WO2003103007A1 - Mass spectrometer - Google Patents

Abstract

An electron capture dissociation system to be coupled with transmission mass analysis means where an ion source (116) injects ions into a reaction/extraction volume (104) where an electron source (102) injects electrons into said volume for collision with said ions.

Description

MASS SPECTROMETER

Cross-Reference to Related Application

This claims the benefit of U.S. Provisional Application No. 60/386,134, filed June 3, 2002, which is incorporated herein by reference.

Field

The invention concerns mass spectrometers, more specifically tandem mass spectrometer systems and methods for using tandem mass spectrometer systems.

Background A mass spectrometer is an instrument that measures the masses of individual molecules and fragments thereof. Mass spectrometry includes a broad range of instruments and methodologies used to elucidate the structural and chemical properties of molecules, to identify the compounds present in physical and biological matter, and to quantify the chemical substances found in samples of such matter. Mass spectrometers can generate useful structural information from minute quantities of pure substances (in typical cases, 1- 20xl0^"12 g, and in favorable cases, l-50xl0^"15 g) and, as a consequence, can identify compounds at very low concentrations (in favorable cases, one part in 10¹²) in chemically complex mixtures. The power of this analytical science is evidenced by the fact that mass spectrometry has become a necessary adjunct to research in every division of natural and biological science and a source of valuable information for a wide range of technologically based professions, such as medicine, law enforcement, process control engineering, chemical manufacturing, pharmacology, biotechnology, food processing and testing, and environmental engineering. In these applications, mass spectrometry is used to identify structures of biomolecules, such as carbohydrates, nucleic acids and steroids; sequence biopolymers, such as proteins and oligosaccharides; study drug adsorption and metabolism; perform forensic analyses (e.g., confirm and quantitate drugs of abuse) analyze environmental pollutants; determine the age and origins of geochemical and archaeological specimens; identify and quantitate components of complex organic mixtures; and perform ultrasensitive multi-element analyses of inorganic materials, such as metal alloys and semiconductors.

A mass spectrometer typically measures the masses of individual molecules that have been converted to gas-phase ions, i.e. to electrically charged molecules in a gaseous state. Conversion to gas-phase ions is an essential prerequisite to the mass sorting and detection processes that occur in a mass spectrometer. The principal parts of any mass spectrometer are the ion source, mass analyzer, detector, and data handling system. Samples, which may be a solid, liquid, or vapor, are introduced into the ion source where ionization and volatilization occur. The phase and state of the sample, and the size and structure of the molecules, determine which physical and chemical processes are necessary to convert the sample into gas-phase ions. For example, electrospray ionization (ESI) is a technique that can produce multiply charged cations or anions of intact biopolymers. In addition, ESI is generally compatible with eluents (e.g., methanol, acetonitrile, and water) and flow rates commonly used in liquid chromatography. These properties make ESI-forms of mass spectrometry especially versatile for applications in the biomolecular sciences and industries.

Ions generated in the source are passed on to the mass analyzer, which uses electromagnetic forces to sort them according to their mass-to-charge (m/z) ratios or a related property, such as velocity, momentum, or energy. After separation by the analyzer, the ions are successively directed to a detector. The detector generates electrical signals whose magnitudes are proportional to the number of ions striking the detector per unit time. The data system records these electrical signals and displays them on a monitor or prints them out in the form of a mass spectrum, i.e. a graph of signal intensity versus m/z. In principle, the pattern of molecular-ion and fragment-ion signals that appear in the mass spectrum of a pure compound constitutes a unique chemical fingerprint from which the compound's molecular mass and, sometimes, its structure can be deduced.

At present, the most widely used mass-selective devices are magnetic sectors, quadrupole mass filters, quadrupole ion traps, Fourier transform ion cyclotron resonance (FT-ICR) cells, and time-of-flight (TOF) tubes. Mass spectrometers based on TOF analysis have been a major factor in the relatively recent, revolutionary expansion of mass spectrometric applications into molecular-biological research and biotechnology. TOF mass analyzers are fundamentally the simplest and the least expensive to manufacture. They separate ions by virtue of their different flight times through a known length of flight tube. To create these different times, an ensemble of ions of like charge are accelerated to equal kinetic energies and, in a brief burst, released from the ion source into the flight tube. Since an ion's kinetic energy is equal to 'Λmv² (where m is its mass and v its velocity) and all ions of like charge have the same energy, light ions will have greater velocities and, consequently, shorter flight times to the detector than heavy ions. The m/z values of each set of ions produced from a given sample of ions in the ion source can be determined by injecting the ions into a TOF tube and measuring their successive transit times from the point of injection through the flight tube to the detector (typically several tens of microseconds for any given ion).

A TOF mass spectrometer has a theoretically unlimited m/z range. With the other four forms of commonly used mass analyzers, the settings of one or more parameters determine the m/z of the ions that are allowed to pass to the detector. In order for ions with a different m/z to be detected, these settings must be increased or decreased. Ultimately, some fundamental or practical characteristic of the mass analyzer limits the extent to which its m/z-determining parameters can be changed to accommodate analysis of increasingly larger ions. In a TOF mass analyzer, increasingly larger ions simply take correspondingly longer times to reach the detector, and there is no fundamental limit to the length of time that can be measured. By virtue of this unique feature, TOF mass analyzers are especially useful for the analysis of large biological molecules.

A TOF mass spectrometer does not acquire a mass spectrum by scanning. Scanning denotes a continuous increasing or decreasing of a mass analyzer's wz/z-determining parameters over a predetermined range so ions over a corresponding range of m/z-values can be detected in succession. The analytical efficiency of a mass spectrometric analysis is greatly reduced by scanning because, while the ions of one particular m/z are being detected, the ions of all other rø/z-values released into the analyzer are irretrievably lost in the instrument. With TOF mass analyzers, by contrast, all of the ions released in a single burst into the flight tube are detected and recorded without changing any instrumental parameters. Consequently, TOF mass spectrometers are particularly fast, sensitive instruments. Full mass spectra can be obtained without losing spectral information or sensitivity at an ion sampling frequency of 5-10 kHz. This high spectral acquisition rate is particularly powerful when mass spectrometry is performed in conjunction with gas or liquid chromatography. This combination makes it possible to (1) acquire accurate mass spectrometric information from essentially all substances that elute from a given chromatographic column; (2) improve the signal-to-noise ratio and, in turn, the sensitivity by intensive data averaging; and (3) accurately monitor fast separations that result in the production of extremely narrow chromatographic peaks. The utility of mass spectrometry can be significantly enhanced by performing multiple stages of analysis in tandem (MSⁿ). A tandem instrument may exist in two general forms: transmission instruments and ion-trapping instruments. A transmission tandem mass spectrometer comprises two or more mass-selective devices, which can be operated independently, arranged one after another with each mass analyzer separated from the preceding one by a region in which ion-dissociation may be induced. An ion-trapping tandem mass spectrometer comprises a single cell that functions (for one or more cycles) alternately as a mass-selective device, a region for inducing ion-dissociation, and then a mass selective device again. Independent operation of the mass analyzers in an MS" system makes it possible to perform analyses based on changes in mass, charge, or reactivity, and on the ability of one or more of the mass analyzers to register those changes. MSⁿ also can be used to substantially improve signal-to-background ratios and, thus, sensitivity by eliminating interferences in certain types of analyses when the ion signal at the m/z of interest is produced by more than one compound. Increasingly, novel instruments are appearing that make it possible to use MS" to probe more precisely into problems of ion structure as well as to increase resolution in analyses of complex mixtures.

Standard modes of tandem mass spectrometric analysis have evolved over the past two decades. These standard modes are described in text books (e.g., E. De Hoffmann, J. Charette, and N. Stroobant, Mass Spectrometry Principles and Applications, John Wiley & Sons: New York, 1996, and J.T. Watson, Introduction to Mass Spectrometry, 3^rd Edition, Lippincott-Raven Publishers: Phihdelphia, 1997.) and in reference books (e.g., Methods in Enzymology,. J. A. McCloskey, Ed., Academic Press, Inc.: San Diego, Vol. 193, 1990). The most frequently practiced form of tandem mass spectrometry is MS² (or MS MS).

Briefly, tandem mass spectrometry may be performed in one of three scanning modes: product-ion scans, precursor-ion scans, and neutral-loss scans. In a product-ion scan, a first mass analyzer (MSI) is set to transmit ions formed in the instrument's ion source at only one particular m/z value. The selected ions, which are called precursor ions, are passed into a region where they are induced to dissociate into fragments. The charged fragments, which are called product ions, are passed into a second mass analyzer (MS2) that is programmed to scan over a range that covers the various .w/z-values of all the product ions. Only mass peaks corresponding to the charged fragments of the precursor ion selected by MSI appear in the resulting product-ion mass spectrum. If the sample is a pure compound and fragment-forming ionization has been used, individual fragment ions originating in the ion source can be selected as precursor ions; their product-ion spectra (which may be thought of as mass spectra within a mass spectrum) can provide much additional structural information about the analyte. If the sample is a mixture and nonfragment-forming ionization is used to produce predominantly molecular ions, the second stage of mass analysis can provide an identifying mass spectrum for each component in the mixture.

In a precursor-ion scan, MSI is programmed to scan over a range that covers the various .H/z-values of all the ions formed in the instrument's ion source while MS2 is set to transmit product ions at only one particular m/z value. In this manner, only mass peaks corresponding to precursor ions that can fragment into a product ion with the specific m/z value transmitted by MS2 appear in the resulting precursor-ion mass spectrum.

In a neutral-loss scan, MSI is programmed to scan over a range that covers the various .w/z-values of all the ions formed in the instrument's ion source while MS2 is programmed to simultaneously scan over a range that is offset from MS 1 ' s mass-range by a constant mass-value. In this manner, only mass peaks corresponding to precursor ions that lose a neutral fragment (e.g. a water or carbon monoxide molecule) whose mass is equal to the constant mass-offset between the scanning ranges of MSI and MS2 appear in the resulting neutral-loss mass spectrum. The utility of a tandem mass spectrometer depends on the types and configurations of the individual analyzers of which it is composed. Recently, two forms of tandem mass spectrometers have independently emerged to set new standards for qualitative mass spectrometry. The first of these is a hybrid configuration that combines the power of quadrupole and TOF analyzers (quadrupole-time-of-flight hybrid) (see, for example, Dodonov et al., USSR Patent No. 1681340A1, February 1987; Mirgorodskaya et al.,

Analytical Chemistry, 66: 99-107, 1994; Verentchikov et al., Analytical Chemistry, 66: 126- 133, 1994; Morris et al., Rapid Communications in Mass Spectrometry, 10, 889-896, 1996; Shevchenko et al., Rapid Communications in Mass Spectrometry, 11, 1015-1024, 1997; Krutchinsky et al., Rapid Communications in Mass Spectrometry, 12, 508-518, 1998; Smirnov et al., in Proceedings ofTJie 47th ASMS Conference on Mass Spectrometry and Allied Topics, Dallas, TX, 1999; Cotter, Analytical Chemistry, 71, 445A-451A, 1999; Chernushevich et al., Analytical Chemistry, 71, 452A-461A, 1999; Blackburn et al., American Pharmaceutical Review, 2: 49-59, 1999; Lazar et al., American Laboratory, February 2000, 110-119; Verentchikov et al., in Proceedings of Hie 48th ASMS Conference on Mass Spectrometry and Allied Topics, Long Beach, CA, 2000; and Guilhaus et al., Mass Spectrometry Reviews, 19, 65-107, 2000, each of which are incorporated by reference herein). Q-TOF systems, which can be used with electrospray ionization (ESI), matrix- assisted laser desorption/ionization MALDI) and other ionization processes, provide high resolution analyses of both precursor- and product-ions while retaining the excellent sensitivity (femtomole levels) and mass range inherent in TOF analyzers. The excellent sensitivity, speed, resolution, mass accuracy, and stability in mass calibration of the orthogonal acceleration that the TOF analyzer brings to the quadrupole-time-of-flight hybrid configuration is particularly advantageous when analyzing the product ions of an induced dissociation process. In protein research, a quadrupole-time-of-flight hybrid instrument can attain resolving powers of 7,000 or better and mass accuracies approaching 5 ppm, which is sufficient to unequivocally determine the charge states of multiply-charged ions and the elemental compositions of most tryptic peptides. Thus, these instruments have the sensitivity to detect proteins in 2-D gel spots, the resolution to distinguish among peptides of similar molecular weights, and the mass range to examine non-covalent interactions. For projects involving identification of metabolites or discovery of natural products, quadrupole-time-of-flight hybrid systems can greatly reduce ambiguity.

The second form of tandem mass spectrometer to appear recently is the tandem TOF (time-of-flight-time-of-flight) (see, for example, Piyadasa et al., Rapid Communications in Mass Spectrometry, 12: 1655-1664, 1998; Vestal et al., An Improved Delayed Extraction MALDI-TOFfor PSD and CID, 46^th ASMS Conference on Mass

Spectrometry and Allied Topics, Orlando, Florida, 1998; Katz and Barofsky, A New Design or a MALDI Tandem Time-of-Flight Mass Spectrometer, 47^th ASMS Conference on Mass Spectrometry and Allied Topics, Dallas, Texas, 1999; Medzihradszky et al., Analytical Chemistry, 72: 552-558, 2000; Barofsky et al., Tandem Time-of Flight Mass Spectrometer, U.S. Patent Application No. 09/405,208; and Vestal, A Tandem Time-of-Flight Mass

Spectrometer with Delayed Extraction and Method for Use, PCT Publication WO 99/40610, each of which is incorporated by reference herein). Novel design and operation of the velocity selector used in the first TOF analyzer of these instruments makes it possible to achieve resolving powers up to 5,000 in MSI. The use of time-lagged focusing of ions selected in MS 1 makes it possible to achieve high resolution mass spectra in MS2 that cover the entire mass range of the product-ions and their precursor-ions without stepping the ion- reflector's voltage (which is an exceptionally inefficient, tedious, manual form of scanning). Both of these features are achieved while retaining the advantages of sensitivity, unlimited mass range, and fast nonscanning data acquisition inherent to TOF mass analyzers. The methods used to induce fragmentation of precursor ions in MS" experiments vary. Forcing precursor ions to collide at low or high kinetic energies with inert gas atoms is the most common method used to induce fragmentation. This technique, which is known as collisionally induced (or activated) dissociation (CID or CAD), is a powerful tool for acquiring amino acid sequence data from peptides and may be used to identify proteins and sites of modification on proteins .

The effect of low-energy activation on an ionized organic molecule, such as a peptide, is markedly different than that of high-energy activation. In the former case, energy is incrementally imparted to the ion through a succession of collisions that occur on the average every few microseconds or so while, in the latter case, energy is transferred to the precursor ion in a single, fast, low-scattering angle collision. It is not surprising, therefore, that the energy-mode used to collisionally excite an ion strongly influences the pathways through which a given molecular structure dissociates into fragments. Low-energy CID permits facile variation of the ion energy in an MS/MS experiment; this added dimension of analytical specificity can be used in some instances to differentiate isomeric structures. However, fundamental restrictions on the amount of energy that can be deposited internally into an ionic molecule limit low-energy CID MS/MS analyses to organic and organometallic compounds with molecular weights less than -3,000 Daltons. High-energy CID is typically more reproducible, generally results in more fragmentation, and can fragment much larger precursor ions than the low-energy version of CID.

Nevertheless, CID techniques do have shortcomings, especially in regard to their use in the analysis of proteins and oligopeptides. The amino acid residues in a protein can fragment at several different positions. Even more problematic, fragmentation generally does not occur at every residue; therefore, gaps appear in the sequence-data. Furthermore, the fragment peak intensity can vary from almost background level to that of the most intense signal in the spectrum. Consequently, CID mass spectra can be much more difficult and tedious to interpret than to acquire.

Recently, electron capture dissociation (ECD) has been shown to be an effective means for inducing ESI-precursor fragmentation, including fragmentation of biomolecular cations formed by ESI (see, for example, Zubarev et al., Journal of the American Chemical Society, 120: 3265-3266, 1998; Zubarev et al., Journal of the American Chemical Society, 121: 2857-2862, 1999; Zubarev et al., Analytical Chemistry. 72: 563-573, 2000; Axelsson et al., Rapid Communications in Mass Spectrometry, 13: 474-477, 1999; Mirgorodskaya et al., Analytical Chemistry, 71: 4431-4436, 1999; Kelleher et al., Analytical Chemistry, 71, 4250- 4253, 1999; Horn et al., Journal of the American Society for Mass Spectrometry, 11, 320- 332, 2000; and McLafferty et al., Journal of the American Society for Mass Spectrometry, 12, 245-249, 2001). In this method, radical-site dissociation of multiply charged cations (i.e., positive ions having an ionic charge greater than 1) is induced through electron capture. Since the products of ECD must also be ions to be mass analyzed, only multiply charged cations, which yield cationic products on reduction, can serve as precursors. One advantage of ECD for protein analysis is that it predominantly leads to cleavage of the peptide backbone to form a- and y-type product ions (the nomenclature used in this document to name sequence-specific ion-fragments complies with the conventional rules introduced by Roepstorff and Fohlman, Biomedicαl Mass Spectrometry, 11, 601, 1984, and subsequently modified by Biemann, in Methods in Enzymology, J. A. McCloskey, Ed., Academic Press, Inc.: San Diego, Vol. 193, pp. 886-887, 1990). Furthermore, a greater percentage of a given peptide's amino acid sequence is generally revealed by ECD than by low energy CID. Consequently, ECD spectra are usually easier to interpret than CID spectra, and ECD sequence-data tend to complement CID sequence-data. Presently, despite its advantages, ECD MS" can only be performed with Fourier transform ion-cyclotron-resonance (FT-ICR) mass spectrometers (i.e. it can only be practiced with an ion-trapping type mass spectrometer). This significantly limits the opportunity for application of the technique. While FT-ICR instruments are arguably the most powerful (in terms of sensitivity, resolution, and mass accuracy) mass spectrometers in existence, they also are difficult and expensive to operate and among the most expensive to purchase. Most likely for these reasons, their use has not become widespread. Furthermore, the method by which ECD-FT-ICR analyses are conducted place fundamental and practical limits on their applications. The methods used to produce low-energy electrons and expose precursor ions to such electrons limit control over electron energy and ionization efficiency. Results are spectacular when they are obtained, but that occurrence cannot be reliably predicted. Consequently, experiments are difficult to conduct and reproduce.

Summary

An electron capture dissociation system that is compatible with transmission mass spectrometers is described. A transmission mass spectrometer that can perform electron capture dissociation (ECD) analysis also is described. Li one embodiment, the mass spectrometer is a tandem mass spectrometer that includes an electron capture dissociation cell, a low-energy collisional dissociation cell, and a high-energy collisional dissociation cell, arranged to enable a number of previously unknown modes of analysis, including transmission ECD-MS".

Brief Description of the Figures

FIG. 1 is a schematic drawing of an apparatus for transmission electron capture dissociation. FIG. 2 is a block diagram showing ion flow in an embodiment of a transmission mass spectrometer system that incorporates a first collisionally induced (low or high energy) dissociation cell, a transmission electron capture dissociation system, and a second collisionally induced (high energy) dissociation cell. FIG. 3 is a schematic drawing of an embodiment of a quadrupole/time-of-flight hybrid mass spectrometer system incorporating a low-energy CID cell, a transmission electron capture dissociation system, and a high-energy CID cell.

Detailed Description

A transmission electron capture dissociation system, comprising a pulsed ion accelerator and a source of electrons positioned to deliver electrons to an extraction volume of the pulsed ion accelerator is disclosed. This may further comprise a source of ions positioned to deliver ions to the extraction volume of the pulsed ion accelerator in a direction that is substantially orthogonal to the direction in which ions are accelerated by the pulsed ion accelerator. In some embodiments, the electron source and the ion source are positioned to deliver intersecting flows of electrons and ions to the extraction volume of the pulsed ion accelerator. The intersecting flows of electrons and ions may be substantially orthogonal to each other, opposite to each other, or the flows may intersect at any other angle, such as any non-zero angle within 4π steradians. In particular embodiments the electron source is an electron monochromator and the pulsed ion accelerator is a multiple- stage pulsed ion acceleration system capable of time-lag focusing ions onto a space focal plane.

In another aspect, a mass spectrometer that includes a transmission electron capture dissociation system is disclosed. In one embodiment, the mass spectrometer includes an electron source positioned to deliver electrons to an extraction volume of the pulsed ion accelerator, an ion source positioned to deliver ions to the extraction volume of the pulsed ion accelerator in a direction substantially orthogonal to the direction in which ions are accelerated by the pulsed ion accelerator, and a mass selective device positioned to receive ions accelerated by the pulsed ion accelerator. According to the disclosure, the mass selective device may be a monopole or multipole mass filter (e.g. a quadrupole, hexapole, octapole or decapole mass filter), a magnetic sector analyzer, a time-of-flight analyzer, or a tandem mass-selective device (e.g. a time-of-flight-time-of-flight or quadrupole-time-of- flight hybrid device). The ion source may be one based on electrospray, atmospheric pressure chemical, corona discharge thermospray, matrix-assisted particle-induced desorption, fast atom bombardment, fast ion bombardment, ²⁵³Cf plasma desorption, electron impact, chemical, laser desorption, or matrix-assisted laser desorption ionization.

In some embodiments, the disclosed mass spectrometers may also include either or both of a high-energy or low-energy CID cell. In other embodiments the mass spectrometer may also include a second mass-selective device (e.g. a quadrupole mass filter or any of the other types of mass selective devices mentioned above) positioned between the ion source and the transmission electron capture dissociation system. Furthermore is also possible to position a CID cell between the second mass selective device (e.g. a quadrupole mass filter) and the transmission electron capture dissociation system. In particular embodiments, such a CID is a low-energy CID cell.

A transmission tandem mass spectrometer system which comprises the following components also is disclosed: (1) an ion source, (2) an electron source comprising an electron monochromator, (3) an electron capture dissociation chamber fluidly coupled to the ion source and the electron source, the electron capture chamber comprising a pulsed ion accelerator, a first input positioned to deliver ions from the ion source to an extraction volume of the pulsed ion accelerator in a direction substantially orthogonal to the direction ions are accelerated by the pulsed ion accelerator, a second input positioned to deliver electrons from the electron source to the extraction volume of the pulsed ion accelerator in a direction that intersects the ions delivered through the first input, and an output positioned to permit ions accelerated by the pulsed ion accelerator to exit the electron capture chamber, (4) a quadrupole mass filter fluidly coupled to the output of the electron capture chamber and positioned to accept ions exiting the electron capture chamber, (5) a CID cell fluidly coupled to the quadrupole mass filter and positioned to accept ions transmitted through the quadrupole mass filter; and (6) a time-of-flight mass analyzer fluidly coupled to the CTD cell and positioned to accept ions transmitted through the CID cell. The ion source may be any of those described above capable of producing ions having plural positive charges from substances of interest, but in particular embodiments is an electrospray ion source or any of its variants. The time-of-flight analyzer may be any type thereof, but, in particular embodiments, is a one that incorporates a reflection. In yet other embodiments an additional mass-selective device may be fluidly coupled to the ion source and the electron capture dissociation chamber and positioned to transmit ions from the ion source to the electron capture dissociation chamber.

In a particularly disclosed embodiment, a mass spectrometer comprises the following components: (1) an ion source, (2) a first mass-selective device downstream from the ion source (3) a first CID cell downstream from the first mass-selective device, (4) a transmission electron capture dissociation system downstream from the first dissociation cell, the transmission electron capture dissociation system comprising a pulsed ion accelerator oriented to accelerate ions in a direction substantially orthogonal to the direction from which the ions enter an extraction volume of the pulsed ion accelerator and a source of electrons positioned to deliver electrons to the extraction volume of the pulsed ion accelerator along a path that intersects the ions entering the extraction volume at some nonzero angle within 4π steradians of the direction of the ions, (5) a second mass-selective device downstream from the electron capture dissociation chamber, (6) a second CID cell downstream from the second mass-selective device, and (7) a third mass-selective device downstream from the second dissociation cell. The first and second CID cells may be a low-energy CID cell, a high-energy CID cell, or a chamber for surface-collisionally induced dissociation. In more particular embodiments the mass spectrometer may include an off- axis detector positioned between the first collision-induced dissociation cell and the transmission electron capture dissociation system. In other more particular embodiments, the source of electrons may be an electron monochromator, the second mass-selective device may be a TOF device comprising a velocity selector (e.g. a velocity selector that operates by a 180° change in the direction of an applied electric field), the second mass- selective device may be a magnetic sector analyzer, the second CID cell may be a high- energy CID cell, and the third mass-selective device may be a time-of-flight mass analyzer (e.g. a reflection) or a magnetic sector analyzer.

Also disclosed is a kit for converting an existing orthogonal extraction quadrupole- time-of-flight hybrid mass spectrometer to an ECD-quadrupole-time-of-flight hybrid system. Such a kit may comprise, in packaged combination, an electron source and electron deflecting optics configured to deliver electrons to an extraction volume of a pulsed ion accelerator that is a component of the existing orthogonal extraction quadrupole-time-of- flight hybrid mass spectrometer.

Also disclosed is a method of stimulating electron capture dissociation that is compatible with transmission mass spectrometer systems. In this method multiply charged cations are directed to an extraction volume of a pulsed ion accelerator and electrons are directed to intersect the multiply charged cations within the extraction volume of the pulsed ion accelerator.

Several methods for analyzing a sample are disclosed, including a method comprising: ionizing a sample to produce a first set of ions, accelerating the first set of ions in a first direction, using an ion accelerator to accelerate the first set of ions into a time-of- flight mass analyzer, the time-of-flight mass analyzer having a flight-axis that is substantially orthogonal to the first direction, using a velocity selector in the time-of-flight mass analyzer to select a second set of ions from the first set of ions, the second set of ions comprising ions that have the same mass-to-charge ratio, and using a high-energy CID cell to fragment at least a portion of the second set of ions and produce a third set of ions, which may be detected and analyzed to determine their mass-to-charge ratios. Also disclosed is a method for analyzing a sample that includes ionizing a sample to produce a first set of ions, accelerating the first set of ions in a first direction, selecting a second set of ions from the first set of ions using a quadrupole mass filter, the second set of ions having the same mass-to-charge ratio, using an ion accelerator to accelerate the second set of ions into a time-of-flight mass analyzer, the time-of-flight mass analyzer having a flight-axis that is substantially orthogonal to the first direction, and using a high-energy CTD cell to fragment at least a portion of the second set of ions to produce a third set of ions.

Methods for analyzing a sample that include electron capture dissociation are disclosed. For example, in one disclosed method a sample is ionized to produce a first set of ions, the first set of ions comprising ions having plural positive charges. The first set of ions are accelerated in a first direction and a second set of ions is selected from the first set of ions using a quadrupole mass filter. The second set of ions comprises ions having the same mass-to-charge ratio. At least a portion of the second set of ions are allowed to react with electrons in an extraction volume of an ion accelerator to produce a third set of ions. The ion accelerator is used to accelerate the third set of ions into a time-of-flight mass analyzer, the time-of-flight mass analyzer having a flight-axis that is substantially orthogonal to the first direction. A mass spectrum may be recorded using the time-of-flight mass analyzer, but resolution may be increased by further using a velocity selector to select a fourth set of ions from the third set of ions, the fourth set of ions having the same mass-to-charge ratio. Once selected, the fourth set of ions also may be passed through a high-energy CID cell to fragment at least a portion of the fourth set of ions to produce a fifth set of ions which are recorded by the time-of-flight analyzer. Alternatively, the high-energy CTD cell may be used to fragment at least a portion of the third set of ions to produce a fourth set of ions without first using a velocity selector. In some embodiments, neutral loss or precursor ion spectra may be recorded by scanning the quadrupole mass analyzer across a range of mass- to-charge ratios to select, at any given time, a second set of ions comprising ions having the same mass-to-charge ratio.

Another disclosed method for analyzing a sample that includes electron capture dissociation is a method that includes ionizing a sample to produce a first set of ions, the first set of ions comprising ions having plural positive charges; accelerating the first set of ions in a first direction; selecting a second set of ions from the first set of ions using a quadrupole mass filter; the second set of ions comprising ions having the same mass-to- charge ratio; using a low energy CID cell to fragment at least a portion of the second set of ions to produce a third set of ions; allowing at least a portion of the third set of ions to react with electrons in an extraction volume of an ion accelerator to produce a fourth set of ions; and using the ion accelerator to accelerate the fourth set of ions into a time-of-flight mass analyzer, the time-of-flight mass analyzer having a flight-axis that is substantially orthogonal to the first direction. Other more particular embodiments include using a velocity selector to select a fifth set of ions from the fourth set of ions, the fifth set of ions having the same mass-to-charge ratio and then possibly using a high-energy CID cell to fragment at least a portion of the fifth set of ions selected using the velocity selector to produce a sixth set of ions. Alternatively, a high-energy CTD cell may be used to fragment at least a portion of the fourth set of ions to produce a fifth set of ions without first using a velocity selector. Again, neutral loss and precursor spectra may be recorded by scanning the quadrupole mass analyzer across a range of mass-to-charge ratios to select, at any given time, a second set of ions comprising ions having the same mass-to-charge ratio.

Another disclosed electron capture dissociation method is comprised of the following operations: (1) ionizing a sample to produce a first set of ions, the first set of ions comprising ions having plural positive charges, (2) accelerating the first set of ions in a first direction, (3) using a low energy CID cell to fragment at least a portion of the first set of ions to produce a second set of ions, (4) allowing at least a portion of the second set of ions to react with electrons in an extraction volume of an ion accelerator to produce a third set of ions, and (5) using the ion accelerator to accelerate the third set of ions into a time-of-flight mass analyzer, the time-of-flight mass analyzer having a flight-axis that is substantially orthogonal to the first direction. This method may also include using a velocity selector to select a fourth set of ions from the third set of ions, the fourth set of ions having the same mass-to-charge ratio and/or using a high-energy CID cell to fragment at least a portion of the third or fourth sets of ions to produce either a fourth or fifth set of ions.

Disclosed Apparatus

A representative embodiment of an apparatus for transmission electron capture dissociation is illustrated in FIG. 1. In this particular embodiment, transmission electron capture dissociation system 100 comprises an electron source 102 and an electron capture chamber 104. The electron capture chamber 104 has at least a first input 106 and a second input 114 and at least one output system 124. The first input 106 receives electrons from the electron source 102 and transmits them through energy-retarding optics 108 into the extraction volume of output system 124. Optics 108 may comprise a set of focusing/steering electrodes 110 and an energy-retarding grid 112. The second input 114 receives a flow of ions (which when the system is functioning to stimulate electron capture dissociation is a flow of positively charged gaseous ions of charge +2 or greater) from the ion source 116 and transmits them through energy-retarding optics 118 into extraction volume of output system 124. The intersecting flows of elections and ions, from inputs 106 and 114, respectively, may be substantially orthogonal to each other, opposite to each other as depicted in FIG. 1, or the flows may intersect at any other angle, such as any non-zero angle within 4π steradians. Optics 118 may comprise a set of focusing/steering electrodes 120 and an energy-retarding grid 122. Fundamentals of energy-retarding optical systems, which can be applied equally to electrons and ions, and examples of how those systems are used in conjunction with various ion sources and orthogonal extraction of ions are described, for example, in Guilhaus et al., Mass Spectrometry Reviews, 19, 65-107, 2000, which is incorporated by reference herein. Standard ion optical simulation programs designed to study and analyze ion optics in both two and three dimensional modes and views, for example the SIMION 3D Ion and Electron Optics Program available from Scientific Instrument Services, Inc. (Ringoes, NJ), may be used to customize energy- retarding optics 108 and 118 to the dimensions, configuration, and performance requirements of any particular embodiment.

The illustrated output system 124 comprises an ion accelerator that can be pulsed to extract ions from electron capture dissociation chamber 104 in a direction that is orthogonal to the direction that the ions are received from the ion source. In the particular embodiment illustrated by FIG. 1, a standard two-stage ion accelerator, a repeller electrode 126, a draw- out (or acceleration) grid 128, and an exit grid 130 are shown. Fundamentals of pulsed optical systems used for orthogonal extiaction and examples of those systems in operation with various ion types of ion beams and time-of-flight analyzers are described, for example, in Guilhaus et al., Mass Spectrometry Reviews, 19, 65-107, 2000, which is incorporated by reference herein. The terms "extraction volume" and "reaction/extraction volume" refer to the region of space from which the ion accelerator of output system 124 can accelerate ions and in which elections may be allowed to react with ions when the system is functioning to stimulate election capture dissociation.

The electron source 102 in FIG. 1 may, in certain embodiments, be an electron monochromator. An electron monochromator is a device that can supply large numbers of electrons for electron capture at a precisely defined energy in the range 0-10 eV (See, for example, U.S. Patent Nos. 5,340,983 and 5,493,115 to Deinzer and Laramee and Laramee et al., Mass Spectrometry Reviews, 15:15-42 (1996), each of which are incorporated by reference herein). Briefly, the electron monochromator focuses electrons emitted from a filament into a deflection region defined by crossed magnetic (B) and electric (E) fields. The electric field, perpendicular to the electron beam, together with the magnetic field, parallel to the electron beam, cause the electrons to undergo trochoidal motion. The axis along which the trochoidal motion occurs drifts in a direction perpendicular to both fields at a constant velocity. The electron energy can be varied by varying the filament potential. The intensity of an electron beam produced by a trochoidal monochromator is approximately constant over the electron energy range 0-lOeV. Election monochromators are available commercially from JEOL (Peabody, MA). In other embodiments, the electron source is a directly-heated filament or an indirectly heated surface and a system that uses an electric field, a magnetic field, or a combination of both to precisely define the average energy and the standard deviation in energy of the electrons. The pulsed ion accelerator comprising the output system 124 illustrated by FIG. 1 may be configured for orthogonal extraction of ions (relative to the direction such ions are received from the ion source 116) from the electron capture dissociation chamber 104. The ion accelerator may be any multiple-stage pulsed ion acceleration system capable of time- lag focusing the ions onto a space focal plane some distance from the exit electrode 130. Time-lag focusing is a conventional technique. Descriptions of time-lag focusing and associated terminology are given, for example, in R. J. Cotter, Time-of-Flight Mass Spectrometry, American Chemical Society: Washington, DC, 1997; pp. 23-38; Chernushevich et al., Analytical Chemistry, 71, 452A-461A, 1999; Cotter, Analytical Chemistry, 71, 445 A-451 A, 1999; and Weickhardt et al., Mass Spectrometry Reviews, 15, 139-162, 1996, each of which is incorporated by reference herein. Fundamentals and applications of time-lag focusing in pulsed devices used for orthogonal extraction and examples of those devices in operation are described, for example, in Guilhaus et al., Mass Spectrometry Reviews, 19, 65-107, 2000; Shevchenko et al., Analytical Chemistry, 72, 2132- 2141, 2000; Cotter, Analytical Chemistry, 71, 445A-451A, 1999; Chernushevich et al., Analytical Chemistry, 71, 452A-461A, 1999; Krutchinsky et al., Rapid Communications in Mass Spectrometry, 12, 508-518, 1998; Shevchenko et al., Rapid Communications in Mass Spectrometry, 11, 1015-1024, 1997; Morris et al., Rapid Communications in Mass Spectrometry, 10, 889-896, 1996; Verentchikov et al., Analytical Chemistry, 66:126-133, 1994; Mirgorodskaya et al., Analytical Chemistry, 66: 99-107, 1994; Dawson et al., Rapid Communications in Mass Spectrometry, 3: 155-159, 1989; Sin et. al., Analytical Chemistry, 63: 2897-2900, 1991; and, Boyle and Whitehouse, Analytical Chemistry, 64: 2084-2089, 1992; all of which are incorporated herein by reference.

The transmission electron capture dissociation system 100 of FIG. 1 may be incorporated into mass spectrometer systems that match the block diagram shown in FIG. 2. The mass spectrometer system includes an ion source 200, a mass-selective device 202, a first collisional dissociation cell 204, a transmission electron capture dissociation system 100, a second mass-selective device 206, a second collisional dissociation cell 208, and a third mass-selective device 210. Such a system is capable of performing a wide variety of MS² and MS³ types of tandem experiments that are useful, in general, for analyzing organic molecules and, in particular, for sequencing biopolymers, such as proteins and nucleic acids. For any of the modes of CTD-analysis that are possible on the system diagramed in FIG. 2, the ion source 200 may be any ion source that can produce positive or negative gaseous ions. For any of the ECD-modes of analysis that are possible on the system diagrammed in FIG. 2, the ion source 200 may be any ion source that can produce gaseous cations bearing a charge equal to or greater than +2. Examples of ion sources that can provide such ions, include, without limitation, electiospray ionization (ESI) sources and variations thereof (e.g. ionspray (IS) or pneumatically assisted electiospray sources, ultraspray or ultrasonically assisted electiospray (US) sources, thermally assisted electiospray (TES) sources, and nanospray (NS) sources), atmospheric pressure chemical ionization (APCI) sources, corona discharge thermospray (CTS) sources, all variations of matrix-assisted particle-induced desorption/ionization sources (e.g. fast atom bombardment (FAB) sources, fast ion bombardment or secondary ion sources, and ²⁵²Cf plasma desorption sources), election impact (El) ionization sources, chemical ionization (CI) sources, laser desorption/ionization (LDI) sources, and matrix-assisted laser desorption/ionization (MALDI) sources.

In one embodiment, the ion source 200 is an ESI source. Electiospray ionization sources and methods of operating such sources are well known in the art and are described, for example, in Lazar et al., American Laboratory (February), 110-119, 2000; Smith et al., Analytical Chemistry, 60: 436-441, 1988; Smith et al., Analytical Chemistry, 60: 1948-1952, 1988, Gale and Smith, 7: 1017-1021, 1993; Verentchikov et al., Analytical Chemistry, 66: 126-133, 1994; U.S. Pat No. 5,015,845 to Allen, et al.; and U.S. Pat. Nos. 5,844,237 and 6,060,705 to Whitehouse et al.; all of which are incorporated by reference herein. Briefly, ESI is achieved by spraying a sample solution [which may, for example, be effluent out of a directly coupled high performance liquid chromatography (HPLC), capillary zone electiophoresis (CZE), or supercritical fluid chromatography (SFC) column] from a needle (held at a high potential) in the direction of an orifice that is the entrance to the interface with the mass-selective device 202. Heat and gas phase collisions may be used to internally energize the ions as they traverse the interface so that fully desolvated, multiply charged ions, such as multiply charged cations, are introduced into the mass-selective device 202. Typically, the number of charges acquired by a molecule tends to increase as the molecular weight of the molecule increases.

The ion source 200 may be provided as an ESI source and may include associated transfer optics, such as multipole ion guides. Various forms of ESI sources and associated transfer optics are available, for example, as integral components of complete mass spectrometer systems manufactured by Applied Biosystems (Foster City, CA), Bruker Daltonics (Billerica, MA), Kratos Analytical (Chestnut Ridge, NY), Micromass, Ltd. (Manchester, UK), and or as stand-alone items manufactured by Advion BioSciences (Ithaca, NY), Analytica of Branford (Branford, CT), Applied Biosystems (Foster City, CA), and New Objective (Cambridge, MA). The mass-selective device 202 of FIG. 2 may be any known or commercially available mass filter. In certain embodiments, the mass-selective device is a monopole or multipole (e.g. quadrupole, hexapole, octapole or higher multipole) mass filter. In other embodiments, the mass-selective device 202 may be a one- or three- dimensional quadrupole ion trap, a sector-field mass analyzer, or a time-of-flight (TOF) analyzer.

Quadrupole mass filters are known in the art and are commercially available for example, as integral components of complete mass spectrometer systems manufactured by Applied Biosystems (Foster City, CA), Bruker Daltonics (Billerica, MA), Kratos Analytical (Chestnut Ridge, NY), Micromass, Ltd. (Manchester, UK), and Thermo Finnigan (San Jose , CA) or as stand-alone units manufactured by ABB Extrel (Pittsburgh, PA). The construction of and operation of these commercially available multipole mass filters is also known in the art (See for example U.S. Pat No. 5,298,745 to Kernan, et al.). Briefly, DC (direct current) and RF (radio frequency) potentials can be superimposed on the multipole (e.g., quadrupole) rods that comprise the mass filter in a way that permits only ions of a selected mass-to-charge ratio to be tiansmitted. At this setting of the DC and RF potentials, ions with other mass-to-charge ratios have trajectories through the multipole mass filter that eventually cause them to collide with the rods. A description of the theory behind quadrupole mass filters may be found in March et al., "Quadrupole Storage Mass Spectrometry," Chemical Analysis, volume 102, Wiley, 1989. The first collisional dissociation cell 204 of FIG. 2 may provide low-energy collisionally induced dissociation if the mass-selective device 202 is a multipole mass filter or a quadrupole ion trap or provide high energy collisionally induced dissociation if the mass-selective device 202 is a time-of-flight or sector-field analyzer. The second collisional dissociation cell 208 of FIG. 2 may provide high-energy collisionally induced dissociation. Collisionally induced dissociation (CID, also known as collisionally activated dissociation or CAD) is a process whereby precursor ions of a mass-to-charge ratio are collided with neutral target atoms or molecules (typically noble gases, for example helium, argon, xenon, and mixtures thereof). As a result of the collisions, some of any given precursor ion's kinetic energy is converted into internal energy. If there is enough excess internal energy to break chemical bonds, the ion will dissociate into a set of two or more smaller fragment (product) species, at least one of which must be an ion. If a precursor ion is accelerated to a kinetic energy of approximately one kilovolt or higher prior to entering the CID cell (high energy CID), any resulting collision with a target atom will result in an electronic excitation of the precursor ion that dissipates relatively quickly into an almost uniform a distribution of internally excited vibrational and rotational states. In this state of approximately uniform internal excitation, virtually all structurally possible fragmentations of the ion have some probability of occurring. If a precursor ion possess a kinetic energy in the range of 1 eV to several hundred eV (for example, 500 eV or less) prior to entering the CID cell (low energy CID), it may acquire sufficient vibrational excitation after a succession of collisions with target atoms to induce fragmentation. Vibrational excitation resulting from these successive low energy collisions in the gas phase typically results in a non- uniform internal energy distribution that favors fragmentation pathways in the precursor ion that generally are markedly different than those followed after a single high energy collision in the gas phase. In the low energy case, the fragmentation pattern depends strongly on the average kinetic energy of the collisions. For example, the fragmentation pattern produced after precursor ions of a given structure are subjected to 20 eV collisions may be very different from the fragmentation pattern produced after precursors with identical structures are subjected to 50 eV collisions. A low energy fragmentation pattern will also depend strongly on the mass, pressure, and temperature of the target gas. For these reasons, low energy CTD is more difficult to reproduce than high energy CTD.

CID cells, as well as their construction and operation, are known in the art. Low energy CID cells typically are more complex than high-energy CID cells due to the requirement that ions entering the dissociation cell may need to be decelerated to the appropriate energies and that a long flight path through the cell must be provided to increase the likelihood that each ion experiences several collisions in transit. The construction and operation of low energy CID cells are described, for example, in U.S. Pat No. 5,248,875 to Douglas, et al. and Thomson et al., "Experimental Collision Cell for the API HI", Research Report KE109202, PE Sciex (now a division of Applied Biosystems), 1992. Such cells are commercially available, for example, as integral components of complete mass spectiometer systems manufactured by Applied Biosystems (Foster City, CA), Bruker Daltonics (Billerica, MA), Kratos Analytical (Chestnut Ridge, NY), Micromass, Ltd. (Manchester,

UK), and Thermo Finnigan (San Jose , CA), or as stand-alone units manufactured by ABB

Extrel (Pittsburgh, PA). High energy CID cells are much simpler in design, basically consisting of a rectangular or cylindrical chamber into which the target gas is introduced and through which the ions can drift without the aid of electric or magnetic fields. Illustrations of such cells and descriptions of their use can be found, for example, in Bricker and Russell,

Journal of the American Chemical Society, 108: 6174-6179, 1986 and Medzihradszky et al.,

Analytical Chemistry, 72: 552-558, 2000. The mass-selective devices 206 and 210 of FIG. 2 may be any type of mass analyzer compatible with the transmission system including, without limitation, time-of-flight mass analyzers, quadrupole mass analyzers and magnetic sector mass analyzers. The combination of mass-selective device 206, collision dissociation cell 208, and mass-selective device 210 in a series as shown in FIG. 2 compose a high- energy-CID-MS² tandem mass spectrometer. In particular embodiments, the mass spectrometer system incorporating the transmission electron capture dissociation system 100 of FIG. 1 is a quadrupole/time-of- flight/time-of-flight (Q/TOF/TOF) instrument with the configuration illustrated in FIG. 3. In FIG. 3 the transmission electron capture dissociation system again appears with reference number 100. The Q/TOF/TOF mass spectrometer system of FIG. 3, including transmission election capture dissociation system 100, provides unprecedented flexibility with regard to its operation as well as an unprecedented number of modes of analysis.

In the embodiment illustrated in FIG. 3, ion source 300, like ion source 200 of FIG. 2, may be any type already discussed above with reference to FIG. 2. In general, ion source 300 may be any ion source that can produce positive or negative gaseous ions for CID analyses and, in particular, any ion source that can produce gaseous cations bearing a charge equal to or greater than +2 for any of the possible modes of ECD-analysis. However, in more particular embodiments, the ion source 300 is either an ESI source or an APCI source. Downstream of the ion source 300 are tiansfer optics 302. These optics transform the dynamic state of the ions as they leave the source into one that matches the entrance requirements along the optical axis (i.e. the x-axis in FIG. 3) of the quadrupole mass filter 304.

The transfer optics 302 may incorporate a vacuum interface and multipole ion guides, as for example when the ion source 300 is an ESI or APCI source, that respectively transfers the ions from atmospheric pressure into vacuum and collisionally cools them before they are focused and transported into the mass filter. Various forms of transfer optics for ESI and other sources, as well as their construction and operation, are known in the art [Guilhaus et al., Mass Spectrometry Reviews, 19, 65-107, 2000; Shevchenko et al., Analytical Chemistry, 72, 2132-2141, 2000; Verentchikov et al., in Proceedings of The 48th ASMS Conference on Mass Spectrometry and Allied Topics, Long Beach, CA, 2000; Cotter, Analytical Chemistry, 71, 445A-451A, 1999; Chernushevich et al., Analytical Chemistry, 71, 452A-461A, 1999; Smirnov et al., in Proceedings of The 47th ASMS Conference on Mass Spectrometry and Allied Topics, Dallas, TX, 1999; Krutchinsky et al., Rapid Communications in Mass Spectrometry, 12, 508-518, 1998; Shevchenko et al., Rapid Communications in Mass Spectrometry, 11, 1015-1024, 1997; Morris et al., Rapid Communications in Mass Spectrometry, 10, 889-896, 1996; Verentchikov et al., Analytical Chemistry, 66:126-133, 1994; and Mirgorodskaya et al., Analytical Chemistry, 66: 99-107, 1994; all of which are incorporated by reference herein]. Suitable tiansfer optics 302 are commonly provided with commercially available mass spectrometers that use ion sources, such as electiospray ionization sources. Downstream from the quadrupole mass filter is a low-energy CID cell 306. An off-axis detector 308 may be included, making it possible to record mass spectra using only the quadrupole mass filter. Although shown downstream of the low-energy CTD cell 306, the off-axis detector 308 may be located either upstream or downstream of the low-energy CID cell 306. The components indicated as being part of MSI in FIG. 3 may be any existing or future linear-quadrupole/gas collision cell mass- spectiometer system capable of a accepting and ionizing a sample to provide positive or negative gaseous ions, in general, for CID-analyses and gaseous cations bearing a charge equal to or greater than +2, in particular, for ECD-analyses including, for example, systems incorporating ESI sources and APCI sources. Particularly useful as MSI is any existing or future linear-quadrupole/gas collision cell mass spectiometer system with an upper mass-to- charge ratio limit of at least 3,000 and a mass-to-charge ratio resolution of one or less across the entire mass-to-charge ratio range. Such existing or future linear-quadrupole/gas collision cell mass spectiometer systems may be modified to include an off-axis detector 308, if this feature is desired.

Downstream of the low-energy CID cell is a transmission electron capture dissociation system 100. In the embodiment of FIG. 3, the transmission electron capture dissociation system 100 includes ion optics 310 and election optics 314. In this embodiment, ion optics 310 are configured like the energy-retarding optics 118 depicted in FIG. 1, and electron optics 314 are configured like the energy-retarding optics 108 depicted in FIG. 1. The optics may serve to slow or prevent the passage of ions and electrons, respectively, as they enter the extiaction volume of ion accelerator 312. They also may serve to direct ions and electrons along intersecting paths so they collide and react within the extraction volume of ion accelerator 312. More specifically, optics 310 and 314 may be used to determine the ions' and elections' respective entrance times into and residence times within the reaction/extraction volume of transmission electron capture dissociation system 100. In addition, ion optics 310 may be used to determine the kinetic energy of the ions in the reaction/extraction volume of the transmission election capture dissociation system 100. If election source 316 is not an election monochromator, the election optics 314 may be configured like the energy-retarding optics 118 depicted in FIG. 1 so that the electron optics 314 can be used to determine both the timing and energy of the elections' entry into the reaction/extraction region of the transmission electron capture dissociation system 100. In the embodiment of FIG. 3, the ion source 300 and the electron source 316 are arranged to produce opposing flows of ions and electrons along the instrument's x-axis. In other embodiments, the two are arranged to provide a flow of ions along the x-axis into the transmission election capture dissociation system 100 and a cross-flow of elections along a path that intersects the x-axis at some non-zero angle within 4π steradians (i.e. an entire sphere), hi particular embodiments, the cross-flow of elections may have a path contained in the yz-plane (marked A in FIG. 3) that intersects the flow of ions along the x-axis. The incident flows of positive ions and electrons may be in the form of continuous beams that intersect where the x-axis and plane marked A in FIG. 3 meet, or in the form of packets (bundles), supplied in periodic pulses (bursts), that are timed so that elections and positive ions arrive at the intersection of the x-axis and the plane marked A in FIG. 3 at substantially the same time.

Ion accelerator 312, which is part of the transmission electron capture dissociation system 100, may be pulsed periodically or non-periodically to extract ions from the system in a direction that is substantially orthogonal to the direction from which they enter system 100, and into one or more mass selective devices (e.g. TOF mass spectiometer systems like MS2 and MS3 in FIG. 3). The ion accelerator 312 may be any existing or future plural stage, time-lag focusing configuration of electrodes (for example, a 2-stage Wiley/Mclaren time-lag focusing configuration). Orthogonal pulsed accelerators were discussed and examples of particular embodiments were referenced in a preceding paragraph with respect to FIG. 1. The accelerator is modified with electron optics 314 (as described in preceding paragraphs with respect to FIG. 1 and FIG. 3) to accept electrons from the electron source 316, which, in some embodiments, is an electron monochromator as described in a preceding paragraph with respect to FIG. 1. Downstream from the transmission election capture dissociation system 100 and along the z-axis contained in the plane marked A, is velocity selector 318. In some operating modes, ion accelerator 312 may serve as the ion source for velocity selector 318, in which case these two components comprise the TOF mass spectrometer labeled MS2 in FIG. 3. The velocity selector 318 (also known as a timed ion selector or TIS) may be any known or future velocity selector. For example, velocity selector 318 may be a single set of deflection plates (see, for example, Schey et al, International Journal of Mass Spectrometry and Ion Processes, 11: 49, 1987 or Jardine et al., Organic Mass Spectrometry, 27: 1077, 1992), a single set of interleaved wire-combs (see, for example, Bradbury, Physical Review, 44: 1129, 1993), a pair of deflection plates (see, for example, Haberland, Review of

Scientific Instruments, 62: 2368, 1991), a pair of wire-comb deflectors (see, for example, Stoermer et al., Review of Scientific Instruments, 69: 1661, 1998), a multideflector as described in U.S. Pat No. 5,696,375 to Park et. al., an extended Bradbury-Nielson gate as described in U.S. Pat. No. 5,986,258 to Park, or a form of dynamic ion deflector as described by Piyadasa et al., Rapid Communications in Mass Spectrometry, 12: 1655-1664, 1998. In particular embodiments, the velocity selector 318 is the dual ion deflector as described by Piyadasa et al., Rapid Communications in Mass Spectrometry, 12: 1655-1664, 1998 and in U.S. Patent No. 6,489,610, both of which are incorporated herein by reference. The latter velocity selector achieves significantly improved resolution by reversing the polarity of the electric field that is applied to deflect the ions. To maximize the mass resolution of the velocity selector 318, the velocity selector 318 may be centered on the space focal plane of the ion accelerator 312.

Downstream of velocity selector 318 is a high-energy CID cell 320. High-energy CID cells are discussed above with reference to FIG. 2. Downstream from the high-energy CID cell 320 is the TOF mass spectrometer labeled MS3 in FIG. 3. MS3 comprises ion accelerator 312, ion accelerator 322, ion detector 324, reflection 326, and ion detector 328. When MS2 is inoperative and MS3 is operative in a low-resolution mode, ion accelerator 312 acts as MS3 's ion source. In this case, ion accelerator 322, which is located so that its space focal plane (the xy-plane marked C in FIG. 3) substantially coincides with the object plane of reflection 326 (plane C in FIG. 3), functions as a third stage of time-lagged focusing in the ion source. When both MS2 and MS3 are operative, a plane located substantially at the center of velocity selector 318 (the xy-plane marked B in FIG. 3) and ion accelerator 322 comprise MS3's ion source. In this case, the ions appear to originate from plane B, and ion accelerator 322 serves as the time-lagged focusing stage for this virtual source of ions. Pulsed, linear, ion accelerators were discussed in a preceding paragraph with reference to FIG. 1. Reflection 326 may be any single or plural stage, linear or non-linear reflection now in existence or developed in the future.

MS2 and MS3 of the embodiment of FIG. 3 comprise a time-of-flight-time-of-flight mass spectiometer, which in particular embodiments is a commercially available time-of- flight-time-of-flight instrument [available, for example, from Applied Biosystems (Foster City, CA) and Bruker Daltonics (Billerica, MA)] or a custom built time-of-flight-time-of- flight instrument constructed from an existing orthogonal extraction, ion-reflector TOF tube with a resolving power of at least 10,000 up to an m/z limit of at least 10,000. In the latter case, the analyzer may be converted to the configuration shown in FIG. 3 by the addition of ion optics 310 and electron optics 314, electron source 316, velocity selector 318, high- energy CID cell 320, and time-lagged ion accelerator 322. In some embodiments of such a conversion, election source 316 is desirably an electron monochromator.

Ion detectors 324 and 328 are positioned, respectively, to detect ions that either pass directly through the reflection 326 or are reflected by the reflection 326. Ion detectors 324 and 328 may be any device based on continuous or discrete election multiplication now in existence or developed in the future. Such ion detectors, which are integral to all existing commercial time-of-flight mass spectrometer systems, are currently available, for example, from Detector Technology, Inc. (Palmer, MA), ETP Electron Multipliers Pty Ltd. (Ermington, NSW, Australia) and Burle Electro-Optics, Inc. (Sturbridge, MA). Since the transmission electron capture systems described herein are compatible with all types of transmission mass-selective devices (e.g. quadrupole, sector, and TOF devices), it may be incorporated in any combination with any number of such transmission mass-selective devices. Given this flexibility and the relative ease with which transmission mode ECD may be performed using the disclosed transmission electron capture systems, it may be desirable to include the systems in pre-existing mass spectrometers by adding an electron source and electron deflecting optics to an orthogonal extiaction chamber. Thus, a kit, comprising in packaged combination the components required to convert the orthogonal extraction chamber of an orthogonal extraction system into a transmission electron capture dissociation system, is provided. For example, such a kit might comprise an electron source and electron deflecting optics. The advantage of such a kit is that orthogonal extraction quadrupole-time-of-flight hybrid systems with CTD capability are already in widespread use for protein sequencing and could be inexpensively converted to ECD-quadrupole-time-of- flight hybrid systems that provide enhanced sequencing capabilities. Operation of the Apparatus Embodiments of the disclosed transmission electron capture dissociation system enable ECD to be controllably and reproducibly applied to the analysis of organic and biological molecules using transmission mass spectrometers of various types. Furthermore, certain embodiments enable different fragmentation processes such as low energy CID, high energy CID and ECD, in combination or alone, on a single instrument.

The tiansmission mass spectiometer systems described in the preceding sections with reference to FIG. 2 and FIG. 3 may be operated in a variety of MS¹, MS², and MS³ modes. These operating modes provide a level of flexibility that is unprecedented relative to known systems. Furthermore, the transmission electron capture dissociation system of the disclosed mass spectiometer systems facilitates several new MS² and MS³ techniques for analyzing molecules. These election capture methods overcome significant disadvantages, such as unpredictability and expense, associated with electron capture methods performed on ion-trapping Fourier-transform ion cyclotron resonance instruments. Furthermore, when the transmission electron capture dissociation system includes an election monochromator as the election source, it is possible to attach an electron to any one of the carbonyl groups located along the backbone of an peptide or protein, resulting in sequence specific cleavage of the biopolymer. In addition, use of an electron monochromator as electron source makes it possible to induce specific fragmentations by attaching electrons according to the different resonance energies of the aromatic side chains of phenylalanine, tyrosine, tryptophan, and histidine. Similarly, selective reduction of disulfide linkages and specific electron capturing sites such as phospate, sulfate, and nitro groups on the side chains of amino acids is possible, greatly aiding detection of post-translational modifications of proteins.

The various MS¹, MS², and MS³ techniques (old and new) that may be performed on embodiments described in preceding sections are delineated below with reference to FIG. 3, but it should be recognized that similar operating modes are possible with alternative embodiments of the disclosed mass spectrometer systems.

A. MS¹ Modes In some instances, it may be preferable to operate the mass spectiometer of FIG. 3 in a customary low-to-medium resolution, low-to-medium mass range or a single- quadrupole mode. In this mode of operation, ions generated in ion source 300, such as ions generated in an electiospray ionization source, are transferred along the x-axis, through the interface optics 302, and into the quadrupole mass filter 304. Mass filter 304 is operated in a combined DC/RF mode, and the transmitted ions are registered by the off-axis detector 308. The DC/RF potentials may be varied (scanned) to produce a mass spectrum of a sample. This mode of operation is widely known and practiced by those of ordinary skill in the conventional art of quadrupole and quadrupole-time-of-flight hybrid mass spectrometry. In other instances, it may be preferable to operate the mass spectrometer of FIG. 3 in a customary orthogonal extiaction, low-to-medium resolution, high-mass range, linear TOF mode. In this mode, ions generated in the ion source 300 are transferred along the x- axis, through the interface optics 302, through the quadrupole mass filter 304 (which in this operating mode functions as an RF only ion guide), through the low-energy CID cell 306 (which in this operating mode is nonfunctional), through the optics 310 (which in this operating mode are nonfunctional), and into the reaction/extraction volume of tiansmission electron capture dissociation system 100 (which in this operating mode is not functioning to stimulate ECD). The first stage of ion accelerator 312 is used to periodically (for example, at a rate of up to about 1 kHz) accelerate bunches or packets of ions off the x-axis and onto the z-axis, which lies orthogonal to the x-axis in the plane marked A. The second stage of ion accelerator 312 is used to time-lag focus the extiacted ions onto a space-focal plane that substantially coincides with the entrance plane of detector 324. In this operating mode, velocity selector 318, high-energy CID cell 320, and ion accelerator 322, and reflection 326 are nonfunctional. Before the ions register at detector 324, they drift and separate according to mass-to-charge ratio in the space separating ion accelerator 312 and detector 324. The signals generated by detector 324 as it is struck by ion packets of different mass-to-charge ratio appear in a mass spectrum as peaks located at positions corresponding to the ion packets' respective flight times. This mode of operation is widely known and practiced by those of ordinary skill in the conventional art of quadrupole-time-of-flight hybrid mass spectrometry. In other embodiments, it is preferable to operate the mass spectiometer of FIG. 3 in a customary orthogonal extraction, high resolution, high-mass range, reflecting TOF mode. In this mode, ions generated in the ion source 300 are transferred along the x-axis, through the interface optics 302, through quadrupole mass filter 304 (which, in this operating mode functions as an RF-only ion guide), through low-energy CID cell 306 (which in this operating mode is nonfunctional), through ion optics 310 (which in this operating mode are nonfunctional), and into the reaction/extraction volume of the transmission electron capture dissociation system 100 (which in this operating mode is not functioning to stimulate ECD). The first stage of ion accelerator 312 is then used to periodically accelerate ions onto the z- axis. The second stage of ion accelerator 312 is used to time-lag focus the extracted ions onto a space-focal plane (marked C in FIG. 3) that substantially coincides with the object plane of reflection 326. In this operating mode, velocity selector 318, high-energy CID cell 320, and ion accelerator 322 are nonfunctional. The ions, which drift and separate according to mass-to-charge ratio in the space separating the ion accelerator 312 and the detector 328, are reflected and=focused onto detector 328, which is placed at the focal plane of the reflection 326.

The signals generated by detector 328 as it is struck by ion packets of different mass-to-charge ratio appear in a mass spectrum as peaks located at positions corresponding to the ion packets' respective flight times. This mode of operation is widely known and practiced by those of ordinary skill in the conventional art of quadrupole-time-of-flight hybrid mass spectrometry.

B. Product Ion MS² Modes

In some instances, it may be preferable to operate the mass spectrometer of FIG. 3 in a customary low-energy-CID, MS², product-ion mode. In this mode, ions generated in the ion source 300 are tiansferred along the x-axis, through the interface optics 302, through the quadrupole mass filter 304 (which is operating in a combined DC/RF mode to selectively transmit precursor ions of one particular mass-to-charge ratio), through the low- energy CID cell 306 (which in this operating mode is functional), through the ion optics 310 (which in this operating mode are nonfunctional), and into the reaction/extraction volume of the transmission election capture dissociation system 100 (which in this operating mode is not functioning to stimulate ECD). The first stage of ion accelerator 312 is then used to periodically (for example, at a rate of up to about 1 kHz) accelerate bunches or packets of ions off the x-axis onto the z-axis, which lies orthogonal to the x-axis in the plane marked A. The second stage of ion accelerator 312 is used to time-lag focus the extiacted ions onto a space-focal plane that substantially coincides either with the entrance plane of detector 324 or the object plane of reflection 326 depending respectively on whether the ions are mass analyzed in MS3 in a linear TOF mode using detector 324 or in a reflecting TOF mode using reflection 326 and detector 328. In this operating mode, velocity selector 318, high- energy CID cell 320, and ion accelerator 322 are nonfunctional. The signals generated by detector 324 or 328, whichever is the case, as it is struck by ion packets of different mass-to- charge ratio appear in a product ion mass spectrum as peaks located at positions corresponding to the ion packets' respective flight times. This mode of operation is widely known and practiced by those of ordinary skill in the conventional art of quadrupole-time- of-flight hybrid tandem mass spectrometry. In other instances, it may be preferable to operate the mass spectiometer of FIG. 3 in a high-energy-CTD, MS², product-ion mode. In this mode, ions generated in the ion source 300 are transferred along the x-axis, through the interface optics 302, through the quadrupole mass filter 304 (which is either operating in a combined DC/RF mode to selectively tiansmit precursor ions of one particular mass-to-charge ratio or is functioning as an RF-only ion guide), through the low-energy CID cell 306 (which in this operating mode is nonfunctional), through the ion optics 310 (which in this operating mode are nonfunctional), and into the reaction/extraction volume of the transmission electron capture dissociation system 100 (which in this operating mode is not functioning to stimulate ECD). The first stage of ion accelerator 312 is then used to periodically accelerate ions onto the z- axis as in the low-energy-CID, MS², product-ion mode. The second stage of ion accelerator 312 is used to time-lag focus the extracted ions onto a space-focal plane (marked B in FIG. 3) that substantially coincides with the center of the velocity selector 318 (which is either nonfunctional or functional depending respectively on whether or not the quadrupole mass filter 304 is operating to select the precursor ions or is functioning only as an ion guide). The precursor ions, whether selected by the quadrupole mass filter 304 or by the velocity selector 318, drift from the velocity selector 318 to the high-energy CID cell 320 where they collide with target atoms or molecules. The fragment ions resulting from these collisions and any undissociated precursor ions drift into the ion accelerator 322 where they are simultaneously accelerated to an energy that is higher than they had when they entered ion accelerator 322 and time-lag focused onto a space-focal plane (marked C in FIG. 3) that substantially coincides with the object plane of reflection 326. From this object plane, the ions, which drift and separate according to mass-to-charge ratio in the space separating the object plane of reflection 326 and the detector 328, are reflected and focused onto detector 328, which is placed at the focal plane of the reflection 326. The signals generated by detector 328 as it is struck by ion packets of different mass-to-charge ratio appear in a high- energy CID-product ion mass spectrum as peaks located at positions corresponding to the ion packets' respective flight times. This mode of operation is novel for those of ordinary skill in the conventional art of quadrupole-time-of-flight hybrid tandem mass spectrometry, but analogous modes of operation are known and practiced by those of ordinary skill in the conventional art of four-sector and time-of-flight-time-of-flight mass spectrometry.

In yet other instances, the mass spectrometer of FIG. 3 may be operated to provide mass spectral analysis of the product ions generated in the transmission electron capture dissociation system 100. In this mode (a limited variation of which is currently available only on ion-trapping FT-ICR instruments), ions generated in the ion source 300 are transferred along the x-axis, through the interface optics 302, through the quadrupole mass filter 304 (which is operating in a combined DC/RF mode to selectively tiansmit precursor ions of one particular mass-to-charge ratio), through the low-energy CID cell 306 (which in this operating mode is nonfunctional), through the ion optics 310 (which in this operating mode are functional), and into the reaction/extraction volume of the transmission election capture dissociation system 100 (which in this operating mode is functioning to stimulate ECD). Voltages may be applied to the ion optics 310 either continuously or in a sequence of timed pulses so that a continuous beam or a series of packets, respectively, of precursor ions enter the ECD-cell with an average kinetic energy equal to or greater than 0.025eV (0.025 is the average kinetic energy per molecule of a gas at 20 °C). This step, which may include focusing, ensures that the precursor ions approach plane A along the x-axis at or slightly above a thermal energy. In any specific embodiment, the geometrical configuration and dimensions of the reaction/extraction volume in transmission election capture dissociation system 100 determine the actual values and timing sequence, if the latter is applicable, of the voltages that must be applied to ion optics 310. In the particular embodiment of FIG. 3, the bulk of the precursor ions entering the reaction/extiaction volume of the transmission election capture dissociation system 100 encounter a counter- current of low-energy elections at the intersection of the x-axis and plane A. The elections originate in the election source 316, which advantageously is an election monochromator, and enter the reaction/extraction volume of the transmission election capture dissociation system 100 from the side opposite that where the ions enter. In other embodiments, the election source 316 may be arranged to permit the electrons to flow into the reaction/extraction volume along a path contained in plane A so that they encounter the flow of precursor ions from a direction orthogonal to the x-axis. The elections are introduced into the ECD-cell as a continuous beam or a series of packets by applying voltages to electron optics 314, respectively, either continuously or in a sequence of timed pulses. For particular embodiments in which both precursor ions and electrons are introduced into the reaction/extraction volume of the transmission electron capture dissociation system 100 as packets, their entry may be timed so that a cluster of ions and a cluster of electrons arrive substantially simultaneously at substantially the intersection of the x-axis and plane A (i.e., substantially in the center of the reaction/extiaction volume). Since the electron source (advantageously an electron monochromator) may be floated at a potential very near that of plane A, the electrons (whether introduced continuously or in pulses) can be made to arrive at the intersection of the x-axis and plane A with any desired average energy. In some embodiments, the electron source is an electron monochromator, and the mean of the electrons' energy distribution is precisely selected (to within 0.01 eV) by adjusting the energy setting of the election monochromator. In such embodiments, a relatively large population of elections of precisely defined kinetic energy is available to react with a relatively large population of low energy ions in the center of the ECD chamber. After a dwell time, which may typically be between about 1 μs and about 1 ms, the undissociated precursor ions and their charged ECD products are extracted out of the reaction/extiaction volume of tiansmission election capture dissociation system 100 and onto the z-axis of MS2 by pulsed ion accelerator 312. Actual dwell times are selected empirically to maximize ECD yields, while minimizing spreading of the ion-ensemble. The TOF analyzer of MS3 may be operated in either a low-to-medium resolution linear or high resolution reflector mode. Operating under low resolution, the second stage of ion accelerator 312 is used to time-lag focus the extracted ions onto a space-focal plane that substantially coincides with the entrance plane of detector 324; whereas under high resolution, ion accelerator 312 is used to time-lag focus the extiacted ions onto a space-focal plane that substantially coincides with the object plane of reflection 326. Regardless of which resolution MS3 is operated at, velocity selector 318, high-energy CID cell 320, and pulsed, linear time-lagged ion accelerator 322 are nonfunctional. The signals generated by detector 324 or 328, whichever is the case, as it is struck by ion packets of different mass-to-charge ratio appear in an ECD-product ion mass spectrum as peaks located at positions corresponding to the ion packets' respective flight times. This mode of operation is unprecedented in the art of quadrupole-time-of-flight hybrid tandem mass spectrometry or any other form of mass spectrometry other than ion-trapping FT-ICR.

In still further instances, the mass spectrometer may be operated to provide mass spectral analysis of the product ions generated in the low-energy CTD cell 306 and further in the ECD-cell 100. In this mode, ions generated in the ion source 300 are transferred along the x-axis, through the interface optics 302, through the quadrupole mass filter 304 (which is operating in a combined DC/RF mode to selectively transmit precursor ions of one particular mass-to-charge ratio), through the low-energy CID cell 306 (which in this operating mode is functional), through the ion optics 310 (which in this operating mode is either continuously or periodically functional), and into the reaction/extraction volume of the tiansmission electron capture dissociation system 100 (which in this operating mode is functioning to stimulate ECD) either as a continuous beam or a series of packets of precursor ions. As described above, electrons are introduced into the ECD-cell 100 from electron source 316 so that they interact with the CID-precursor ions. The ions produced by combined low-energy CID and ECD are subsequently analyzed in MS3 in either a low-to- medium resolution linear-TOF or a high resolution reflector-TOF mode as described in preceding sections. The signals generated by detector 324 or 328, whichever is the case, as it is struck by ion packets of different mass-to-charge ratio appear in a low-energy- CID/ECD-product ion mass spectrum as peaks located at positions corresponding to the ion packets' respective flight times. This mode of operation is unprecedented in the art of quadrupole-time-of-flight hybrid tandem mass spectrometry or any other form of tandem mass spectrometry other than ion-trapping FT-ICR, where it is restricted to ions with mass- to-charge ratios > 500.

C. Precursor-Ion and Neutral-Loss MS~ Modes

In some instances, it may be preferable to operate the mass spectrometer of FIG. 3 in a low-energy-CID, MS², precursor-ion or neutral-loss mode. In this mode, precursor ions generated in the ion source 300 are tiansferred along the x-axis, through the interface optics 302, through the quadrupole mass filter 304 (which is operating in a combined DC/RF mode to scan slowly, for example at a rate > 1 m/z-umt per second, through a range of wz/z-values specified by the user), through the low-energy CID cell 306 (which in this operating mode is functional), through the ion optics 310 (which in this operating mode are nonfunctional), and into the reaction/extraction volume of the transmission election capture dissociation system 100 (which in this operating mode is not functioning to stimulate ECD). The fragment ions produced by low-energy CID are subsequently extracted into MS3 (for example, at a rate of about 1 kHz or greater) and analyzed in either a low-to-medium resolution linear-TOF or a high-resolution reflector-TOF mode as described in preceding sections. Signals generated by detector 324 or 328, whichever is the case, as it is struck by product ion packets of only one particular mass-to-charge may be recorded simultaneously with the time-varying voltage used to control mass filter 304' s scan; since these ion-signals only occur for those particular precursor ions that dissociate into a fragment ion with the specific mass-to-charge ratio being recorded, a precursor-ion spectrum is generated when they are plotted on mass filter 304's .n/z-scale. Alternatively, signals generated by detector 324 or 328, whichever is the case, as it is struck by product ion packets, whose mass-to- charge ratio at any given instant is always smaller by a fixed amount than mass filter 304's m/z-setting, may be recorded simultaneously with the time-varying voltage used to control mass filter 304's scan; since these signals only occur for those particular precursor ions that lose a specific neutral fragment (e.g. a water or carbon monoxide molecule), a neutral-loss spectrum is generated when they are plotted versus m/z (relative to mass filter 304). These two modes of operation can be performed exactly as described on conventional quadrupole- time-of-flight hybrid tandem mass spectrometers and in an analogous manner on triple quadrupole tandem mass spectrometers. h other instances, it may be preferable to operate the mass spectrometer of FIG. 3 in an MS², precursor-ion or neutial-loss mode using ECD exclusively, hi this mode, ions generated in the ion source 300 are transferred along the x-axis, through the interface optics 302, through the quadrupole mass filter 304 (which is operating in a combined DC/RF mode to scan slowly, for example at a rate > 1 m/z-unit per second, through a range of nz/z-values specified by the user), through the low-energy CID cell 306 (which in this operating mode is nonfunctional), through the ion optics 310 (which in this operating mode are either continuously or periodically functional), and into the reaction/extraction volume of the tiansmission election capture dissociation system 100 (which in this operating mode is functioning to stimulate ECD). The fragment ions produced by ECD are subsequently extracted into MS3 (for example, at a rate of about 1 kHz or greater) and analyzed in either a low-to-medium resolution linear-TOF or a high resolution reflector-TOF mode as described in preceding sections. A precursor-ion spectrum or, alternatively, a neutial-loss spectrum may be generated as described in the preceding section on low-energy-CID precursor-ion/neutial-loss analysis. This ECD-mode of operation is unprecedented in the art of quadrupole-time-of-flight hybrid tandem mass spectrometry or any other form of tandem mass spectrometry. In yet other instances, it may be preferable to operate the mass spectiometer of FIG.

3 in an MS², precursor-ion or neutial-loss mode using low-energy CID in combination with ECD. In this mode, ions generated in the ion source 300 are transferred along the x-axis, through the interface optics 302, through the quadrupole mass filter 304 (which is operating in a combined DC/RF mode to scan slowly, for example at a rate > 1 m/z-unit per second, through a range of m/z-values specified by the user), through the low-energy CID cell 306 (which in this operating mode is functional), through the ion optics 310 (which in this operating mode are either continuously or periodically functional), and into the reaction/extiaction volume of the transmission electron capture dissociation system 100 (which in this operating mode is functioning to stimulate ECD). The fragment ions produced through the combined processes of low-energy CTD and ECD are subsequently extiacted into MS3 (for example, at a rate of about 1 kHz or greater) and analyzed in either a low-to-medium resolution linear-TOF or a high resolution reflector-TOF mode as described in preceding sections. A precursor-ion spectrum or, alternatively, a neutral-loss spectrum may be generated as described in the preceding section on low-energy CID precursor-ion/neutial-loss analysis. This combined low-energy CID/ECD-mode of operation is unprecedented in the art of quadrupole-time-of-flight hybrid tandem mass spectrometry or any other form of tandem mass spectrometry.

In still further instances, it may be preferable to operate the mass spectrometer of FIG. 3 in a high-energy-CTD, MS², precursor-ion or neutial-loss mode. In this mode, precursor ions generated in the ion source 300 are tiansferred along the x-axis, through the interface optics 302, through the quadrupole mass filter 304 (which is operating in a combined DC/RF mode to scan slowly, for example at a rate > 1 m/z-unit per second, through a range of /z-values specified by the user), through the low-energy CID cell 306 (which in this operating mode is nonfunctional), through the ion optics 310 (which in this operating mode are nonfunctional), and into the reaction/extraction volume of the tiansmission electron capture dissociation system 100 (which in this operating mode is not functioning to stimulate ECD). Then, as described in the preceding section on high-energy- CID, MS², product-ion analysis, the first stage of ion accelerator 312 is used to periodically accelerate ions onto the z-axis, and the second stage of ion accelerator 312 is used to time- lag focus them onto a space-focal plane (marked B in FIG. 3) that substantially coincides with the center of the velocity selector 318 (which in this operating mode is nonfunctional). The ions then drift from the velocity selector 318 to the high-energy CID cell 320 where they collide with target atoms or molecules. The fragment ions resulting from these collisions and any undissociated precursor ions drift into the ion accelerator 322 where they are simultaneously accelerated to an energy that is higher than they had when they entered ion accelerator 322 and time-lag focused onto a space-focal plane (marked C in FIG. 3) that substantially coincides with the object plane of reflection 326. From this object plane, the ions are analyzed at high resolution in MS3 by using reflection 326 as described in a preceding section. A precursor-ion spectrum or, alternatively, a neutial-loss spectrum may be generated as described in the preceding section on low-energy-CID precursor- ion/neutial-loss analysis. This mode of operation is unprecedented in the art of quadrupole- time-of-flight hybrid tandem mass spectrometry, but an analogous mode of operation is known and practiced on four-sector tandem mass spectrometers.

D. Product Ion MS¹ Modes

In some instances, it may be preferable to operate the mass spectrometer of FIG. 3 in an MS³ product-ion mode that combines low-energy-CID and high-energy-CID. Li this mode, ions generated in the ion source 300 are transferred along the x-axis, through the interface optics 302, through the quadrupole mass filter 304 (which is operating in a combined DC/RF mode to selectively tiansmit precursor ions of one particular mass-to- charge ratio), through the low-energy CID cell 306 (which in this operating mode is functional), through the ion optics 310 (which in this operating mode are nonfunctional), and into the reaction/extraction volume of the tiansmission electron capture dissociation system 100 (which in this operating mode is not functioning to stimulate ECD). Then, as described in a preceding section on high-energy-CID, MS², product-ion analysis, the first stage of ion accelerator 312 is used to periodically accelerate the ions in the transmission electron capture dissociation system from the x-axis onto the z-axis, and the second stage of ion accelerator 312 is used to time-lag focus them onto a space-focal plane (marked B in FIG. 3) that substantially coincides with the center of the velocity selector 318 (which in this operating mode is functioning to selectively transmit low-energy CTD product ions of one particular mass-to-charge ratio). The selected ions then drift from the velocity selector 318 to the high-energy CID cell 320 where they collide with target atoms or molecules. The fragment ions resulting from these collisions and any undissociated low-energy CID precursor ions drift into the ion accelerator 322 where, as described in a preceding section, they are simultaneously accelerated and time-lag focused onto the object plane (marked C in FIG. 3) of reflection 326. From this object plane, the ions are analyzed at high resolution in MS3 by using reflection 326, as described in a preceding section, to produce a low-energy- CTD/high-energy-CID product-ion spectrum. This mode of operation is unprecedented in the art of quadrupole-time-of-flight hybrid tandem mass spectrometry or any other form of tandem mass spectrometry.

In other instances, it may be preferable to operate the mass spectrometer of FIG. 3 in an MS³ product-ion mode that combines ECD and high-energy-CID. In this mode, ions generated in the ion source 300 are transferred along the x-axis, through the interface optics 302, through the quadrupole mass filter 304 (which is operating in a combined DC/RF mode to selectively transmit precursor ions of one particular mass-to-charge ratio), through the low-energy CID cell 306 (which in this operating mode is nonfunctional), through the ion optics 310 (which in this operating mode are either continuously or periodically functional), and into the reaction/extraction volume of the transmission electron capture dissociation system 100 (which in this operating mode is functioning to stimulate ECD). Then, as described in a preceding section on high-energy-CID, MS², product-ion analysis, the first stage of ion accelerator 312 is used to periodically accelerate the ions in the transmission election capture dissociation system from the x-axis onto the z-axis, and the second stage of ion accelerator 312 is used to time-lag focus them onto a space-focal plane (marked B in FIG. 3) that substantially coincides with the center of the velocity selector 318 (which in this operating mode is functioning to selectively tiansmit ECD product ions of one particular mass-to-charge ratio). The selected ions then drift from the velocity selector 318 to the high-energy CID cell 320 where they collide with target atoms or molecules. The fragment ions resulting from these collisions and any undissociated ECD precursor ions drift into the ion accelerator 322 where, as described in a preceding section, they are simultaneously accelerated and time-lag focused onto the object plane (marked C in FIG. 3) of reflection 326. From this object plane, the ions are analyzed at high resolution in MS3 by using reflection 326, as described in a preceding section, to produce an ECD/high-energy-CID product-ion spectrum. This mode of operation is unprecedented in the art of quadrupole- time-of-flight hybrid tandem mass spectrometry or any other form of tandem mass spectrometry.

In yet other instances, it may be preferable to operate the mass spectiometer of FIG. 3 in an MS³ product-ion mode that combines low-energy-CID, ECD, and high-energy-CID. In this mode, ions generated in the ion source 300 are tiansferred along the x-axis, through the interface optics 302, through the quadrupole mass filter 304 (which is operating in a combined DC/RF mode to selectively tiansmit precursor ions of one particular mass-to- charge ratio), through the low-energy CID cell 306 (which in this operating mode is functional), through the ion optics 310 (which in this operating mode are either continuously or periodically functional), and into the reaction/extiaction volume of the tiansmission electron capture dissociation system 100 (which in this operating mode is functioning to stimulate ECD). Then, as described in a preceding section on high-energy-CID, MS², product-ion analysis, the first stage of ion accelerator 312 is used to periodically accelerate the ions in the tiansmission election capture dissociation system from the x-axis onto the z- axis, and the second stage of ion accelerator 312 is used to time-lag focus them onto a space-focal plane (marked B in FIG. 3) that substantially coincides with the center of the velocity selector 318 (which in this operating mode is functioning to selectively transmit low-energy CED/ECD product ions of one particular mass-to-charge ratio). The selected ions then drift from the velocity selector 318 to the high-energy CID cell 320 where they collide with target atoms or molecules. The fragment ions resulting from these collisions and any undissociated low-energy CID precursor ions drift into the ion accelerator 322 where, as described in a preceding section, they are simultaneously accelerated and time-lag focused onto the object plane (marked C in FIG. 3) of reflection 326. From this object plane, the ions are analyzed at high resolution in MS3 by using reflection 326, as described in a preceding section, to produce a low-energy-CID/ECD/high-energy-CID product-ion spectrum. This mode of operation is unprecedented in the art of quadrupole-time-of-flight hybrid tandem mass spectrometry or any other form of tandem mass spectrometry. Although the invention has been described with reference to specific embodiments, it should be apparent to those of ordinary skill in the art that the arrangement and details disclosed herein may be modified without departing from the spirit and scope of the invention. Therefore, we claim all such modifications that fall within the scope and spirit of the following claims, and all equivalents thereto.

Claims

We Claim:

1. A transmission election capture dissociation system, comprising: a pulsed ion accelerator; and a source of elections positioned to deliver electrons to an extraction volume of the pulsed ion accelerator.

2. The system of claim 1 further comprising a source of ions positioned to deliver ions to the extraction volume of the pulsed ion accelerator in a direction that is substantially orthogonal to the direction in which ions are accelerated by the pulsed ion accelerator.

3. The system of claim 2 where the electron source and the ion source are positioned to deliver intersecting flows of elections and ions.

4. The system of claim 3 where the intersecting flows of elections and ions intersect at any angle within 4π steradians.

5. The system of claim 4 where the intersecting flows of electrons and ions are substantially orthogonal to each other.

6. The system of claim 4 where the intersecting flows of elections and ions are substantially opposite each other.

7. The system of claim 1 where the electron source comprises an electron monochromator.

8. The system of claim 1 where the pulsed ion accelerator is a multiple-stage pulsed ion acceleration system capable of time-lag focusing ions onto a space focal plane.

9. A mass spectrometer, comprising: a tiansmission election capture dissociation system, the tiansmission election capture dissociation system comprising a pulsed ion accelerator and an election source, the election source positioned to deliver elections to an extraction volume of the pulsed ion accelerator; an ion source positioned to deliver ions to the extraction volume of the pulsed ion accelerator in a direction substantially orthogonal to the direction in which ions are accelerated by the pulsed ion accelerator; and a first mass selective device positioned to receive ions accelerated by the pulsed ion accelerator.

10. The mass spectiometer of claim 9 where the first mass-selective device comprises a multipole mass filter.

11. The mass spectiometer of claim 10 where the multipole mass filter is a quadrupole mass filter.

12. The mass spectiometer of claim 9 where the first mass-selective device comprises a magnetic sector analyzer.

13. The mass spectiometer of claim 9 where the first mass-selective device comprises a time-of-flight analyzer.

14. The mass spectiometer of claim 13 where the time-of-flight analyzer comprises a reflection.

15. The mass spectiometer of claim 9 where the first mass-selective device comprises a tandem mass-selective device.

16. The mass spectiometer of claim 15 where the tandem mass-selective device is a time-of-flight-time-of-flight device.

17. The mass spectrometer of claim 15 where the tandem mass-selective device is a quadrupole-time-of-flight hybrid device.

18. The mass spectiometer of claim 9 where the pulsed ion accelerator is a pulsed ion acceleration system capable of time-lag focusing ions onto a space focal plane.

19. The mass spectiometer of claim 9 further comprising a second mass- selective device positioned between the ion source and the transmission electron capture dissociation system.

20. The mass spectiometer of claim 19 where the second mass-selective device is a quadrupole mass filter.

21. The mass spectrometer of claim 19 further comprising a CTD cell positioned between the quadrupole mass filter and the tiansmission electron capture dissociation system.

22. The mass spectiometer of claim 21 where the CTD cell is a low-energy CID cell.

23. The mass spectiometer of claim 22 where the first mass selective device is a tandem mass spectrometer comprising a time-of-flight-time-of-flight device, the time-of- flight-time-of-flight device including a high energy CID cell positioned between the TOF segments of the time-of-flight-time-of-flight device.

24. The tiansmission tandem mass spectiometer system of claim 9 where the ion source comprises an ion source selected from the group consisting of electiospray, atmospheric pressure chemical, corona discharge thermospray, matrix-assisted particle- induced desorption, fast atom bombardment, fast ion bombardment, ²⁵³Cf plasma desoφtion, election impact, chemical ionization, laser desoφtion, and matrix-assisted laser desoφtion ionization sources.

25. A tiansmission tandem mass spectrometer system, comprising: an ion source; an election source, the electron source comprising an electron monochromator; an election capture dissociation chamber fluidly coupled to the ion source and the election source, the electron capture chamber comprising a pulsed ion accelerator, a first input positioned to deliver ions from the ion source to an extraction volume of the pulsed ion accelerator in a direction substantially orthogonal to the direction ions are accelerated by the pulsed ion accelerator, a second input positioned to deliver elections from the electron source to the extiaction volume of the pulsed ion accelerator in a direction that intersects the ions delivered through the first input, and an output positioned to permit ions accelerated by the pulsed ion accelerator to exit the electron capture chamber; a quadrupole mass filter fluidly coupled between the ion source and the first input of the electron capture chamber; a CID cell fluidly coupled to the quadrupole mass filter and positioned to accept ions transmitted through the quadrupole mass filter; and a time-of-flight mass analyzer fluidly coupled to the output of the electron capture chamber and positioned to accept ions accelerated out of the election capture chamber in a direction substantially along the axis of the time-of-flight analyzer.

26. The tiansmission tandem mass spectiometer system of claim 25 where the ion source is selected from the group consisting of electiospray, atmospheric pressure chemical, corona discharge thermospray, matrix-assisted particle-induced desoφtion, fast atom bombardment, fast ion bombardment, ²⁵³Cf plasma desoφtion, electron impact, chemical ionization, laser desoφtion, and matrix-assisted laser desoφtion ionization sources.

27. The tiansmission tandem mass spectiometer system of claim 25 where the time-of-flight mass analyzer comprises a reflection.

28. The tiansmission tandem mass spectrometer system according to claim 25 further comprising a mass-selective device fluidly coupled to the ion source and the electron capture dissociation chamber and positioned to transmit ions from the ion source to the election capture dissociation chamber.

29. A mass spectiometer, comprising: an ion source; a first mass-selective device downstream from the ion source; a first CID cell downstream from the first mass-selective device; a tiansmission election capture dissociation system downstream from the first dissociation cell, the tiansmission electron capture dissociation system comprising a pulsed ion accelerator oriented to accelerate ions in a direction substantially orthogonal to the direction from which the ions enter an extraction volume of the pulsed ion accelerator and a source of electrons positioned to deliver elections to the extiaction volume of the pulsed ion accelerator along a path that intersects the ions entering the extiaction volume at some angle within 4π steradians of the direction of the ions; a second mass-selective device downstream from the electron capture dissociation chamber; a second CID cell downstream from the second mass-selective device; and a third mass-selective device downstream from the second dissociation cell.

30. The mass spectiometer of claim 29 where the first mass-selective device comprises a mass filter selected from the group consisting of monopole and multipole mass filters.

31. The mass spectiometer of claim 30 where mass filer is a multipole mass filter and is selected from the group consisting of quadrupole, hexapole, octapole and decapole mass filters.

32. The mass spectrometer of claim 31 where the multipole mass filter is a quadrupole mass filter.

33. The mass spectiometer of claim 29 where the first mass-selective device comprises a magnetic sector analyzer.

34. The mass spectiometer of claim 29 where the first mass-selective device comprises a time-of-flight analyzer.

35. The mass spectrometer system of claim 29 where the first CTD cell comprises a low-energy CID cell.

36. The mass spectiometer of claim 29 where the first CTD cell comprises a high-energy CID cell.

37. The mass spectrometer of claim 29 where the first CTD cell comprises a chamber for surface-collisionally induced dissociation.

38. The mass spectrometer of claim 29 further comprising an off-axis detector positioned between the first collision-induced dissociation cell and the transmission electron capture dissociation system.

39. The mass spectrometer of claim 29 where the source of electrons is an election monochromator.

40. The mass spectiometer of claim 29 where the second mass-selective device is a TOF device comprising a velocity selector.

41. The mass spectiometer of claim 40 where the velocity selector operates by a 180° change in the direction of an applied electric field.

42. The mass spectiometer system of claim 29 where the second mass-selective device comprises a magnetic sector analyzer.

43. The mass spectiometer of claim 29 where the second CTD cell a high- energy CID cell.

44. The mass spectrometer of claim 29 where the third mass-selective device comprises a time-of-flight mass analyzer.

45. The mass spectiometer of claim 44 where the time-of-flight analyzer comprises a reflection.

46. The mass spectiometer of claim 29 where the third mass-selective device comprises a magnetic sector analyzer.

47. A kit for converting an existing orthogonal extiaction quadrupole-time-of- flight hybrid mass spectiometer to an ECD-quadrupole-time-of-flight hybrid system, comprising, in packaged combination, an election source and electron optics configured to deliver elections to an extraction volume of a pulsed ion accelerator that is a component of the existing orthogonal extiaction quadrupole-time-of-flight hybrid mass spectrometer.

48. A method of stimulating election capture dissociation that is compatible with tiansmission mass spectrometer systems, comprising: directing multiply charged cations to an extiaction volume of a pulsed ion accelerator; and directing electrons to intersect the multiply charged cations within the extiaction volume of the pulsed ion accelerator.

49 A method for analyzing a sample, comprising: ionizing a sample to produce a first set of ions; accelerating the first set of ions in a first direction; using an ion accelerator to accelerate the first set of ions into a time-of-flight mass analyzer, the time-of-flight mass analyzer having a flight-axis that is substantially orthogonal to the first direction; using a velocity selector in the time-of-flight mass analyzer to select a second set of ions from the first set of ions, the second set of ions comprising ions that have the same mass-to-charge ratio; using a high-energy CTD cell to fragment at least a portion of the second set of ions and produce a third set of ions.

50. A method for analyzing a sample, comprising: ionizing a sample to produce a first set of ions; accelerating the first set of ions in a first direction; selecting a second set of ions from the first set of ions using a quadrupole mass filter, the second set of ions having the same mass-to-charge ratio; using an ion accelerator to accelerate the second set of ions into a time-of-flight mass analyzer, the time-of-flight mass analyzer having a flight-axis that is substantially orthogonal to the first direction; using a high-energy CTD cell to fragment at least a portion of the second set of ions to produce a third set of ions.

51. A method for analyzing a sample, comprising: ionizing a sample to produce a first set of ions, the first set of ions comprising ions having plural positive charges; accelerating the first set of ions in a first direction; selecting a second set of ions from the first set of ions using a quadrupole mass filter; the second set of ions comprising ions having the same mass-to-charge ratio; allowing at least a portion of the second set of ions to react with elections in an extiaction volume of an ion accelerator to produce a third set of ions; and using the ion accelerator to accelerate the third set of ions into a time-of-flight mass analyzer, the time-of-flight mass analyzer having a flight-axis that is substantially orthogonal to the first direction.

52. The method of claim 51 further comprising using a velocity selector to select a fourth set of ions from the third set of ions, all members of the fourth set of ions having the same mass-to-charge ratio.

53. The method of claim 52 further comprising use of a high-energy CTD cell to fragment at least a portion of the fourth set of ions to produce a fifth set of ions.

54. The method of claim 51 further comprising use of a high-energy CTD cell to fragment at least a portion of the third set of ions to produce a fourth set of ions.

55. The method of claim 51 where selecting a second set of ions comprises scanning the quadrupole mass analyzer across a range of mass-to-charge ratios to successively select, over a range of time, different second sets of ions comprising ions having the same mass-to-charge ratio.

56. A method for analyzing a sample, comprising: ionizing a sample to produce a first set of ions, the first set of ions comprising ions having plural positive charges; accelerating the first set of ions in a first direction; selecting a second set of ions from the first set of ions using a quadrupole mass filter; the second set of ions comprising ions having the same mass-to-charge ratio; using a low energy CTD cell to fragment at least a portion of the second set of ions to produce a third set of ions; allowing at least a portion of the third set of ions to react with elections in an extiaction volume of an ion accelerator to produce a fourth set of ions; and using the ion accelerator to accelerate the fourth set of ions into a time-of-flight mass analyzer, the time-of-flight mass analyzer having a flight-axis that is substantially orthogonal to the first direction.

57. The method of claim 56 further comprising using a velocity selector to select a fifth set of ions from the fourth set of ions, the fifth set of ions having the same mass-to-charge ratio.

58. The method of claim 57 further comprising using a high-energy CID cell to fragment at least a portion of the fourth set of ions to produce a fifth set of ions.

59. The method of claim 56 further comprising using a high-energy CTD cell to fragment at least a portion of the fourth set of ions to produce a fifth set of ions.

60. The method of claim 56 where selecting a second set of ions comprises scanning the quadrupole mass analyzer across a range of mass-to-charge ratios to select, over a range of time, different second sets of ions comprising ions having the same mass-to- charge ratio.

61. A method for analyzing a sample, comprising: ionizing a sample to produce a first set of ions, the first set of ions comprising ions having plural positive charges; accelerating the first set of ions in a first direction; using a low energy CID cell to fragment at least a portion of the first set of ions to produce a second set of ions; allowing at least a portion of the second set of ions to react with elections in an extraction volume of an ion accelerator to produce a third set of ions; and using the ion accelerator to accelerate the third set of ions into a time-of-flight mass analyzer, the time-of-flight mass analyzer having a flight-axis that is substantially orthogonal to the first direction.

62. The method of claim 61 further comprising using a velocity selector to select a fourth set of ions from the third set of ions, the fourth set of ions having the same mass-to-charge ratio.

63. The method of claim 62 further comprising using a high-energy CID cell to fragment at least a portion of the fourth set of ions to produce a fifth set of ions.

64. The method of claim 61 further comprising using a high-energy CID cell to fragment at least a portion of the fourth set of ions to produce a fifth set of ions.