WO2013061145A1

WO2013061145A1 - Method and apparatus for suspending ion-ion reactions

Info

Publication number: WO2013061145A1
Application number: PCT/IB2012/002157
Authority: WO
Inventors: John L. CAMPBELL
Original assignee: Dh Technologies Development Pte. Ltd.
Priority date: 2011-10-26
Filing date: 2012-10-26
Publication date: 2013-05-02

Abstract

A method and apparatus are provided for analyzing a sample of molecules. The method includes generating a first population of precursor ions and reagent ions; transmitting desired precursor ions or reagent ions from the first population into a reaction volume; applying a notched tailored waveform to the reaction volume; subjecting all the ions in the reaction volume to the waveform with the exception of the desired precursor ions and reagent ions whose frequencies exist within the notches of the tailored waveform; and introducing the first population of reagent ions or precursor ions into or through the reaction volume wherein a reaction between the reagent ions and the desired precursor ions occurs forming product ions.

Description

METHOD AND APPARATUS FOR SUSPENDING ION-ION REACTIONS

RELATED APPLICATION [0001] This application claims priority to U.S. Provisional Application No. 61/551 ,716 filed October 26, 201 1, which is incorporated herein by reference in its entirety.

FIELD [0002] The applicant's teachings relate to a method and apparatus of analyzing a sample of molecules, and, in particular, a method and apparatus for suspending ion-ion reactions.

INTRODUCTION [0003] Ion/ion reactions, such as electron-transfer dissociation (ETD), electron capture dissociation (ECD), negative ion ETD and ECD, and proton-transfer reactions (PTRs), have been employed as analytical tools for probing the structure and sequences of ionized biomolecules, such as peptides, proteins, and oligonucleotides. For example, ECD and ETD are well known to fragment ionized peptides by cleaving the peptide back-bone bonds while preserving labile post-translational modifications (PTMs) (e.g., phosphorylation, glycosylation). The most informative product ions produced in both ECD and ETD are the complementary c- and z-type fragment ions. This method differs from traditional ion fragmentation methods, such as collision-induced dissociation or infrared multi-photon dissociation that yield b- and y-type fragment ions for ionized peptides. In addition, the latter dissociation methods often cleave the aforementioned labile PTMs from the peptide ions, losing valuable information. In the field of proteomics, ETD and ECD have been used for sequencing applications in both "bottom up" (sequencing peptides from digested proteins) and "top down" (sequencing of intact proteins) scenarios.

[0004] While ion/ion reactions are renowned for their utility, there can be some drawbacks to their implementation. It has been well documented that the rate of an ion/ion reaction, such as those involved in ETD, is dependent upon the total charge state of the reacting system; the reaction rate increases as the square of the sum of all charges involved (both precursor and reagent ions). This has implications for the analysis of highly charged (z > 5) precursor ions using ion/ion reactions. For example, the analysis of a peptide ion with z = 3 by ETD provides a simple product ion spectrum containing mostly fragments with z = 1 charge state. In this case, one electron-transfer event occurs with the z = 3 [(M+3H)³⁺] precursor ion, forming a charge-reduced species of z = 2 [(M+3H)^2+"], which rapidly fragments to yield c- and z-type ions. The fragment ions are of a sufficiently low charge state that the rate of a secondary electron-transfer reaction is too slow for the time-scale of the experiment such that the reaction period can be terminated prior to this secondary reaction occurring.

[0005] However, when the precursor ion in an ETD reaction has a much higher charge state (e.g., z > 5), control over subsequent electron-transfer events (e.g., secondary, tertiary, ETD reactions) becomes much more tedious. These subsequent ETD reactions (first-generation fragment ions reacting again with ETD reagent anions) have reaction rates that are both very fast and very close in rate to the resulting precursor ions than in the aforementioned case of the lower charge state precursor (e.g., z = 3). The result of these unwanted subsequent ETD reactions is a convoluted product ion spectrum that contains many variants of the expected c- and z-type fragment ions, making interpretation of the resultant ETD product ion mass spectrum very difficult.

[0006] Controlling such unwanted subsequent ion/ion reactions can be accomplished in different ways. In some cases, the difference in the reaction rate between the precursor ion and the fragment ions is sufficient that a simple truncation of the ETD reaction period will limit the amount of subsequent ETD reactions. This is the case when the precursor ions are of lower charge states (e.g., z = 3). However, when the precursor ions have much higher charge states (e.g., z > 5), the difference in the reaction rates of the precursor ions and the fragment ions is much closer, as shown in Figure 1, and simple truncation of the reaction time may be insufficient to stop these fast, convoluting reactions. For example, the difference in the rate of an ion/ion reaction involving a z = 2 ion is -55% faster than that of a z = 1 ion. However, this rate difference drops to only -20% for a z = 8 ion and a z = 7 ion; hence, the ETD fragment ions of a z = 8 precursor ion would react almost as quickly as the precursor ion, resulting in an unnecessarily convoluted ETD mass spectrum.

[0007] One method for impeding such subsequent ion/ion reactions is termed ion parking. Normally, when precursor ions are subjected to PTRs, ions with higher charge states are charge reduced to lower charge states. For example, a precursor ion of z = 5 will have its charge reduced by subsequent PTRs to the z = 4, 3, 2, and 1 charge states (including total charge removal to z = 0). However, during ion parking, a waveform is applied to the ion/ion reaction volume that is at the frequency of one of the charge-reduced species (e.g., the z = 4 ion), and the ion/ion reaction is impeded at that specific charge state (frequency). Subsequent ion/ion reactions (e.g., PTRs) will not occur (i.e., no ions of z = 3, 2, or 1 will appear during or after the ion/ion reaction) and the ion/ion reaction products is efficiently funnelled into this one particular ion.

[0008] Accordingly, other methods for impeding subsequent ion/ion reactions are disclosed herein.

SUMMARY

[0009] In accordance with an aspect of the applicant's teachings, a method of analyzing a sample of molecules is provided. In various embodiments, the method can comprise generating a first population of precursor and reagent ions; transmitting desired precursor ions or reagent ions from the first population into or through a reaction volume; applying a notched tailored waveform to the reaction volume; subjecting all ions in the reaction volume to the waveform with the exception of the desired precursor ions and reagent ions whose frequencies exist within the notches of the tailored waveform; and introducing the first population of reagent ions or precursor ions into or through the reaction volume wherein a reaction between the reagent ions and desired precursor ions occurs forming product ions. In various embodiments, the waveform is applied during a reaction period for impeding subsequent ion- ion reactions. In various aspects, the reaction period occurs from the time of introducing the reagent ions to the end or completion of the reaction. In various embodiments, a second population of ions is introduced into or through the reaction volume wherein a physical overlap of the first and second ion population occurs.

[0010] In accordance with the applicant's teachings, an apparatus for analyzing a sample of molecules is provided. In various embodiments, the apparatus can comprise an ion source for generating a first population of precursor and reagent ions; a reaction volume for receiving or transmitting desired precursor ions and reagent ions; and a waveform generator for applying a notched tailored waveform to the reaction volume during a reaction period and subjecting all ions in the reaction volume to the waveform with the exception of the desired precursor ions and reagent ions whose frequencies exist within the notches of the tailored waveform. In various aspects, the reaction period occurs from the time of introducing the reagent ions to the completion of the reaction. In various embodiments, the reaction volume receives or transmits a second population of ions wherein a physical overlap of the first and second ion populations occurs.

[0011] These and other features of the applicants' teachings are set forth herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The skilled person in the art will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the applicants' teachings in anyway.

[0013] Figure 1 shows a plot of the difference in ion/ion reaction rates for ions of adjacent charge.

[0014] Figure 2 shows a notched tailored waveform applied in accordance with the applicant's teachings.

[0015] Figure 3 shows a sustained off-resonance irradiation (SORI) waveform applied in accordance with the applicant's teachings.

DESCRIPTION OF VARIOUS EMBODIMENTS

[0016] It should be understood that the phrase "a" or "an" used in conjunction with the applicants' teachings with reference to various elements encompasses "one or more" or "at least one" unless the context clearly indicates otherwise.

[0017] In accordance with the applicant's teachings, a method is provided for impeding subsequent ion/ion reactions that can use similar excitation as ion parking but without applying a specific frequency for impeding the ion/ion reaction of an ion selected a priori. In various embodiments, the method can comprise generating precursor ions and reagent ions and injecting an initial population of ions into the reaction volume so that a desired first population is transmitted to and stored in the reaction volume. In various embodiments, a notched tailored waveform can be applied to the ion/ion reaction volume prior to the mixing of the precursor ions and reagent ions. In various aspects, the notched tailored waveform applied to the reaction volume can subject all the ions in the reaction volume to the waveform and can excite all secular frequencies except the desired precursor ions and reagent ions whose frequencies exist within the notches of the tailored waveform. In various aspects, reagent ions can be introduced into or through the reaction volume wherein a reaction between the reagent ions and desired precursor ions can occur forming product ions.

[0018] In various embodiments, a second population of ions can be injected or introduced into or through the reaction volume such that a physical overlap of the first and the second ion populations occurs, with either storage or transmission of the precursor ions or reagent ions occurring; and impeding ion/ion reactions subsequent to the desired reactions via application of the notched tailored waveform. In various embodiments, the waveform can be applied during a reaction period for impeding subsequent ion-ion reactions. In various aspects, the reaction period can vary and can occur from the time of introducing the precursor ions or reagent ions to the completion of the reaction.

[0019] In accordance with the applicant's teachings, an apparatus for analyzing a sample of molecules is provided. In various embodiments, the apparatus can comprise an ion source for generating precursor and reagent ions. In various aspects, any suitable ion source can be used, including, but not limited to, electrospray (ESI), matrix-assisted laser desorption ionization (MALDI), laser desorption ionization (LDI), electron ionization (EI), atmospheric pressure chemical ionization (APCI), photospray, chemical ionization (CI), secondary ion mass spectrometry (SIMS), fast atom bombardment (FAB), laserspray, and inlet ionization.

[0020] In various aspects, the apparatus can comprise a reaction volume for receiving or transmitting desired precursor ions and reagent ions. In various aspects, the reaction volume can include, but is not limited to, a linear ion trap, a quadrupole ion trap, and an ion cyclotron trap. In various embodiments, the apparatus can comprise a waveform generator for applying a notched tailored waveform to the reaction volume during a reaction period and subjecting all ions in the reaction volume to the waveform with the exception of the desired precursor ions and reagent ions whose frequencies exist within the notches of the tailored waveform. For example, in various embodiments, the notched tailored waveform can be applied to opposing rods in a quadrupole linear ion trap for dipolar excitation of all ions except the desired precursor and reagent ions.

[0021] In various embodiments, a second population of ions can be injected into the reaction volume such that a physical overlap of the first and the second ion populations occurs, with either storage or transmission of the precursor ion or reagent ions occurring; and impeding ion/ion reactions subsequent to the desired reactions via application of the notched tailored waveform. In various embodiments, the waveform can be applied during a reaction period for impeding subsequent ion-ion reactions. In various aspects, the reaction volume receives or transmits a second population of ions wherein a physical overlap of the first and second ion populations occurs. In various aspects, the reaction period can vary and can occur from the time of introducing the precursor ions or reagent ions to the completion of the reaction.

[0022] In various aspects, the waveform can be composed of the secular frequencies present in the mass-to-charge domain of the experiment, with the exception of the omitted (or notched) frequencies. As shown in Figure 2, the first omitted (or notched) frequency corresponds to that of the reagent ion; the second omitted (or notched) frequency corresponds to that of the selected or desired precursor ions. When the precursor ions interact with the reagent ions, the desired ion/ion reaction will take place (e.g., ETD, PTR). However, as product ions are formed, they will immediately experience the influence of the broadband waveform at their particular secular frequency. This will result in an increase in kinetic energy and radial amplitude within the reaction volume for this product ion, which in turn impedes any further ion/ion reactions (e.g., secondary ETD reactions) from taking place.

[0023] In accordance with the applicant's teachings, a method for impeding ion/ion reactions is provided subjecting the precursor ions to sustained off-resonance irradiation (or SORI). In various embodiments, a waveform can be applied to the reaction volume with a frequency slightly different than the secular frequency of the precursor ion. Upon the addition of the reagent ion into or through the reaction volume, no reaction will occur. However, when the waveform is removed for a given period of time, the ion/ion reaction will proceed. For example, Figure 3 displays the effect of applying SORI to a precursor ion of m/z 380 (z = 3) during a reaction time totaling 300 ms. When the SORI waveform is applied for all but 5 ms of the reaction time (mass spectrum in front row, colored presently in red), few ETD fragments are observed (m/z 385 and 387); in contrast, when this waveform is turned off for progressively longer fractions of the total reaction time (rows of mass spectra leading toward the back part of this 3D plot), more and more ETD product ions are formed. However, this method will only influence the reaction of the precursor ion subjected to SORI; any ion/ion reaction product ions can undergo subsequent ion/ion reactions unimpeded.

[0024] All literature and similar material cited in this application, including, but not limited to, patents, patent applications, articles, books, treatises, and web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety. In the event that one or more of the incorporated literature and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls. [0025] While the applicants' teachings have been particularly shown and described with reference to specific illustrative embodiments, it should be understood that various changes in form and detail may be made without departing from the spirit and scope of the teachings. Therefore, all embodiments that come within the scope and spirit of the teachings, and equivalents thereto, are claimed. The descriptions and diagrams of the methods of the applicants' teachings should not be read as limited to the described order of elements unless stated to that effect.

[0026] While the applicants' teachings have been described in conjunction with various embodiments and examples, it is not intended that the applicants' teachings be limited to such embodiments or examples. On the contrary, the applicants' teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art, and all such modifications or variations are believed to be within the sphere and scope of the invention.

Claims

A method of analyzing a sample of molecules, the method comprising:

generating a first population of precursor and reagent ions;

transmitting desired precursor ions or reagent ions from the first population into a reaction volume;

applying a notched tailored waveform to the reaction volume;

subjecting all the ions in the reaction volume to the waveform with the exception of the precursor ions and reagent ions whose frequencies exist within the notches of the tailored waveform; and

introducing the first population of reagent ions or precursor ions into or through the reaction volume wherein a reaction between the reagent ions and the desired precursor ions occurs forming product ions; the waveform being applied during a reaction period for impeding subsequent ion-ion reactions.

The method of claim 1 wherein the reaction period occurs from the time of introducing the precursor ions or reagent ions to the completion of the reaction.

The method of claim 1 wherein a second population of ions is introduced into or through the reaction volume wherein a physical overlap of the first and second ion populations occurs.

An apparatus for analyzing a sample of molecules, the apparatus comprising:

an ion source or sources for generating a first population of precursor and reagent ions; a reaction volume for receiving or transmitting desired precursor ions and reagent ions; and

a waveform generator for applying a notched tailored waveform to the reaction volume during a reaction period and subjecting all the ions in the reaction volume to the waveform with the exception of the precursor and reagent ions whose frequencies exist within the notches of the tailored waveform.

The apparatus of claim 4 wherein the reaction period occurs from the time of introducing the precursor ions or reagent ions to the completion of the reaction. The apparatus of claim 4 wherein the reaction volume receives or transmits a second population of ions wherein a physical overlap of the first and second ion populations occurs.