Patent Application
20150129762
The invention generally relates to mass spectrometry, and more particularly to methods and apparatus for reducing interference in isobaric tag-based quantifications using mass spectrometry.
Mass spectrometry (MS) is an analytical technique for determining the elemental composition of test substances with both qualitative and quantitative applications. For example, MS can be useful for identifying unknown compounds, determining the isotopic composition of elements in a molecule, determining the structure of a particular compound by observing its fragmentation, and quantifying the amount of a particular compound in a sample.
MS analysis of complex samples (e.g., biological matrices) can sometimes result in inaccurate and/or non-specific detection as a result of interfering species contained within the sample that are not sufficiently resolved from the analyte of interest. Despite advances in MS that have enabled high-resolution mass analyzers to distinguish target species from interfering species within about 0.01 Th, it is not always feasible or possible to use a high-resolution mass analyzer or high-resolution analysis, for example, due to availability, cost, and/or experimental conditions.
While isobaric tagging techniques such as isobaric tags for relative and quantitative quantification (iTRAQ) and tandem mass tags (TMTs) provide a powerful tool in quantitative proteomics, such techniques might not lend themselves to high-resolution isolation by mass analyzers. In iTRAQ techniques, for example, an analyte of interest is labeled with a labeling reagent having a known molecular mass. The labeling reagent includes a reporter group, a balance group, and a reactive group that non-specifically binds the labeling reagent to the analyte of interest. Upon isolation and fragmentation of a labeled analyte of interest, the reporter group is cleaved (e.g., through collision induced dissociation, fragmentation) to enable quantification of the analyte of interest based on detection of the reporter group's characteristic signature. In multiplexing experiments, different isobaric labeling reagents can be mixed with various samples such that a particular analyte of each sample can be quantified in a single run of the MS based on the difference in mass of the unique reporter groups associated with each of the isobaric labeling reagents.
As noted above, sample peptides labeled with isobaric tags are typically isolated at resolutions much lower (e.g., ˜1-4 Th) than those achievable by mass analyzers known in the art. Thus, for highly complex samples containing many peptides, the wide isolation window can result in the concurrent isolation of multiple interfering species having similar mass-to-charge ratios to that of the labeled analyte of interest. Because of the non-specific binding of the isobaric tags, the detected quantity of reporter ions might not reflect the true quantity of the specific analyte of interest.
Accordingly, there remains a need for methods and systems for enhanced discrimination between the analyte of interest and interfering species when using mass spectrometry isobaric tagging techniques.
In accordance with one aspect, certain embodiments of the applicants' teachings relate to a method for performing mass spectrometry. According to the method, at least one sample containing or suspected of containing an analyte of interest can be reacted with a labeling reagent to generate a plurality of labeled analytes. The method can also include ionizing the plurality of labeled analytes to generate a plurality of labeled analyte ions, wherein the plurality of labeled analyte ions comprises labeled ions of the analyte of interest, if any, and labeled ions of an interfering species, the labeled ions of the interfering species being characterized by a m/z ratio substantially identical to that of the labeled ions of the analyte of interest. The labeled analyte ions can be reacted with a neutral reagent (i.e., a reagent without a net electrical charge such as a neutral molecule) to change the charge state of at least one of the labeled ions of the analyte of interest and the labeled ions of the interfering species such that a m/z ratio of the labeled ions of the analyte of interest differs from a m/z ratio of the labeled ions of the interfering species. Subsequently, the labeled ions of the analyte of interest can be isolated from the labeled ions of the interfering species based on the difference in m/z ratio, and fragmented so as to cleave a reporter group from the labeled ion of the analyte of interest. The method can also include detecting the reporter group.
In some aspects, reacting the at least one sample with a labeling reagent can comprise reacting each of two or more samples with a different isobaric labeling reagent, the method further comprising mixing the plurality of labeled analytes from the two or more samples. In a related aspect, each of the isobaric labeling reagents can comprise a reporter group with the reporter groups of each of the different isobaric labeling reagents differ in mass. Each of the reporter groups can be detected.
In some aspects, charge states of the labeled ions of the analyte of interest and the labeled ions of the interfering species can differ prior to reacting the labeled analyte ions with a neutral reagent. In various embodiments, the analyte of interest and the interfering species can be peptides.
In various aspects, methods in accordance with applicants' teachings can include transporting the labeled analyte ions through an ion mobility spectrometer prior to reacting the labeled analyte ions with the neutral reagent. The ion mobility spectrometer can be a differential mobility spectrometer, for example.
In some aspects, the method can include reacting the labeled analyte ions with the neutral reagent while transporting said labeled analyte ions through an ion mobility spectrometer. In various aspects, the labeled analyte ions are caused to react with the neutral reagent prior to transporting said labeled analyte ions through an ion mobility spectrometer. In some aspects, the method can include reacting the labeled analyte ions with the neutral reagent subsequent to transporting said labeled analyte ions through an ion mobility spectrometer.
In some aspects, detection of the reporter group can allow for the relative or absolute quantification of the analyte (e.g., peptide) of interest in the one or more samples.
In accordance with one aspect, certain embodiments of the applicants' teachings relate to a method for performing mass spectrometry. According to the method, a sample containing or suspected of containing an analyte of interest can be reacted with a labeling reagent having a reporter group to generate a plurality of labeled analytes. The method can also include ionizing the plurality of labeled analytes to generate a plurality of labeled analyte ions, wherein the plurality of labeled analyte ions comprises labeled ions of the analyte of interest, if any, at a first charge state characterized by a first m/z ratio. The labeled ions of the analyte of interest can be reacted with a neutral reagent so as to change the charge state of the labeled ions of the analyte of interest to a second charge state characterized by a second m/z ratio different from the first m/z ratio. The method can also include fragmenting the labeled ions of the analyte of interest characterized by the second m/z ratio so as to cleave the reporter group from the labeled ions of the analyte of interest. The reporter group can be detected.
In accordance with one aspect, certain embodiments of the applicants' teachings relate to a mass spectrometry system that includes a differential mobility spectrometer for receiving ions from an ion source, the differential mobility spectrometer having an internal operating pressure, electrodes, and at least one voltage source for providing DC and RF voltages to the electrodes. The system also includes a mass spectrometer sealed to, and in fluid communication with, the differential mobility spectrometer for receiving the ions from the differential mobility spectrometer. A vacuum chamber surrounding the mass spectrometer maintains the mass spectrometer at a vacuum pressure lower than the internal operating pressure of the differential mobility spectrometer such that the vacuum chamber is operable to draw a gas flow including the ions through the differential mobility spectrometer and into the vacuum chamber. A reaction chamber connects an outlet of the differential mobility spectrometer to an inlet of the mass spectrometer to define an ion path of travel therebetween, the reaction chamber being configured for reacting ions with a neutral reagent molecule prior to entry into the inlet of the mass spectrometer.
In some aspects, the operating pressure of the differential mobility spectrometer can be about atmospheric pressure. In various embodiments, the ions can be reacted with the neutral reagent ions at about atmospheric pressure. In some aspects, the reaction chamber can include a port for delivering neutral reagent molecules to the reaction chamber. The port can comprise a nozzle for delivering a liquid neutral reagent. In other aspects, the port supplies a gas into the reaction chamber, wherein the gas is configured to be bubbled through a neutral reagent supply prior to introduction into the reaction chamber. In related aspects, the gas port can be adjustable to vary a concentration of the neutral reagent molecule in the reaction chamber. In various aspects, the gas port can be adjustable to vary a gas flow rate through the differential mobility spectrometer. In various aspects, the system can include a supply of neutral reagent molecules, the supply being connected to the port.
These and other features of the applicants' teachings are set forth herein.
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 any way.
It will be appreciated that for clarity, the following discussion will explicate various aspects of embodiments of the applicants' teachings, while omitting certain specific details wherever convenient or appropriate to do so. For example, discussion of like or analogous features in alternative embodiments may be somewhat abbreviated. Well-known ideas or concepts may also for brevity not be discussed in any great detail. The skilled person will recognize that some embodiments of the applicants' teachings may not require certain of the specifically described details in every implementation, which are set forth herein only to provide a thorough understanding of the embodiments. Similarly it will be apparent that the described embodiments may be susceptible to alteration or variation according to common general knowledge without departing from the scope of the disclosure. The following detailed description of embodiments is not to be regarded as limiting the scope of the applicants' teachings in any manner.
Methods and systems for performing mass spectrometry of analytes labeled with isobaric tags are provided herein. In accordance with various aspects of the applicants' teachings, the methods and systems can enable enhanced discrimination between an analyte of interest and one or more interfering species when using isobaric tagging techniques. In some broad aspects, the present teachings relate to utilizing reactions between neutral reagents (e.g., neutral molecules) and ionic species having substantially identical m/z ratios (e.g., ±2 Th) so as to cause sufficient change in their m/z ratios (e.g., via charge transfer reactions) such that those species can be resolved from one another via mass spectrometry (e.g., to allow for their direct or indirect quantification without interference from the other). By way of example, the m/z ratio of at least one of the target species and the interfering species may be changed such that the difference between their m/z ratios is greater than the operative resolution window of the mass analyzer, for example, at least about 2 Th.
With reference now to
Ions provided from an ion source 112 can be emitted into the curtain chamber 122 containing the differential mobility spectrometer 120 via curtain chamber orifice 125. As will be appreciated by a person skilled in the art, the ion source can be virtually any ion source known in the art, including for example, a continuous ion source, a pulsed ion source, an atmospheric pressure chemical ionization (APCI) source, an electrospray ionization (ESI) source, an inductively coupled plasma (ICP) ion source, a matrix-assisted laser desorption/ionization (MALDI) ion source, a glow discharge ion source, an electron impact ion source, a chemical ionization source, or a photoionization ion source, among others. By way of non-limiting example, as shown in
The differential mobility spectrometer 120 can have a variety of configurations, but is generally configured to resolve ions received from the ion source 112 based on their mobility through a fixed or variable electric field. Though generally described herein as a differential mobility spectrometer, the mobility spectrometer 120 can utilize other techniques/systems for performing mobility spectrometry including, for example, an ion mobility spectrometer, FAIMS devices of various geometries such as parallel plate, curved electrode, or cylindrical FAIMS device, among others, and modified in light of the teachings herein.
In the exemplary embodiment depicted in
As shown in
The pressure of the curtain gases in the curtain chamber 122 (e.g., ˜760 Torr) can provide both a curtain gas outflow out of curtain gas orifice 125, as well as a curtain gas inflow into the differential mobility spectrometer 120, which becomes the drift gas that carries the ions through the differential mobility spectrometer 120 and into a reaction chamber 130 that defines a path of travel for the ions between the differential mobility spectrometer 120 and the mass spectrometer 140 contained within the vacuum chamber 142. In some embodiments, for example, the outlet 126 of the differential mobility spectrometer 120 can be aligned with the inlet 144 of the mass spectrometer 140 to define the ion path of travel therebetween, while the walls 131 of the reaction chamber 130 are spaced from this path of travel to provide an increased reaction volume.
As shown in
In the embodiment of
A person skilled in the art will appreciate that the neutral reagent molecule can be any molecule that facilitates ion/molecule reactions such as ion-molecule adduct formation, condensation reactions, displacement/elimination reactions, clustering reactions, charge exchange reactions and proton transfer reactions, all by way of non-limiting examples.
As will be appreciated by a person skilled in the art, the vacuum chamber 142 can be maintained at a much lower pressure than the curtain chamber 122. For example, the vacuum chamber 142 can be maintained at a pressure of about 2.3 Torr by a vacuum pump (not shown), while the curtain chamber 122 and an internal operating pressure of the differential mobility spectrometer 120 can be maintained at a pressure of 760 Torr. As a result of the significant pressure differential between the curtain chamber 122 and the vacuum chamber 142, the drift gas can be drawn through the differential mobility spectrometer 120, the reaction chamber 130 and, via vacuum chamber inlet 144, into the vacuum chamber 142 and mass spectrometer 140. As shown, the mass spectrometer 140 can be sealed to (or at least partially sealed), and in fluid communication with the differential mobility spectrometer 120, via the reaction chamber 130, to receive the ions transmitted by the differential mobility spectrometer 120.
Though only mass spectrometer 140 is shown, a person skilled in the art will appreciate that the mass spectrometry system 100 can include additional mass analyzer elements downstream from the vacuum chamber 142. As such, ions transported through vacuum chamber 142 can be transported through one or more additional differentially pumped vacuum stages containing one or more mass analyzer elements. For instance, in one embodiment, a triple quadrupole mass spectrometer may comprise three differentially pumped vacuum stages, including a first stage maintained at a pressure of approximately 2.3 Torr, a second stage maintained at a pressure of approximately 6 mTorr, and a third stage maintained at a pressure of approximately 10−5 Torr. The third vacuum stage can contain, for example, a detector, as well as two quadrupole mass analyzers (e.g., Q1 and Q3) with a collision cell (Q3) located between them. It will be apparent to those skilled in the art that there may be a number of other ion optical elements in the system. This example is not meant to be limiting as it will also be apparent to those of skill in the art that the differential mobility spectrometer/mass spectrometer coupling described above can be applicable to many mass spectrometer systems that sample ions from elevated pressure sources. These can include time of flight (TOF), ion trap, quadrupole, or other mass analyzers, as known in the art.
As will be appreciated by a person skilled in the art, the vacuum chamber inlet 144 can be an orifice, or, alternatively, may be a capillary, heated capillary, or an ion pipe. In some embodiments of the present teachings, it can be advantageous to provide a braking potential (e.g., by providing a DC offset voltage to either the electrode plates 128 of the differential mobility spectrometer 120 relative to the declustering or inlet potential provided to the vacuum chamber inlet 144) to slow down the ions transmitted into the reaction chamber 130 from the differential mobility spectrometer 120. By slowing down the ions prior to entering the vacuum chamber 142, the exposure of the ions to the neutral reagent can be increased, thereby increasing the chemical reaction, and ultimately, increasing the sensitivity of detection by the mass spectrometer 140.
Though
Moreover, it will be appreciated that a neutral reagent can be introduced into a reaction chamber for reaction with the analyte ions between a differential mobility spectrometer and downstream mass spectrometer elements without the addition of a throttle gas as described above with reference to
The applicants' teachings can be even more fully understood with reference to the exemplary experiment presented in
In an exemplary operation, two or more samples (e.g., protein digestion, blood, serum, plasma, urine, saliva, etc.) can be obtained, prepared in accordance with standard techniques known in the art, and reacted with an isobaric labeling reagent such as an iTRAQ or TMT labeling reagent such that the various peptides within each sample become associated with a reporter group that expresses a characteristic MS signal. By way of example, with specific reference to the experiment depicted in
In accordance with various aspects of the applicants' teachings, the labeled analyte ions (Mn*+, X*+, and Y*+) can be subjected to an initial mobility-based separation, for example, by being introduced into a differential mobility spectrometer. As will be appreciated by a person skilled in the art, the conditions of the ion mobility separation can be selected so as to transmit through the differential mobility spectrometer only the analyte of interest, and in some cases analytes having similar mobilities under the particular differential mobility spectrometer conditions. By way of example, the SV and CV can be selected based on historical data such that the labeled analyte ions of interest (Mn*+) are preferentially transmitted by the differential mobility spectrometer. As shown in
As discussed above, though a high-resolution mass analyzers may be able to discriminate between a target species exhibiting a m/z that differs from that of an interfering species by as little as 0.1 Th, mass spectrometric analysis of compounds that have been labeled with isobaric tags is generally performed at lower resolution (e.g., ˜1-4 Th). Moreover, as will be appreciated by a person skilled in the art, a target species and an interfering species can exhibit identical m/z ratios.
By way of non-limiting example, though the labeled analyte ions of interest (Mn*+) and the labeled analyte ions (X*+) present in the ionized, labeled sample can have different masses, each can exhibit ˜m/z of 500. For example, the labeled analyte ions of interest (Mn*+) can have a molecular mass of 2000 Da at a +4 charge state while the labeled analyte ions (X*+) can have a molecular mass of 1500 Da at a +3 charge state. As such, in a standard MS/MS analysis, a low-resolution MS isolation in Q1 would allow both the labeled analyte ions of interest (Mn*+) and the labeled interfering analyte ions (X*+) to be transmitted for downstream MS analysis. Subsequent fragmentation of the precursor ions and detection of the child reporter ions would not accurately reflect the quantity of the analyte of interest (M) present in a sample and/or the ratio of the quantity of the analyte of interest (Mn) present in various multiplexed samples.
In order to effect the separation of the labeled analyte ions of interest (Mn*+) and the labeled interfering analyte ions (X*+), ion/molecule reactions can be performed to alter one or both of the charge state and/or mass of the labeled analyte ions (Mn*+, X*+) such that the various species would become resolvable due to difference in their modified m/z. By way of example, as discussed above with reference to
As a result of the difference in the modified m/z, a low-resolution MS isolation can be effective to isolate the target labeled analyte ions [(Mn*−N)+] from the interfering analyte ions [(X*−N)+], as indicated in
Other embodiments of the applicants' teachings will be apparent to those skilled in the art from consideration of the present specification and practice of the present teachings disclosed herein. It is intended that these examples be considered as exemplary only.
As such, methods in accord with various aspects of the teachings herein enable improved specificity in discriminating between the analyte of interest and interfering species when using mass spectrometry isobaric tagging techniques. Moreover, in various aspects, the applicants' teachings enable separations based on differential mobility to reduce advantageously the population of ions subject to ion/molecule reactions, reactions which can be difficult to control as the number of ions increases. Further, use of the differential mobility spectrometer to isolate the target analyte ions from interfering ions can advantageously prevent the depletion of the neutral reagent molecules in downstream ion/molecule reactions.
The section headings used herein are for organizational purposes only and are not to be construed as limiting. While the applicants' teachings are described in conjunction with various embodiments, it is not intended that the applicants' teachings be limited to such embodiments. On the contrary, the applicants' teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.
This application claims the benefit of priority from U.S. Provisional Application Ser. No. 61/649,055, filed on May 18, 2012, the entire contents of which is incorporated by reference herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2013/000963 | 5/17/2013 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61649055 | May 2012 | US |
