This application may relate to a method and apparatus for identifying components of a biomolecule, and in particular to the identification of proteins.
The dissociation of gaseous polypeptide ions, in a tandem mass spectrometry experiment, plays a role in several commonly used approaches for the identification of proteins. The most commonly used approach for activating polypeptide ions has involved energetic collisions with neutral target gases, and is referred to as collisional activation.
A range of collisional activation conditions have been utilized that include collision energies ranging from a few electron volts to as high as several kilo-electron volts; numbers of collisions ranging from a single collision to many hundreds of collisions; and, time-scales ranging from the time for a single collision to hundreds of milliseconds. In general, collisional activation methods have been useful in deriving primary structure information from peptide and protein ions. However, no single dissociation method has able to provide all structural information of interest. For example, collisional activation often fails to provide complete primary structure information, and often fails to provide information regarding the positions of post-translational modifications.
Approaches other than energetic collisions with gaseous targets have been also examined for dissociating polypeptide ions. These include, for example, collisions with surfaces, referred to as surface-induced dissociation, and a range of photo-dissociation techniques, such as infra-red multi-photon dissociation (IRMPD), black-body infra-red dissociation (BIRD), and single-photon UV-photo-dissociation at one of several wavelengths. In the case of multiply-protonated polypeptides, ion-electron and ion-ion reactions have been used. Electron capture by, or electron transfer to, a multiply-protonated peptide gives rise to fragmentation that is often highly complementary to that resulting from collisional activation. The former is referred to as electron capture dissociation (ECD) and the latter is referred to as electron transfer dissociation (ETD). Both ECD and ETD have proven to be of particular utility for the characterization of post-translationally modified peptide and protein cations.
In the case of an ETD experiment, products from an ion/ion reaction can be allocated into one of three principal categories. These are proton transfer, a competing ion/ion reaction that generally does not lead to fragmentation; electron transfer followed directly by dissociation (i.e., the ETD process); and, electron transfer without subsequent dissociation of the polypeptide product. The partitioning between these three reaction categories is, at least to some extent, particular to each species of reactant ions. For example, the competition between proton transfer and electron transfer is known to depend strongly upon the identity of the reagent anion. The size and charge state of the peptide ion may play a significant role in determining the extent to which ETD occurs relative to electron transfer without dissociation, and the temperature of the bath gas in the electrodynamic ion trap used as a reaction vessel can affect the relative contributions of the total ETD, the relative contributions of individual reaction categories that contribute to ETD, and the extent of electron transfer without dissociation. Doubly-protonated peptides of the size often observed from tryptic digests, for example, usually show significantly less ETD than the triply charged versions of the same peptide.
From an analysis perspective, it may desirable to minimize the competitive proton transfer channel and to maximize ETD relative to electron transfer without dissociation. The selection of the reagent anion may be important in this regard. One technique that may maximize the extent of ETD for species that undergo electron transfer, but do not dissociate, is to subsequently activate these electron transfer products. That is, subsequent activation of the electron transfer (ET) “survivors” can improve the net conversion of precursor ions to structurally informative product ions.
In this regard, it may desirable to maximize the dissociation of the survivors while minimizing dissociation of proton transfer products. The latter species generally give rise to b-and y-type ions that could complicate spectral interpretation and may compromise the quality of data-base matching algorithms that assume the formation of only the c-and z-type ions generally associated with ETD. The use of elevated bath gas temperature is one technique of altering the extent of ETD. However, this approach can affect both the reactant ions and the product ions and, therefore, may not be exclusively an activation method for survivor ions.
The use of elevated bath gas temperatures, for example, has not been shown to consistently provide improved ETD yields relative to the use of room temperature bath gas.
A method of mass spectrometry is disclosed, the method including creating a first ion trapping volume within a chamber of a first ion trap; injecting a first population of ions into the ion trapping volume so that the first population is stored in the trapping volume; injecting a second population of charged ions into the first ion trapping volume such that a physical overlap of the first and the second ion populations occurs; creating a second ion trapping volume within a chamber of a second ion trap; ejecting ions from the first ion trap into the second ion trap; and, performing mass spectrometry on the ions of the second ion trap.
In an aspect, a method of performing mass spectrometry includes: operating a first source of ions; selecting first ions by a mass filter and injecting the selected first ions into a first volume acting as first linear ion trap (LIT); switching off the first source and cooling the selected first ions in the first LIT; operating a second source of ions; selecting second ions by a mass filter and injecting the selected second ions into the first volume acting as a LIT; storing the first and second selected ions in the first LIT; ejecting stored ions from the first LIT to a second LIT; and, analyzing a mass of the ions of the second LIT.
An apparatus for analyzing molecules is disclosed including a first linear ion trap (LIT), configured to accept and store a first population of ions; and, to accept and store a second population of ions. A second linear ion trap (LIT) is configured to accept stored ions ejected from the first LIT by application of a voltage between the first and the second LITs; and, a mass analyzer for analyzing the ions of the second LIT.
Exemplary embodiments may be better understood with reference to the drawings, but these embodiments are not intended to be of a limiting nature. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention which, however, may be practiced without some or all of these specific details. In other instances, well known process operations have not been described in detail in order not to unnecessarily obscure the description.
The Q TRAP electronics were modified to superimpose auxiliary RF signals on the containment lenses IQ2 and IQ3 of the Q2 quadrupole array, which allowed the mutual storage of oppositely charged ions in the Q2 cell. The frequency and amplitude of the auxiliary RF signals applied to the containment lenses IQ2 and IQ3 of the Q2 quadrupole array were optimized for the electron transfer ion/ion reaction experiments. The Q TRAP operated at a drive RF frequency of 650 kHz.
The ion path was based on that of a triple-quadrupole mass spectrometer with the last quadrupole rod array (Q3) configured to operate either as a conventional RF/DC mass filter or as a linear ion trap (LIT) with mass-selective axial ejection (MSAE).
The ions from the sources travel through a curtain gas and differential pumping regions (OR, SK) into a quadrupole ion guide (Q0). The Q0 chamber and the analyzer chamber were separated by a differential pumping aperture IQ1. The analyzer chamber contained three round-rod quadrupole arrays in series: viz., an analyzing quadrupole Q1, a collision-cell quadrupole (Q2), and an analyzing quadrupole (Q3). Each of the quadrupoles was 127 mm in length with a field radius of 4.17 mm. A short RF-only Brubaker lens (ST), located in front of the Q1 quadrupole, was capacitively coupled to the Q1 drive RF power supply.
The Q TRAP electronics were modified to superimpose auxiliary RF signals on the containment lenses IQ2 and IQ3 of the Q2 quadrupole array, which allowed mutual storage of oppositely charged ions in the Q2 cell acting as a linear ion trap (LIT). The frequency and amplitude of the auxiliary RF signals applied to the containment lenses IQ2 and IQ3 of the Q2 quadrupole array were optimized for the electron transfer ion/ion reaction experiments.
The Q3 quadrupole was constructed from round gold-coated ceramic rods. Downstream of Q3 there were two additional lenses, the first with a mesh-covered 8-mm-diameter aperture, and the second with an open 8-mm aperture. These lenses are referred to as the “exit lens” (EX) and “deflector”, respectively. Generally, the deflector was held at about 200 V more attractive with respect to the exit lens in order to extract ions from the Q3 LIT toward the ion detector, an ETP (Sydney, Australia) discrete dynode electron multiplier. The detector was operated in pulse counting mode with the entrance floated to −6 kV for positive ion detection and +4 kV for negative ion detection. An auxiliary RF voltage applied to Q3 was ramped in proportion to mass/charge (m/z) during the analytical scans. The ions trapped within the Q3 LIT were resonantly excited by a 380-kHz signal and mass-selectively ejected axially.
The pulsed dual ionization source was coupled directly to the interface of the QTRAP mass spectrometer, and included a nano-electrostatic-ion (ESI) emitter for the generation of multiply charged peptide cations, and an atmosphere pressure chemical ionization (APCI) needle for the formation of radical anions derived from azobenzene. Sequential pulsing and accumulation of the oppositely charged ions were under the control of the Daetalyst 3.6 software, provided by MDS SCIEX.
The materials used in an experimental example of the method were methanol and glacial acetic acid (Mallinckrodt, Phillipsburg, N.J.); and peptides KGAILKGAILR, LLLLKLLLK and LIAGDGAILR were synthesized from SynPep (Dublin, Calif.). Solutions of peptides were diluted to 20 μM in 50/50/1 (v/v/v) methanol/water/acetic acid for positive nano-electrospray (nano-ESI). Azobenzene was obtained from Sigma-Aldrich (St. Louis, Mo.). The materials were used without further purification.
An example of performing the method using the apparatus of
The method described results in the collisional activation of ions upon transfer from Q2 into Q3 of the apparatus. The collisional activation of ions occurs upon transfer from the relatively high pressure environment of Q2 into the lower pressure environment of Q3, which is operated in the ion trapping mode. Here, Q3 is operated throughout the transfer process so as to collect as wide a range of product ions as possible, rather than initial operation of Q3 at an RF amplitude level sufficiently high to avoid collection of dissociation products lower in mass-to-charge ratio than that of the precursor ion of interest. Operation of Q3 in the latter mode may introduce a delay period before collection of dissociation products, such that fragments from relatively long-lived precursor ions are sampled. In the method described herein, substantially all ETD products formed initially in Q2 are transferred to Q3, while subjecting the ions to relatively mild collisional activation in the transfer process. In this way, relatively fragile species, such as survivor ions from electron transfer, may be induced to fragment while the fragmentation of proton transfer products, as well as of first generation electron transfer dissociation products, may be minimized.
The extent of ETD may be much lower for doubly-protonated peptides with about 10-20 residues than for triply-charged versions of the same peptide. Such peptide ions, therefore, are candidates for application of a post-ion/ion reaction collisional-activation step.
for doubly-protonated species. By excluding the signal due to unreacted doubly-protonated peptides, the % ETD term indicates the percentage of reacting peptide species that give rise to electron transfer fragments. For the data of
The improvement in % ETD for a particular polypeptide reactant depends upon the extent to which ET survivors are formed, and the efficiency with which the survivors can be dissociated. The % ETD may be increased both by the selection of the anionic reagent and by the selection of conditions for subsequent activation of the survivors.
Dissociation of proton transfer product ions and undissociated polypeptide precursor ions may be reduced so as to simplify the data interpretation.
These products may arise from collisional activation of the precursor ion upon injection into Q2 from Q1 as well as from the dissociation of residual reactant ions and the proton transfer product upon transfer from Q2 to Q3.
The data collected at voltage difference of 2 V represents a condition in which almost all of the fragment ions may have been formed in Q2 prior to transfer into Q3. As the voltage difference increases, the changes in fragment ion abundances may be due to dissociation resulting from the Q2 to Q3 transfer process. The results suggest a factor of approximately three increase in the yields of electron transfer dissociation products over the Q2/Q3 voltage difference range (2-12 V). The shape of the b-and y-type fragment curve (Curve 2) differs from that of the electron transfer fragment curve (Curve 1) as the value increases more slowly up until approximately 12 V, and then increases relatively rapidly at high voltage differences. This may be consistent with the stabilities of the electron transfer survivors being lower than those of the proton transfer products and may allow for the establishment of a voltage difference that may preferentially sample electron transfer survivors. At the highest difference voltages in the present experiments, the contribution from first generation electron transfer fragments may decrease, which may arise from further dissociation of the first generation fragments.
Ion-trap collision-induced dissociation of electron transfer survivor ions may yield some products from cleavages of amide bonds that may not be the same as those observed for direct ETD. Similar observations have been made with the method for collisional activation of the survivor ions, as illustrated in
The acceleration of ions from one linear ion trap to another in the presence of a background gas at roughly 1 mtorr may be used to effect collision-induced dissociation. The extent of collision-induced dissociation of species with different stabilities may be controlled. For example, the voltage difference between the two ion traps can be adjusted to maximize dissociation of electron transfer survivor ions while minimizing collision-induced dissociation of proton transfer and residual precursor ions. The use of this method increases the % ETD and extent of structural information from polypeptide ions that tend to yield a significant population of electron transfer survivor ions. The method does not require extensive tuning and the acceleration may not be significantly dependent upon the mass-to-charge ratios of the ions. The method also does not require careful matching of a resonance excitation frequency to the ions of interest when compared to ion trap collisional activation.
This method may be used, for example, in proteomics, where protein identification and characterization is accomplished by dissociation of peptide or protein ions. The method results in activating surviving electron transfer products without heating the vacuum system.
Although only a few examples of this invention have been described in detail above, those skilled in the art will readily appreciate that many modifications are possible without materially departing from the novel teachings and advantages of the invention. Accordingly, all such modifications are intended to be included within the scope of this invention as defined in the following claims.
This application claims the benefit of U.S. provisional application Ser. No. 60/872,357, filed on Dec. 1, 2006, which is incorporated herein by reference.
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