is the case for DNA because of the greater strength of the glucosidic bond in RNA. Hence, while small RNA such as 6-mers have been sequenced using CID MS, the sequencing of larger RNA has not been generally successful using tandem MS.
Currently, IRMPD methods are limited to mass spectrometers based on FTICR and QIT. With FTICR methods the kinetic energy release which accompanies the dissociation event can cause a redistribution of the ions in the trapped ion cell. Upon excitation, these ions can obtain a range of cyclotron radii, which precludes high performance mass measurements. Also, the laser irradiation interval is identical for each ion, which limits the dissociation pathways available to the ion.
FTICR-MS, like ion trap and quadrupole mass analyzers, allows selection of an ion that may actually be a weak noncovalent 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; and Winger et al., J. Am. Soc. Mass Spectrom., 1993, 4, 566-577); Huang and Henion, Anal. Chem., 1991, 63, 732-739), and is compatible with 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; and 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.
Tandem mass spectrometry, as performed using electrospray ionization (EST) on FTICR, triple quadrupole, or ion-trap mass spectrometers, has been found to be a powerful tool for determining the structure of biomolecules. It is known in the art that both small and large ( greater than 3000 kbase) RNA and DNA may be transferred from solution into the gas phase as intact ions using electrospray techniques. Further, it is known to those skilled in the art that these ions retain some degree of their solution structures as ions in the gas phase; this is especially useful when studying noncovalent complexes of nucleic acids and proteins, and nucleic acids and small molecules by mass spectrometric techniques.
A limited use of MS and shutters is known in the art. Certain internal shutters have been used to regulate or monitor ions after they have entered the mass spectrometer. Recently, the stability of an ion source was demonstrated by measuring the current of the electrosprayed ions at the shutter of the Fourier transform ICR mass spectrometer as a function of time (Hannis et al., Rapid Commun. Mass Spectrom. 1998, 12(8), 443-448). The use of an operating arm/shutter has been used for the simultaneous use of an ion collector and as a mass spectrometer connection. This feature allowed a simultaneous readout of real time ion detection and data received by the MS unit (Smith et al., U.S. Pat. No. 5,545,304, Ion Current Detector for High Pressure Ion Sources For Monitoring Separations). Electromechanical shutters have also been used to improve the system design of a mass spectrum. The addition of a downstream mechanical shutter to halt the flow of neutrals to the trapped ion cell during FT-ICR detection allowed for more than 100-fold improvement in pressure drop between the source and mass analyzer chamber to be realized (Guan et al., Rev. Sci. Instrum. 1995, 66(9), 4507-15). A set of electromechanical shutters were also used to minimize the effect of the directed molecular beam produced by the ESI source and were open only during injection (Winger et al., J. Am. Soc. Mass Spectrom. 1993, 4(7), 566-77).
Although improvements have been made in mass spectrometric analysis of biomolecules, especially with the use of mass spectrum shutters, there remains a need for further improved mass spectrometric methods and apparatuses.
In a first embodiment of the invention there are provided methods and apparatuses that selectively isolate ions external to a mass analyzer of a mass spectrometer. Processes and devices are described for effecting ion-molecule and ion-ion reactions on these isolated ions by first injecting the ions into a space and then isolating these ions to prevent the introduction of new ions. Once the ions are isolated, a reactive moiety may be introduced for a time sufficient for at least some of the reactive moiety to react with at least some of the ions to form the reacted ions. The reacted ions formed are subsequently moved into an analyzer for analysis.
In an additional embodiment of the invention, after injecting the ions into a space, the introduction of a physical barrier in operative association with the space allows for the isolation of these ions to prevent the introduction of new ions. The barrier may include a seal to prevent the further introduction of new ions. The barrier may be a shutter where the shutter is connected to and is actuated by a signal from a host computer.
In further embodiment of the invention, the ion-molecule and ion-ion reactions that occur with the ions in the isolated space is effected by the introduction of a reactive moiety. The reactive moiety is introduced as a gas or a plasma and may be either a gas phase deuterated solvent (D2O, ND3 or CH3OD), a gas phase acid (acetic acid, trifluoroacetic acid or hydroiodic acid) or a gas phase base (ammonia, dimethylamine, triethylamine or N,N,Nxe2x80x2,Nxe2x80x2-tetramethyl-1,8-naphthylenediamine). The reactive moiety may also be an isotope such as deuterium, to effect an ion-molecule reaction. Generally, the reactive moiety should be a chemical isotope that is absent from the isotopic species that forms the elemental building blocks of the isolated ions. Ion-ion reactions may be effected by the introduction of a reactive moiety such as perfluoro-1,3-dimethylcyclohexane into the isolated population of ions.
In another embodiment of the invention, a population of ions to be modified, prior to introduction into a mass spectrometer analyzer, are first generated from an ion source. The beam of ions produced are allowed to enter the opening of the instrument. The opening is closed to segregate a population of ions from the further ions that are continually generated. This slice of ions is allowed to react with a reactive moiety and the ions are subsequently analyzed by mass spectrometry.
In a still further embodiment of the invention, a common ion source used is electrospray ionization. The shutter is optimally positioned between the ion source and the inlet capillary opening of the mass spectrometer. The solvated ions generated by the electrospray conditions are selected by the opening and closing of the shutter which allows the ions to accumulate in the instrument. The solvated ions enter the capillary opening of the mass spectrometer and are subsequently desolvated and stored prior to reaction with a reactive moiety. The selected population of ions can be reacted by effecting an isotopic exchange, for example, hydrogen may be exchanged for deuterium.
In another embodiment of the invention, processes of analyzing a population of ions can be accomplished by using an ion source which generates a continuous population of ions, some of which enter the opening of the mass spectrometer and can be stored in an ion reservoir. A physical barrier located between the ion source and the ion reservoir of the instrument allows for the physical interruption of the flow of ions. The isolated population of ions may be stored in the ion reservoir of the instrument for a fixed period of time. Subsequent release of the ions from the ion reservoir allows for analysis of the ions.
A further embodiment of the invention provides processes of analyzing a population of ions in a mass spectrometer that are stored in an ion reservoir which has both an inlet and an outlet. The inlet of the ion reservoir is guarded by a physical barrier. The physical barrier is positioned between the ion source and the inlet of the ion reservoir and allows for the selective accumulation of ions. The outlet of the ion reservoir may be guarded by a barrier. The outlet allows for the analysis of the ions through a mass analyzer. A common mass analyzer used is an FTICR ion mass analyzer. The inlet of the ion reservoir may be guarded by a barrier that has a seal. The inlet and the outlet of the ion reservoir may both be guarded by a barrier that has a seal for optimal performance of the instrument wherein the outlet may be connected to a mass analyzer.
Another embodiment of the invention involves improvements for electrospray ionization which generate a continuous population of solvated ions. The solvated ions can be accumulated into an ion reservoir which serves as a solvent evaporation region for the instrument. Physically interupting the flow of solvated ions between the ion source and the evaporation region affords an isolated population of ions in the ion reservoir.
In a further embodiment of the invention, an electrospray mass spectrometer can be improved by the use of a barrier such as a shutter that is positioned between the ion source and the opening of the solvent evaporation chamber. The solvated ions generated from the electrospray ionization can be blocked by the shutter from entering the solvent evaporation chamber. The barrier may include a seal for preventing the influx of ions from going into the solvent evaporation chamber. The barrier may be a shutter capable of being positioned in the path of the ions between the ion source and the solvent evaporation chamber. The shutter could be actuated by a computer to allow selective entry of a population of ions.
Preferably the population of ions includes 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. More preferably the population of ions includes protein ions, peptide ions, oligonucleotide ions, nucleic acid ions or carbohydrate ions.
In another embodiment of the invention, the vacuum pressure in an ion reservoir of an electrospray mass spectrometer may be modulated by the use of a barrier having a vacuum seal. The attached vacuum seal on the barrier, which is located between the ion source and the ion reservoir, closes the ion reservoir allowing modulation of the vacuum pressure in the ion reservoir.
In a further embodiment of the invention, the barrier is a shutter having a seal and is located between the ion source and the ion reservoir. Another barrier or shutter may be located between the ion reservoir and the mass analyzer and may be used to vacuum seal the ion reservoir. The use of the two described shutters and vacuum seals may be used in conjunction to modulate the vacuum pressure in the ion reservoir.
In another embodiment of the invention, an improvement to an electrospray mass spectrometer is afforded by sealing the ion reservoir using the above described shutters and vacuum seals to modulate the vacuum pressure of the instrument.
In another embodiment of the invention, the ion reservoir of an electrospray mass spectrometer may be isolated by the use of an upstream shutter located between the ion source and the ion reservoir.
In a further embodiment of the invention, the upstream shutter may include a seal or a vacuum seal capable of sealing the ion reservoir from the the ion source. The use of an upstream shutter and downstream shutter equipped with vacuum seals would allow for the modulation of the pressure in the mass spectrometer.
In another embodiment of the invention, an improvement to an electrospray mass spectrometer comprises an ion source,an ion reservoir, an analyzer and an upstream shutter which is capable of limiting the population of solvent and ions entering into the ion resevoir. Further, the ion reservoir is a multipole ion reservoir capable of serving as a desolvation chamber.
In a further embodiment of the invention,an improvement to an electrospray mass spectrometer comprises an ion source,an ion reservoir, an analyzer and an upstream shutter which is capable of limiting the population of -solvent and ions entering into the ion resevoir. Further, a laser that is positioned in operative association with the ion reservoir for exciting the solvated ions to vaporize the solvent and afford the desolvated ions.
The continual accumulation of ions in a multipole ion reservoir affords a weaker ion intensity as seen in the mass analyzer due to the unfocused nature of the ions. By holding a selected population of ions in the multipole for a certain time, for example from about 50 msec to about 200 msec, collisional focusing occurs resulting in a greater ion intensity as seen in the mass analyzer. This collisional focusing is not seen when the ions are continually generated and allowed to enter the multipole and immediately mass analyzed. The time element allotted for collisional focusing allows a greater ion intensity than would otherwise be seen.
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