Patent Grant
6858840
1. Field of the Invention
The invention relates to procedures and devices for fragmenting molecular ions, preferably biomolecular ions in tandem mass spectrometers.
2. Background of the Invention
Over the last decade, mass spectrometry has played an increasingly important role in the identification and characterization of biochemical compounds in research laboratories and various industries. The speed, specificity, and sensitivity of mass spectrometry make spectrometers especially attractive for requiring rapid identification and characterization of biochemical compounds. Mass spectrometric configurations are distinguished by the methods and techniques utilized for ionization and separation of the analyte molecules. The mass separation process can include techniques for ion isolation, subsequent molecular fragmentation, and mass analysis of the fragment ions. The pattern of fragmentation yields information about the structure of the analyte molecules introduced into the mass spectrometer. Increased fragmentation thus increases one's ability to distinguish one mass group from another mass group.
Indeed, ion isolation, molecular fragmentation, and mass analysis have been combined in a technique referred to as tandem mass spectrometry (or MS/MS) to thereby enhance identification of ion species. Tandem mass spectroscopy typically coupled with electrospray ionization (ESI) is a known technique utilized to produce gas phase ions of bio- and chemical molecules. Indeed, ESI is a soft ionization technique which produces multiply-charged molecular ions of large biomolecules. ESI continuously produces ions at normal atmospheric conditions. Once produced, the ions are introduced into a vacuum of a mass spectrometer using an atmospheric pressure interface. Liquid separation techniques such as for example high pressure liquid chromatography (HPLC), charge exchange (CE) generating radical ions of low internal energy, and on-line electrospray ionization mass spectrometry have all contributed to the success of modern biochemistry, pharmacology and health sciences. Even with these advances, the distinction of one large biomolecule from another depends on unique fragmentation patterns characteristic of the particular chemical bonding of the specific biomolecule.
Furthermore, the tandem mass spectrometer concept has been extended to triple quadrupole mass spectrometers. Triple quadrupole mass spectrometers have also been interfaced to electrospray ion sources. Triple quadrupole mass spectrometers offer medium resolution (up to several thousands Da) and low mass range (up to 2000-3000 Da) for MS/MS analysis. Further, systems known as QqTOF (or Q-TOF) combine two quadrupole mass sectors with time-of-flight mass analyzers (TOFMS). QqTOF techniques have been described for example by Morris et al, Rapid Commun. Mass Spectrometry, 1996, 10:889-896, and by Shevchenko et al, Rapid Commun. Mass Spectrom. 1997, 11:1015-1024, the entire contents of which are incorporated herein by reference. The QqTOF configuration can be considered as a replacement of the third quadrupole in a triple quadrupole instrument by a time-of-flight mass analyzer. The benefits of the QqTOF system are high sensitivity, mass resolution and mass accuracy in both precursor (MS) and product ion (MS/MS) modes. A particular advantage for full-scan sensitivity (over a wide mass range) is provided in both modes by the parallel detection feature available in TOF MS.
Fragmentation of ions is achieved in commercial tandem mass spectrometers through collisionally induced dissociation (CID) with buffer gas molecules in a quadrupole collision cell, see for example U.S. Pat. No. 6,285,027, the entire contents of which are incorporated herein by reference. In collisionally induced dissociation or fragmentation, the energy of collision is quickly redistributed over the large number of vibrational degrees of freedom available in large biomolecules. The energy redistribution leads to dissociation of bonds only of the lowest activation energy. Thus, the CID method seldomly provides sufficient MS/MS sequence information for proteins larger than 2 kDa. Since the excitation in CID is not specific, the most labile bonds are typically cleaved (which are often a modifying group) and not necessarily the structurally important bonds. Furthermore, CID requires the presence of the buffer gas at pressures of the order of 10 mTorr or more. Because the subsequent mass analyzer needs a relatively high vacuum for its operation, restricting apertures are introduced between the last fragmenting quadrupole and a second mass analyzer, thus reducing the number of ions transmitted and the overall sensitivity.
Electron capture dissociation (ECD) is a recent fragmentation technique that utilizes an ion-electron recombination reaction, as described by Zubarev et al, J. Am. Chem. Soc. 1998, 120: 3265-3266, the entire contents of which are incorporated herein by reference. The maximum cross section for the ion-electron recombination reaction occurs at very low electron energies (e.g., lower than 0.5 eV) and exceeds the collision cross section with neutral species by about 100 times. To date, ECD has been implemented in ion cyclotron resonance Fourier transform mass spectrometers (ICR-FTMS) with electrons injected directly into ICR cell only. Almost all ECD fragment ions come from a single bond cleavage. This makes electron capture dissociation well suited for protein sequencing. In contrast to CID, ECD is believed to be non-ergodic, i.e., the cleavage happens prior to any intramolecular energy redistribution. As a result, the ECD method cleaves more bonds than a conventional CID technique. Almost all known proteins in vivo contain post-translational modifications, which modulate and often define their biological function. Determination of the sites of these modifications is a top priority in proteomics studies. However, fragmentation using for example low-energy CID has the drawback of fragmenting the most labile bonds at the highest rate, which often are the linker bonds to the modifications. As a result, a modification group is often lost prior to the backbone fragmentation, making it difficult or impossible to determine a prior location of the modification group. In contrast, ECD cleaves specifically N—Cα bonds and imparts only a minimum of the internal energy into the fragments. The latter species, especially the even-electron c ions, i.e. one classification of fragmented peptides, retain the modification groups making their localization straightforward.
Recently, another type of ECD method referred to as “hot” electron capture dissociation (HECD) has been reported by Kjeldsen et al, Chem. Phys. Lett. 2002, 356: 201-206, the entire contents of which are incorporated herein by reference. Besides having a known maximum of electron capture dissociation (ECD) of gas-phase polypeptide polycations at low electron energy, a broad local maximum is found around 10 eV. The existence of this 10 eV maximum can be attributed to an electronic excitation prior to electron capture, a phenomenon similar to that in the dissociative recombination of small cations. In the HECD regime, not only N—Cα bonds are cleaved as in ECD, but secondary fragmentation is also induced due to the excess energy. Beneficially, this fragmentation includes abundant losses of, for example, CH(CH3)2 from Leucine and CH2CH3 from Isoleucine residues terminal to the cleavage site, which allows for distinguishing between these two isomeric residues. Even for larger molecules, the HECD produces abundant secondary fragmentation, despite the presence of substantially more degrees of freedom over which the excess energy could be distributed.
ECD/HECD ion fragmentation of biological molecules has been made in an ion cyclotron resonance Fourier transform mass spectrometer (ICR-FTMS) having electrons injected directly into the ICR cell. U.S. Patent Publication Application No. 2002/0175280, the entire contents of which are incorporated herein by reference, describes the use of electron capture dissociation for ion fragmentation in a three-dimensional ion trap. Since an electric potential inside the ion trap including the central point is time dependent, the electron source is kept at the highest positive potential achieved at the center of the ion trap during the RF cycle. Electrons can reach the ions stored inside the ion trap only during a period of few nanoseconds (or 0.1% of oscillation cycle) when the electric field potential at the center of the ion trap is close to the electron source potential. Together with a small size of aperture for electron beam introduction, this makes the effectiveness of ECD in this arrangement low.
Thus, to date, mass analyzers have not optimized electron capture dissociation to provide improved fragmentation and cleavage of input ionized species.
One object of the present invention is to inject electrons inside multipole ion guides in a mass analyzer sector such that injected electrons can interact with the input ionized species to more fully dissociate the ionized species.
A further object of the present invention is to supplement the dissociation by providing a buffer gas in the region of interaction of the ionized species.
Yet, another object of the present invention is to promote fragmentation via electron capture dissociation via interactions of the injected electrons with the ions present in the mass analyzer sector. As such, according to the present invention, improvements in tandem mass spectrometry capability are realized. Some of the advantages can include: (i) an increase of the mass range for tandem MS analysis; (ii) a simplification of the peptide sequencing process; (iii) a more reliable determination of posttranslational modifications, (iv) resolving the ambiguity arising from Leucine/Isoleucine isobars, and (v) high mass assignment accuracy for fragment ions.
In accordance with the present invention, there is provided a system and method for mass analysis on an ion beam. The system includes a mass selector (although optional in some of the embodiments of the present invention), at least one multipole ion guide, and a mass analyzer. In the present invention, ions are passed through a mass selector to select precursor ions with a desired mass to charge ratio, electrons are injected into the at least one multipole ion guide, the precursor ions are fragmented into product ions inside the multipole ion guide via electron capture dissociation from the injected electrons. The product ions are passed into a mass analyzer for mass analysis/detection.
A more complete appreciation of the present invention and many attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein:
Referring now to the drawings, wherein like reference numerals designate identical, or corresponding parts throughout the several views,
Conventional QqTOF tandem mass spectrometers have been used to improve mass accuracy, mass resolution and sensitivity. For example, U.S. Pat. Nos. 6,285,027 and 6,504,148, the entire contents of which are incorporated herein by reference, describe hybrid MS/MS instruments such as QqTOF instruments in which the final stage of mass analysis (MS2) is accomplished via a non-scanning time-of-flight (TOF) mass spectrometer. The hybrid MS/MS instruments have a duty cycle advantage over traditional MS/MS instruments in that the TOF section in the QqTOF instruments is not a scanning mass spectrometer, and all of the ions in the product ion mode are collected within a few hundred microseconds. These instruments are typically 10-100 times more sensitive than conventional instruments in the product ion scan mode of operation.
The QqTOF instrument 10 of the present invention includes an ion source 12 such as for example an electrospray source. Other suitable ion sources can be utilized, according to the present invention. Ions generated in the electrospray source 12 form a plume 13 from which ions are collected into a differentially pumped region 14, maintained at a pressure of for example a few Torr. The ions are then collected through a skimmer 16 and pass into a first collimating quadrupole 18 operated in RF-only mode. Quadrupole 18 can be operated, for example, at a pressure around 10−2 Torr. Downstream, another chamber 23 includes two main rod sets for quadrupoles 20 and 22. In this embodiment, the downstream chamber 23 is maintained preferably at a relatively low pressure, for example of approximately 10−5 Torr. The rod set for quadrupole 20 can be operated in a mass resolving mode to select ions with a particular m/z ratio. Selected ions then pass from quadrupole 20 into quadrupole 22 where, according to one embodiment of the present invention, the selected ions are subjected to electron capture dissociation (ECD). A housing 24 around quadrupole 22 allows optionally the addition of a buffer gas, for example, at pressures 10−3-10−2 Torr. The buffer gas promotes collisional focusing of product ions. Energy losses to the buffer gas reduces the energy of the ions inside quadrupole 22 allowing the ions, now having a lower energy, to accumulate (i.e., be focused) to the center of the quadrupole. Then, product ions and any remaining precursor ions enter an analyzer such as for example the TOF mass analyzer 26 depicted in FIG. 1. Electron capture dissociation can be facilitated according to the present invention by either introducing electrons into the region between quadrupoles 20 and 22 or by injecting electrons inside quadrupole 22.
As the ions leave quadrupole rod set quadrupole 22, the ions pass through ion focusing optics 28. A near parallel ion beam continuously enters an ion storage area 30 of an accelerator 32. Initially, the ion storage area 30 is field-free, so ions continue to move in their original direction. When a pulsed electric field is applied across the ion storage area 30, ions are deflected in a direction orthogonal to the original trajectory into an accelerating column. Ions exiting the accelerator 32 pass a field-free drift region 36 and then are reflected back in the ion mirror 38. After passing one more time through the field-free drift region 36, the ions strike a detector 40.
In one embodiment of the present invention, electrons are injected from an electron source 34 into a quadrupole ion guide through a slit 42 in one of the quadruple rods, as shown in
The potential distribution inside the ion guide, as calculated by SIMION 7.0 software at different times, is shown in
Electric field intensities inside the quadrupole ion guide Ex and Ey are described by following relations:
where U0 is an amplitude of the RF field, ω represents the frequency, Φ represents the phase, x,y represent the coordinates measured from the center, and r0 represents the inscribed radius. These relations show that electrons deviating from the x axis, located at the center of the slit, will experience a force towards this axis, thus focusing the electron beam toward the ion guide center.
In one preferred embodiment of the present invention having a simplified mechanical design, an alternative configuration of the quadrupole rod set is used having rectangular shaped rods. Electric potentials for a rod set having rectangular shaped rods is shown in
Decreasing the electric potential of the electron-emitting surface by approximately 10 V provides electrons to the center of the ion guide of an energy around 10 eV, thus allowing, according to the present invention, a switch to a “hot” electron capture dissociation (HECD) regime of ion fragmentation.
A regular sine RF waveform depicted in
A more elaborate mechanism for interacting an ion stream with electrons is depicted in FIG. 5. In this embodiment of the present invention, the Einsel lens arrangement, similar to those described in the aforementioned electron gun and electron source patents, results in only DC fields being present at the area where an electron beam is introduced. Further, as depicted in
Thus, the present invention in general provides a system and a method for performing the general process steps depicted in FIG. 6.
In step 600, the ions can be selected by transmitting the ions into a quadrupole mass selector and/or an ion trap mass selector. Prior to the ions being selected, the ions are generated by any number of well known ion production techniques such as for example the above-discussed electrospray ionization and chemical exchange techniques. As shown in
In step 610, the electron beam can be injected through a gap between electrodes of separate multipole ion guides, through a slit in an electrode of the multipole ion guide, and/or through electrodes of the multipole ion guide. The electrodes of the multipole ion guide can be round rods, hyperbolically shaped rods, and/or rectangular rods. An electron beam can be injected, and the electron beam controlled to a given electron beam current and duration of the electron beam. The measuring and control of the electron beam current and duration using any of the techniques known in the art for electron beam production and control. Accordingly, the injected electrons can be injected at an electric DC potential which is a few tenths of 1 V and/or a few volts lower than a potential at a central axis of the multipole ion guide. The electrons, in one preferred embodiment of the present invention, can be injected into a space between two adjacent multipole ion guides.
In step 620, ions are fragmented by electron capture dissociation. As noted previously, low energy electron capture dissociation or “hot” energy electron capture dissociation can be practiced to enhance molecular fragmentation. As such, precursor ions are interacted with electrons having either an energy close to 0 eV (i.e. low energy electron capture dissociation) or an energy sufficient to produce electronic excitation of the precursor ions prior to electron capture dissociation (i.e. hot energy electron capture dissociation). In one preferred embodiment of the present invention, the precursor ions are biomolecules, and the fragmentation of the biomolecules in the multipole ion guide of the present invention by electron capture dissociation facilitates identification of the biomolecular species.
Further, to facilitate fragmentation, the precursor ions and any remaining parent ions can be trapped to increase the probability of electron capture dissociation. In addition, a buffer gas can be added to provide collisional focusing. In step 620, fragmentation can be enhanced by capturing the electrons injected from the multipole ion guide in a space between two adjacent multipole ion guides. Further, the multipole ion guide can be provided with a radio frequency sine waveform and/or a square waveform having zero-voltage windows to increase electron capture. The square waveform having zero-voltage windows provides in the multipole ion guide larger time periods, as compared to the sine waveforms, in which the electric field potential is close to zero, thus increasing electron capture and dissociation.
Further, use of a square waveform having zero voltage windows permits electrons to be injected obliquely, such as for example at 45° angles such as shown in
In step 630, the product ions and any remaining precursor ions can be transmitted into at least one of a co-axial time-of-flight mass spectrometer, an orthogonal time-of-flight mass spectrometer, a quadrupole mass spectrometer, an ion trap mass spectrometer, a magnetic sector mass spectrometer and a Fourier transform mass spectrometer. Trapped ions in a linear ion guide (e.g., in the at least one multipole ion guide) can be periodically released into the time-of-flight mass analyzer for mass analysis. A release of trapped ions and a start of push-pull pulses in the time-of-flight mass analyzer can be delayed to improve a duty cycle of the time-of-flight mass analyzer.
Numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.
| Number | Name | Date | Kind |
|---|---|---|---|
| 4731533 | Vestal | Mar 1988 | A |
| 6285027 | Chernushevich et al. | Sep 2001 | B1 |
| 20020092980 | Park | Jul 2002 | A1 |
| 20020175280 | Franzen | Nov 2002 | A1 |
| Number | Date | Country |
|---|---|---|
| WO 03103007 | Nov 2003 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20040232324 A1 | Nov 2004 | US |
