Patent Application
20090256067
1. Field of the Invention
The present invention relates to a device and method for achieving electron capture dissociation in radiofrequency ion traps in order to effect selective dissociation of molecular ions.
2. Background Information
Mass spectrometry (MS) analysis of proteins and peptides is a critical function in proteomic studies. MS instrumentation enables selective ion fragmentation that can give structure and sequence information of peptides and proteins leading to identification and in some cases the function of protein in cellular processes. The principal means of achieving sequence information is by collision-induced dissociation (CID) using ion trap, triple quadrupole, or fourier transform MS systems. This method delivers moderate energy to ions, which then undergoes energy randomization to create many chemical bonds. Consequently only the weakest bonds tend to dissociate by CID. Also as molecules increase in size (e.g., proteins), energy randomization becomes so extensive that there is not sufficient energy in any particular bond to cause it to break. Although CID is effective at fragmenting peptides, the bond breaking is generally only along the amide N-C(O) bond generating the so-called b (N-terminal) and y (C-terminal) series as illustrated in
Electron capture dissociation (ECD) is a method of fragmenting peptide and protein bonds in a manner that can give information on post-translational modifications (PTMs). Fragmentation by CID often leads to neutral loss of PTM sites complicating the identification of the sequence and the location of the PTM group. ECD is based on the recombination of a low-energy electron (e.g., <1 eV) with a protonated (or multiply protonated) peptide or protein as illustrated in
ECD is difficult to implement in radiofrequency (RF) driven mass analyzers, such as quadrupole and linear ion traps (QIT and LIT, respectively) because the RF fields accelerate the electrons to energies that greatly exceed what is needed for efficient ECD. ECD has therefore been most successfully employed in FT MS systems where ion containment is achieved by magnetic fields, which impart orbital motion on electrons, but do not accelerate them to higher energy. A complementary method called electron transfer dissociation (ETD) has been developed for RF ion traps and is based on delivering a beam of negative ions and allowing them to transfer an electron through recombination with protonated peptides and proteins to induce dissociation. Because of the added complexity and inefficiency of generating a negative ion beam, it is greatly desirable to develop an ECD method for RF ion traps.
There is a great need to develop ECD methods for RF driven ion traps for several reasons: (i) they are commonly used in proteomics research, (ii) they have much faster analysis times than FT MS systems, which is important for high-speed chromatographic techniques, and (iii) they are more economical than FT MS.
There are three prior art techniques for performing ECD in RF ion traps, each of which attempts to inject and maintain low-energy electrons in the trap for sufficient periods of time for useful reaction with positive ions to occur. Silivra et al. injected low-energy electrons into a quadrupole ion trap (QIT) at the beginning of the positive RF semi-period and trapped the electrons with an axial magnetic field by installing permanent magnets inside the ring electrode. The difficulty of this method is that the electrons remain cool for less than one period of the ring electrode RF (i.e., about 1 microsecond) resulting in inefficient ECD and reactions leading to undesired fragmentation.
Ding and Brancia developed a technique for avoiding the accelerating effect of the RF field on the electron energy. They used a square wave generator for the RF ring electrode trapping potential of a QIT, which they termed a digital ion trap. The benefit is that the potential in the ion trap is non-varying except during the step voltage change at each half cycle. These investigators then injected low energy electrons during the low voltage phase of the digital waveform. No magnet is needed by this method; however, the electrons were injected well outside the ion trap, which made it difficult to decelerate and localize the electrons at the center of the ion trap. Consequently, very long ECD interaction times (e.g., 400 ms/scan×250 scans=100 s) were required, which is not amenable to use with chromatography.
U.S. Patent Application 20060169892 filed by Baba et al., achieved ECD using a linear ion trap (LIT) and a magnetic field. Unlike a QIT, the center axis of a LIT is at zero potential. By using a magnetic field and an electron emitter at one end of the linear trap, these investigators succeeded in injecting a sufficiently high density of low-energy electrons to obtain efficient ECD as measured by reaction times of only a few milliseconds. A disadvantage of this method is complexity as it required a second linear ion trap positioned orthogonal to the ion path leading to the TOFMS, as well as the use of a permanent magnet.
Other approaches to ECD in mass spectrometers have been patented. U.S. Pat. No. 6,858,840 issued to Berkout et al. disclose a technique of performing ECD in multiple ion guides, in which electrons are injected into an RF ion guide and the most effective electron energy occurs during a very small fraction of the RF oscillation period. The Berkout patent also discloses using square waves for the RF so that the electric field is constant for a larger fraction of the RF period. This approach is similar to that used by Ding and Brancia in their digital ion trap.
U.S. Pat. Nos. 6,919,562 and 7,049,584 issued to Whitehouse et al. also disclose a technique of performing ECD in multiple ion guides by taking advantage of electron velocity reversals (e.g., slow down and turn around) occurring in the RF field. The electrons then have momentary zero velocity to react with ions by ECD. However, in this method the effective reaction times are very short because of the immediate reacceleration of the electrons in the RF field. These inventors also disclose using a magnet to help contain the electrons in the reactive volume.
U.S. Pat. No. 6,924,478 issued to Zubarev discloses a technique to perform ECD in an ion trap in which electrons are injected and some ion fragmentation occurs and then vibrational excitation is added to achieve further ion fragmentation. The patent discloses that the RF field in the ion trap or ion guide will accelerate the electrons and create high energy electron capture dissociation. However, this method does not give the selective N—Ca bond breakage that gives the informative c and z series of peptide ion fragments shown in
U.S. Pat. No. 7,227,133 issued to Glish et al. disclose a technique for performing ECD in a magnetic trap followed by mass analysis. This patent does not provide disclosures for performing ECD in an RF ion trap or ion guide.
A detector system that has an ion trap coupled to a electron source and a detector. The system includes a controller coupled to the ion trap and the electron source to turn the ion trap and the electron source on and off, such that the electron source is on when the ion trap is off.
Disclosed is a system and method that includes injecting low-energy electrons in a radiofrequency (RF) ion trap in order to dissociate positive ions by electron capture. The system includes an ion trap, a controller to provide RF that can be switched on and off rapidly, and a source of low-energy electrons that can be turned on and off synchronously with the radiofrequency on/off periods. The RF can be applied to the ring electrode of a QIT and to the linear electrodes of a LIT. The disclosed method can be used with other ion multiple guides that operate with RF. It can also be applied to QIT, LIT, and other multiple ion guides that serve for ion storage only and also for mass analysis.
In order to deliver a beam of low-energy electrons into an ion trap it is necessary to have a constant voltage potential in the region of greatest ion density so that the electrons are not accelerated. This can be achieved by turning the RF applied to the ion trap momentarily off and turning on the electron beam during the off period.
The steps involved in an MS/MS scan using ECD are:
(i) Ion collection/isolation: Ions are allowed to enter the QIT while an oscillating voltage is applied to the endcaps 104 to destabilize all but one ion mass-to-charge (m/z) value in a standard QIT mass isolation mode. This MS/MS step is usually preceded by a MS survey scan that determines what ions are present and a determination of what m/z value to store for further dissociation.
(ii) CID or ECD: For standard CID, an oscillating voltage is applied to the endcaps 104 to energize the stored ions and cause them to dissociate. In this case the RF applied to the ring electrode 102 remains on, although its value may be varied from that of the ion collection/isolation step in order to optimize the stabilization of the fragment ions. As shown in
(iii) Ion cool down: This step may or may not be necessary, but there may be a period of subsequent ion cooling prior to mass analysis in the QIT.
(iv) Mass analysis: For conventional QIT MS, the RF on the ring electrode 102 stays on as the ions are scanned out of the QIT. This is achieved by ramping the RF amplitude. Other methods can also be used including an auxiliary field on the endcaps 104 or ring electrodes 102 or a combination of auxillary fields and ramping of the RF amplitude on the ring electrode 102. A similar timing diagram is given in
The disclosed method for performing ECD in a QIT device also enables the combination of ECD and CID to be performed. This can be done on successive scans or it can be done on the same scan by applying CID excitation waveforms to the endcap electrodes 102 by conventional methods.
Also disclosed is a system and method to generate low-energy electrons inside the QIT by photoemission.
All of the means, methods, and embodiments described within for electron interactions with positive ions also apply to the interaction of positron (electron antiparticle) interactions with negative ions.
The disclosed invention has several advantages compared to existing methods including (i) no need for a permanent magnet, (ii) mechanical simplicity, (iii) efficient coupling of the electron source to the center of the QIT, (iv) high density of electrons and positive ions to enhance reaction rate, (v) opportunity to do rapid switching between CID and ECD and even to do both on the same scan cycle. The disclosed invention is applicable to any QIT or LIT instrument, which is the general embodiment described here. However, also described is a particular embodiment for QIT-TOF, which has the advantage of significantly reduced mass analysis times and enables higher repetition rates for MS/MS analysis.
While certain exemplary embodiments have been described and shown in the accompanying drawings, it is to be understood that such embodiments are merely illustrative of and not restrictive on the broad invention, and that this invention not be limited to the specific constructions and arrangements shown and described, since various other modifications may occur to those ordinarily skilled in the art.
