Precursor accumulation in singly charged states in mass spectrometry

Abstract

The ion source ionizes the compound to produce precursor ions with different m / z values. The reactant source supplies charge-reduced reactants. The ion guide is positioned between the ion source and the reactant source and the mass filter. The ion guide applies an AC voltage and a DC voltage to the electrodes of the ion guide to create a pseudopotential to capture precursor ions below a threshold m / z in the ion guide. The AC voltage in turn causes the captured precursor ions to be reduced in charge due to the reactants, so that the m / z value of the captured precursor ions increases to a single m / z value above the threshold m / z. The ion guide applies a DC voltage to the electrodes of the ion guide relative to the DC voltage applied to the electrodes of the mass filter so that the precursor ions with m / z values ​​increased to a single m / z value are continuously transmitted to the mass filter.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 62 / 724,495, filed on August 29, 2018, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The teachings herein relate to a mass spectrometer for reducing the charge of precursor ions with different mass-to-charge ratios (m / z) values ​​of the same compound so as to continuously accumulate and transmit precursor ions with a single m / z value of a preset z charge state (z). More specifically, a Q0 ion guide is positioned between an ion source device and a reactant source device and a Q1 mass filter device. In various embodiments, the ion source device and the reactant source device operate simultaneously or sequentially in time. The Q0 ion guide uses a pseudopotential created by an alternating current (AC) voltage applied to a lens electrode at the exit of the Q0 ion guide or on the Q0 ion guide to capture two or more precursor ions with m / z values ​​below a threshold m / z value and a charge reducing reagent. A DC voltage is also applied to the lens electrode, wherein, for positively (negatively) charged precursor ions and for ions whose charge is reduced, the DC bias on the lens electrode is negative (positive) relative to the DC bias on the Q0 ion guide. In turn, the AC voltage reduces the charge of the two or more captured precursor ions so that their m / z values ​​increase to a single m / z value above a threshold m / z. By applying a direct current (DC) voltage to the Q0 ion guide device relative to the DC voltage applied to the Q1 mass filter device, two or more precursor ions having m / z values ​​increased to the single m / z value are continuously transmitted to the Q1 mass filter device. Q1 selects (or isolates) the species having the single m / z value whose charge is reduced to select a target compound having a preset charge state. Using this method, the intensity of the isolated precursor is the sum of the precursor ions having the original different charge states that are higher than a preset value given by a preset threshold.

[0004] The apparatus and methods disclosed herein may also be combined with a processor, controller, microcontroller or computer system (such as Figure 1 computer system) to execute. Background Art

[0005] Precursor ions with different charge states

[0006] In mass spectrometry, electrospray ionization (ESI), for example, can produce precursor ions with many different charge states. Since the mass-to-charge ratio (m / z) of a precursor ion depends on the charge, this in turn produces precursor ions with a wide range of different m / z values ​​in the case of large biomolecules such as proteins.

[0007] 2 is an exemplary plot 200 of a precursor ion mass spectrum of pure myoglobin, illustrating how electrospray ionization (ESI) can produce precursor ions with many different m / z values. For example, bracket 210 illustrates that ESI of myoglobin can produce at least 17 precursor ions with different m / z values ​​between 771 and 2000.

[0008] In many conventional experiments, a quadrupole ion filter (or Q1) is used to select only one of the precursor ions of myoglobin for analysis. For example, in a mass spectrometry / mass spectrometry (MS / MS) experiment, only the precursor ion of myoglobin with an m / z of 1413.82 is selected for fragmentation. Selecting only one precursor ion means that the remaining precursor ions with different m / z values ​​under bracket 210 are not examined or are lost.

[0009] Selecting only one precursor ion reduces the overall sensitivity of the measurement. Sensitivity is, for example, the observed change in ion current per molecule of interest. By selecting only the precursor ion of myoglobin with an m / z of 1413.82, the ion currents from the remaining 16 precursor ions are lost, reducing the overall sensitivity.

[0010] One way to regain sensitivity is to abandon the isolation of a single precursor ion and apply MS / MS to all precursor ions. In other words, the 17 precursor ions of bracket 210 are selected from the background noise ions outside bracket 210 by Q1 set to broadband transmission to cover the precursor ions in bracket 210. Unfortunately, this approach is often not applicable when the sample contains more than one protein or additional contaminants. In such cases, the precursor ions of the protein of interest may not be distinguished from the precursor ions of other proteins or contaminants.

[0011] Another method to regain sensitivity is to move two or more precursor ions to substantially the same m / z value. McLuckey et al., Anal. Chem. 2002, 74, 336-346 (hereinafter referred to as the "McLuckey paper") provide a method for moving precursor ions, which they call "ion parking". The McLuckey paper describes that prior to the development of ion parking techniques, it was well known that the ionic charge associated with high mass multiply charged ions could be manipulated.

[0012] For example, it is known that ions accumulated in an ion trapping instrument can be mixed with a strong neutral radical gas to produce an ion / molecule reaction that causes the charge state of the ions to decrease. Similarly, it is known that the accumulated ions can also be mixed with ions of opposite charge to produce a proton transfer reaction (PTR) to also cause the charge state of the ions to decrease.

[0013] However, the McLuckey paper introduces a new technique in which the rate of ion / ion PTR is suppressed in a selective manner so that only specific ions are held in the trap. The McLuckey paper refers to this suppression of ion / ion PTR as "ion parking". In order to suppress ion / ion PTR, the technique of the McLuckey paper applies a bipolar resonant excitation voltage to the end cap electrodes of the 3D quadrupole ion trap. The exemplary resonant excitation voltage described in the McLuckey paper has a frequency of about tens of thousands of hertz.

[0014] A resonant excitation AC voltage is applied at the long-term frequency of the target precursor peak at a preset charge state to excite the species; then, PTR is applied to a group of precursor ions with many charge states. Because the PTR reaction rate is reduced by the high kinetic energy of the precursor ions, PTR is stopped when the precursor charge state or m / z reaches the excitation target.

[0015] Unfortunately, this method has not yet been implemented in commercial instruments due to the complex parameter settings required. Another problem with this method is that the resonant excitation of the precursor ions is very likely to cause the precursor ions to lose fragile post-translational modification parts such as glycosylation. In other words, the resonant excitation of the precursor ions can fragment the precursor ions. Yet another problem with this method is that it involves a pulsed release of the PTR ions. The PTR ions are retained in the trap. Then, they are released from the trap at one time for selection and analysis. This pulsed release means that a large number of ions can be released at one time. Releasing a large number of ions at one time can cause the downstream mass analyzer to be saturated due to space charge.

[0016] Mass spectrometry background

[0017] Mass spectrometry (MS) is an analytical technique for the detection and quantification of chemical compounds based on the analysis of the m / z values ​​of ions formed from these compounds. MS involves ionizing one or more compounds of interest from a sample to produce precursor ions, and mass analyzing the precursor ions.

[0018] Tandem mass spectrometry or mass spectrometry / mass spectrometry (MS / MS) involves ionizing one or more compounds of interest from a sample, selecting one or more precursor ions of the one or more compounds, fragmenting the one or more precursor ions into product ions, and mass analyzing the product ions.

[0019] Both MS and MS / MS can provide qualitative and quantitative information. The measured precursor or product ion spectra can be used to identify molecules of interest. The intensities of the precursor ions and product ions can also be used to quantify the amount of compounds present in a sample.

[0020] Fragmentation technology background

[0021] Electron-based dissociation (ExD), collision-induced dissociation (CID), and ultraviolet (UV) or infrared (IR) light dissociation are often used as fragmentation techniques for tandem mass spectrometry (MS / MS). ExD may include, but is not limited to, electron capture dissociation (ECD), electron transfer dissociation (ETD), and electron impact excitation (EIEIO) of ions from organic matter. CID is the most common technique for dissociation in tandem mass spectrometers. Summary of the invention

[0022] Disclosed is an apparatus, method and computer program product for reducing the charge of precursor ions of the same compound with different m / z values ​​so as to continuously accumulate and transmit precursor ions of a single m / z value. The apparatus includes an ion source device, a reactant source device, a mass filter device and an ion guide device.

[0023] The ion source device ionizes the compound of the sample. This produces two or more precursor ions of the compound with different m / z values. The reactant source device supplies a charge-reducing reactant.

[0024] The ion guide apparatus is positioned between both the ion source apparatus and the reactant source apparatus and the mass filter apparatus. The ion guide apparatus receives two or more precursor ions from the ion source apparatus and receives a charge-reduced reactant from the reactant source apparatus.

[0025] The ion guide device applies an AC voltage and a DC voltage to one or more electrodes of the ion guide device that create a pseudopotential to capture two or more precursor ions received with m / z values ​​below a threshold m / z in the ion guide device. The AC voltage in turn causes the captured two or more precursor ions to be reduced in charge due to the received charge-reducing reactant, so that the m / z values ​​of the two or more precursor ions are increased to a single m / z value above the threshold m / z. The ion guide device also applies a DC voltage to one or more electrodes of the ion guide device relative to the DC voltage applied to the electrodes of the mass filter device so that the two or more precursor ions with m / z values ​​increased to the single m / z value are continuously transmitted to the mass filter device.

[0026] These and other features of the applicant's teachings are set forth herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Those skilled in the art will appreciate that the drawings described below are for illustration purposes only and are not intended to limit the scope of the present teachings in any way.

[0028] Figure 1 is a block diagram illustrating a computer system upon which embodiments of the present teachings may be implemented.

[0029] 2 is an exemplary plot of a precursor ion mass spectrum of pure myoglobin, illustrating how electrospray ionization (ESI) can produce precursor ions with many different mass-to-charge ratio (m / z) values.

[0030] Figure 3 is a schematic diagram of an apparatus for reducing the charge of precursor ions of the same compound having different m / z values ​​in order to sequentially accumulate and transmit precursor ions of a single m / z value using an ion guide in which sample ions and reactants are simultaneously received through different ports according to various embodiments.

[0031] Figure 4 is a schematic diagram of a Chimera device configured as a collision chamber in accordance with various embodiments.

[0032] Figure 5 is a three-dimensional perspective view of a chimera device according to various embodiments.

[0033] Figure 6 is an exemplary plot of a precursor ion of pure myoglobin, according to various embodiments, hypothetically illustrating how to use Figure 3 The device increases the m / z values ​​of these precursor ions.

[0034] Figure 7 According to various embodiments, the assumptions Figure 3 Example hypothetical plot of pure myoglobin precursor ions transmitted by the Q0 ion guide device to the Q1 mass filter device.

[0035] Figure 8 It is assumed that according to various embodiments Figure 3 Exemplary hypothetical plot of the Q1 mass filter device selecting and transmitting a pure myoglobin precursor ion.

[0036] Fig. 9 According to various embodiments Figure 3 Schematic diagram of an apparatus in which a Q0 ion guide device that simultaneously receives sample ions and reactants through different ports is replaced by a Q0 ion guide device that separately and sequentially receives sample ions and reactants through the same port.

[0037] Fig.10is a flow chart illustrating a method for reducing the charge of precursor ions of the same compound having different m / z values ​​in order to continuously accumulate and transmit precursor ions of a single m / z value, according to various embodiments.

[0038] Fig.11 is a schematic diagram of a system including one or more different software modules that implement a method for reducing the charge of precursor ions of the same compound having different m / z values ​​in order to continuously accumulate and transmit precursor ions of a single m / z value according to various embodiments.

[0039] Before describing one or more embodiments of the present teaching in detail, those skilled in the art will understand that the present teachings are not limited to the details of the arrangement of components, arrangement of steps and construction as described in the following detailed description or illustrated in the accompanying drawings. In addition, it is to be understood that the words and terms used herein are for the purpose of description and should not be considered as limiting. DETAILED DESCRIPTION

[0040] Computer-implemented systems

[0041] Figure 1 1 is a block diagram illustrating a computer system 100 on which embodiments of the present teachings may be implemented. The computer system 100 includes a bus 102 or other communication mechanism for communicating information, and a processor 104 coupled to the bus 102 for processing information. The computer system 100 also includes a memory 106, which may be a random access memory (RAM) or other dynamic storage device, coupled to the bus 102 for storing instructions to be executed by the processor 104. The memory 106 may also be used to store temporary variables or other intermediate information during the execution of instructions to be executed by the processor 104. The computer system 100 also includes a read-only memory (ROM) 108 or other static storage device coupled to the bus 102 for storing static information and instructions for the processor 104. A storage device 110, such as a magnetic disk or optical disk, is provided and coupled to the bus 102 for storing information and instructions.

[0042] The computer system 100 may be coupled to a display 112, such as a cathode ray tube (CRT) or a liquid crystal display (LCD), via the bus 102 for displaying information to a computer user. An input device 114, including alphanumeric and other keys, is coupled to the bus 102 for communicating information and command selections to the processor 104. Another type of user input device is a cursor control 116, such as a mouse, trackball, or cursor direction keys, for communicating direction information and command selections to the processor 104 and for controlling cursor movement on the display 112. Such input devices typically have two degrees of freedom in two axes, a first axis (i.e., x) and a second axis (i.e., y), which allows the device to specify a position in a plane.

[0043] The computer system 100 can perform the present teachings. Consistent with certain implementations of the present teachings, the computer system 100 provides results in response to the processor 104 executing one or more sequences of one or more instructions contained in the memory 106. Such instructions can be read into the memory 106 from another computer-readable medium such as the storage device 110. The execution of the sequence of instructions contained in the memory 106 causes the processor 104 to perform the processing described herein. Alternatively, hard-wired circuitry can be used instead of software instructions or in combination with software instructions to implement the present teachings. Therefore, implementations of the present teachings are not limited to any specific combination of hardware circuitry and software.

[0044] In various embodiments, computer system 100 can be connected to one or more other computer systems (like computer system 100) across a network to form a networked system. The network can include a private network or a public network such as the Internet. In a networked system, one or more computer systems can store data and supply data to other computer systems. In a cloud computing scenario, one or more computer systems that store and supply data can be referred to as servers or clouds. For example, one or more computer systems can include one or more web servers. For example, other computer systems that send data to a server or cloud and receive data from a server or cloud can be referred to as clients or cloud devices.

[0045] As used herein, the term "computer-readable medium" refers to any medium that participates in providing instructions to processor 104 for execution. Such media may take many forms, including, but not limited to, non-volatile media, volatile media, and transmission media. Non-volatile media include, for example, optical or magnetic disks, such as storage device 110. Volatile media include dynamic memory, such as memory 106. Transmission media include coaxial cables, copper wire, and fiber optics, including the wires that comprise bus 102.

[0046] Common forms of computer readable media or computer program products include, for example, floppy disks, flexible disks, hard disks, magnetic tapes or any other magnetic media, CD-ROMs, digital video disks (DVDs), Blu-ray disks, any other optical media, thumb drives, memory cards, RAM, PROMs and EPROMs, FLASH-EPROMs, any other memory chips or cartridges or any other tangible media from which a computer can read.

[0047] Various forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to processor 104 for execution. For example, the instructions may initially be carried on a disk of a remote computer. The remote computer may load the instructions into its dynamic memory and send the instructions over a telephone line using a modem. A modem local to computer system 100 may receive the data on the telephone line and use an infrared transmitter to convert the data into an infrared signal. An infrared detector coupled to bus 102 may receive the data carried in the infrared signal and place the data on bus 102. Bus 102 carries the data to memory 106, from which processor 104 retrieves and executes the instructions. The instructions received by memory 106 may optionally be stored on storage device 110 before or after execution by processor 104.

[0048] According to various embodiments, instructions configured to be executed by a processor to perform the method are stored on a computer readable medium. A computer readable medium may be a device that stores digital information. For example, a computer readable medium includes a compact disk read-only memory (CD-ROM) for storing software as known in the art. The computer readable medium is accessed by a processor adapted to execute the instructions configured to be executed.

[0049] For the purpose of illustration and description, the following description of various implementations of this teaching has been given. It is not exhaustive and does not limit this teaching to the precise form disclosed. Modifications and variations are possible in view of the above teachings, or can be obtained from the practice of this teaching. In addition, the described implementations include software, but this teaching can be implemented as a combination of hardware and software or only as hardware. This teaching can be implemented with both object-oriented and non-object-oriented programming systems.

[0050] Pseudopotential ion accumulation and charge reduction

[0051] As described above and shown in Figure 2, electrospray ionization (ESI) can enable precursor ions of, for example, proteins to have many different charge states. Since the mass-to-charge ratio (m / z) of a precursor ion depends on the charge, this in turn can enable precursor ions of a single protein to have a large number of different m / z values.

[0052] In many experiments, only one of the precursor ions of the compound of interest is selected for analysis. However, this reduces the overall sensitivity of the analysis. One way to regain sensitivity is to move two or more precursor ions to essentially the same m / z value. The McLuckey paper provides a method for moving precursor ions known as ion parking. In this method, ion / ion proton transfer reactions (PTRs) are suppressed at selected charge states or m / z values ​​by applying a resonant excitation voltage to the end cap electrodes of a 3D quadrupole ion trap. Unfortunately, this method requires complex parameter settings, can fragment precursor ions, and can cause saturation problems due to the pulsed release of charge-reduced precursor ions.

[0053] In various embodiments, precursor ions are accumulated in the same charge state in an ion guide without using resonant excitation. Instead, an additional alternating current (AC) voltage is applied to all rods of the ion guide or to an exit aperture or lens of the ion guide to create a pseudopotential voltage barrier through which only precursor ions that have reached a certain m / z value can be transmitted.

[0054] In the McLuckey paper, the additional AC resonant excitation applied to the ion trap was given a frequency corresponding to the m / z value that suppressed charge reduction. This frequency caused the ions of this m / z value to be excited with higher kinetic energy, thus preventing them from reacting with the charge-reducing reactants. Unfortunately, this higher kinetic energy can also fragment these precursor ions.

[0055] In contrast, in various embodiments, an additional AC voltage applied to the ion guide creates a pseudopotential barrier that prevents precursor ions having m / z values ​​below a threshold m / z value from moving in the axial direction between the Q0 ion guide and the Q1 filter. This allows them to continue to react with the charge-reducing reactant. The amplitude of the additional AC voltage is proportional to the square root of the threshold m / z value. Thus, decreasing the amplitude of the AC voltage decreases the threshold m / z value.

[0056] In the case of applying ion parking to a linear RFQ such as Q0, an AC voltage is applied in the radial direction to excite the long-term frequencies of the species whose charge is reduced. In contrast, in various embodiments, an AC voltage is applied in the axial direction, which does not cause resonant excitation in the radial direction. Instead, it creates a potential barrier between the Q0 ion guide rods at the exit of the Q0 ion guide. There are at least two options for applying an AC voltage to the Q0 ion guide. One is to apply an AC voltage to the Q0 rods to apply an AC field between the Q0 rod group and the lens electrode placed at the exit of the Q0 (or IQ1 electrode) ion guide. Another option is to apply an AC voltage at the IQ1 electrode.

[0057] To generate the mass selection threshold, a DC bias is applied between the IQ1 electrode and the Q0 ion guide. For positively charged precursor ions, IQ1 is set to negative relative to the Q0 ion guide. For negatively charged precursor ions, IQ1 is set to positive relative to the Q0 ion guide.

[0058] For example, in a quadrupole ion guide, an appropriate radio frequency (RF) voltage is applied to opposing pairs of electrodes within the ion guide to radially confine the ions. In various embodiments, an additional AC voltage is superimposed on the RF voltage to create a pseudopotential barrier in the axial direction at the exit of Q0. Background information on pseudopotentials can be found in Gerlich, "The Encyclopedia of Mass Spectrometry" (Vol. 1, 182-194 (2003)) on RF ion guides, which is incorporated herein by reference.

[0059] U.S. Patent No. 7,456,388, issued on November 25, 2008 and incorporated herein by reference (hereinafter referred to as the "'388 patent"), for example, describes an ion guide for focusing ion packets. The '388 patent provides an apparatus and method that allows, for example, ions to be analyzed over a wide m / z range with little transmission losses. The ejection of ions from the ion guide is affected by creating conditions in which all ions (regardless of m / z) can be caused to arrive at a specified point in space, such as an extraction region or accelerator of a time-of-flight (TOF) mass analyzer, in a desired order or at a desired time and with approximately the same energy. The ions bunched in this manner can then be manipulated as a group, for example by being extracted using a TOF extraction pulse and propelled along a desired path so as to arrive at the same point on a TOF detector.

[0060] In order to eject ions from the ion guide so that all ions arrive at the desired location at the desired time and with approximately the same energy, the '388 patent applies an additional AC voltage to the ion guide. The additional AC voltage creates a pseudopotential barrier. In the '388 patent, first, the amplitude of the AC voltage is set to allow only ions with the largest m / z value to be ejected. Then, the amplitude of the AC voltage is gradually reduced to change the depth of the pseudopotential well and allow ions with increasingly smaller m / z values ​​to be ejected from the ion guide. In other words, in the '388 patent, the AC voltage amplitude is scanned.

[0061] In various embodiments, the AC voltage applied to the ion guide is not scanned. One AC voltage amplitude is set to correspond to the m / z threshold. In addition, the AC voltage is not used to sequentially eject ions of different m / z values. Instead, the AC voltage is used to create a potential barrier through which ions that reach the threshold m / z value after charge reduction due to PTR are sequentially ejected.

[0062] Figure 3 is a schematic diagram 300 of an apparatus for reducing the charge of precursor ions of the same compound having different m / z values ​​to sequentially accumulate and transmit precursor ions of a single m / z value using an ion guide in which sample ions and reactants are simultaneously received through different ports in accordance with various embodiments. Figure 3 The device of 310 includes an ion source device 311, a reactant source device 312, a Q1 mass filter device 318 and a Q0 ion guide device 315. The device is, for example, part of a mass spectrometer 310.

[0063] The ion source device 311 ionizes the compound of the sample to generate two or more precursor ions of the compound with different m / z values. For example, the two or more precursor ions are received by the Q0 ion guide device 315 through the orifice and skimmer 313 and the QJet 314. For example, two or more precursor ions of the compound myoglobin with different m / z values ​​are shown in FIG.

[0064] return Figure 3 , the reactant source device 312 supplies the PTR reactants to the Q0 ion guide device 315. Two or more precursor ions and the PTR reactants are simultaneously and continuously supplied to the Q0 ion guide device 315. This is possible because the Q0 ion guide device 315 includes at least two separate inlet ports.

[0065] Q0 ion guide device 315 is also Figure 4 and Figure 5 The chimera device shown in FIG. The chimera device includes eight L-shaped electrodes, thereby providing four branches. A pair of aligned branches receives the two or more precursor ions from the ion source device 311. At the same time, another pair of aligned branches receives the PTR reactants from the reactant source device 312.

[0066] Figure 4 is a schematic diagram 400 of a chimera device configured as an ExD chamber according to various embodiments. The chimera device for ExD includes an electron emitter or filament 410 and an electron gate 420. Electrons are emitted perpendicular to the ion flow 430 and parallel to the magnetic field direction 440.

[0067] return Figure 3 , the Q0 ion guide device 315 is not used for fragmentation, so the chimera device does not need to include an electron source or any other equipment necessary to perform ExD.

[0068] Figure 5 is a three-dimensional perspective view 500 of a chimera device according to various embodiments. Figure 5The direction 510 of sample compound ion flow through the chimera device is shown. Figure 5 It is also shown that a PTR reactant can be added to the chimera device in direction 520 .

[0069] return Figure 3 , two or more precursor ions and PTR reactants are supplied to a Q0 ion guide device 315 composed of a chimera structure so that the charge state of the two or more precursor ions is reduced. However, in the absence of some kind of trapping force, the two or more precursor ions will simply pass through the Q0 ion guide device 315. In order to trap the two or more precursor ions in the Q0 ion guide device 315, an AC voltage is applied to all rods of the Q0 ion guide device 315, for example, using an AC voltage source 322. In various alternative embodiments, the AC voltage is applied to an electrode of an exit aperture or an IQ1 lens 316. As described above, the AC voltage creates a pseudopotential encountered by the two or more precursor ions.

[0070] Plot 340 depicts the potentials encountered by different precursor ions at different locations in mass spectrometer 310. For example, line 341 depicts the DC potential encountered by all precursor ions between Q0 ion guide device 315 and mass filter device 318. Line 342 depicts the combined AC and DC (pseudo) potentials encountered by precursor ions having m / z values ​​below a threshold m / z value. Line 342 shows that there is a potential barrier that prevents these ions from moving to Q1 mass filter device 318.

[0071] Line 343 depicts the combined AC and DC (pseudo) potential encountered by precursor ions having m / z values ​​above a threshold m / z value. Line 343 shows that there is no barrier preventing these ions from moving to the Q1 mass filter device 318. Reactant ions having opposite charge signs are always trapped in Q0 regardless of their m / z values ​​because the DC potential acts as a trapping barrier.

[0072] Plot 340 shows that while the AC voltage traps precursor ions with m / z values ​​below the threshold m / z value, it also allows precursor ions with m / z values ​​above the threshold m / z value to continue to move to mass filter device 318. Since the AC voltage traps precursor ions with m / z values ​​below the threshold m / z value and Q0 ion guide device 315 is supplied with PTR reactants, these trapped precursor ions are reduced in charge by the PTR reactants until their m / z values ​​increase above the threshold m / z. In this way, the AC voltage limits the PTR.

[0073] The PTR reactants may include, for example, negatively charged ions. Alternatively, the PTR reactants may include neutral charge scavenger ions such as ammonia or acetone. In this case, mutual capture is not required.

[0074] For example, the DC potential 341 in plot 340 is created by setting the DC voltage of the exit aperture or IQ1 lens 316 to be lower than the DC voltage of the rods of the Q0 ion guide device 315. Additionally, the DC voltage of the optional ST1 ion guide device 317 is set to be lower than the exit aperture or IQ1 lens 316, and the DC voltage of the Q1 mass filter device 318 is set to be lower than the DC voltage of the rods of the Q0 ion guide device 315. By coupling the pseudopotential created by the AC voltage near the exit aperture or IQ1 lens 316 with the DC voltage, the Q0 ion guide device 315 performs a high m / z filter extraction.

[0075] Due to the PTR, the charge state of the precursor ions in the Q0 ion guide device 315 is continuously reduced and their m / z values ​​are increasing. When the m / z value of the precursor ions reaches an m / z higher than the m / z extraction threshold, the ions are extracted from the Q0 ion guide device 315. Because there are no PTR reactants outside of the Q0 ion guide device 315, further charge reduction is stopped. This means that the charge state of the precursor ions accumulates at a single value determined by the high m / z extraction threshold.

[0076] Figure 6 is an exemplary plot 600 of precursor ions of pure myoglobin, hypothetically illustrating how to use Figure 3 The bracket 210 again delimits, for example, at least 17 precursor ions of myoglobin having different m / z values ​​between 771 and 2000. However, if an AC voltage is applied to Figure 3 2 to create an m / z threshold of about 1413 Da, then the 12 precursor ions in bracket 210 with m / z below 1413 are reduced in charge. This increases the m / z values ​​of these 12 precursor ions to a single m / z of 1413.82, for example, Figure 6 As shown by the arrow in . Therefore, at this time, the intensity of a total of 13 precursor ions is found at 1413.83. If this m / z value is selected and used for mass analysis or fragmentation at this time, the sensitivity is greatly improved.

[0077] However, bracket 620 shows that setting the m / z threshold at 1413 does not merge all precursor ions at 1413.82 Da. Four precursor ions have m / z values ​​above this value and are transmitted without charge reduction. If only the single m / z of 1413.82 is selected by the mass filter device, the ion current contributions from these four precursor ions are not included. In other words, not setting the m / z threshold to an m / z value close to the precursor ion with the highest m / z value may cause some precursor ions to not be used.

[0078] This is not a significant problem because collecting the ion current from many precursor ions with lower m / z values ​​provides a high percentage of the possible total ion current. In addition, setting the threshold m / z value too high may cause other problems. For example, the mass filter device may not be able to select the ion with the highest m / z value of the precursor ion. Similarly, reaching higher and higher m / z values ​​requires longer PTRs in time. In some experiments, there may not be enough time to wait for the PTR to move the precursor ion with the lowest m / z value to the m / z value of the precursor ion with the highest m / z value. Another problem can be the low dissociation efficiency after electron capture in an ExD experiment when the precursor charge state is too high.

[0079] Figure 7 According to various embodiments, the assumptions Figure 3 An exemplary hypothetical plot 700 of pure myoglobin precursor ions transmitted by a Q0 ion guide device to a Q1 mass filter device. Figure 7 Shows Figure 3 The Q0 ion guide device 315 acts as a high m / z extraction filter or a high m / z pass filter. Figure 7 Only precursor ions with m / z values ​​higher than the m / z threshold of about 1413 are now shown. The precursor ion with an m / z value of 1413.82 now includes ion currents from 13 precursor ions (the original precursor ion and the 12 precursor ions that were reduced in charge and moved to this m / z value). Bracket 620 shows that the four precursor ions with m / z values ​​above the m / z threshold are still there as well. In addition, there are some other high m / z ions that may be protein precursor ions, precursor ions from other proteins, or contaminant precursor ions. Therefore, using Figure 3 The Q1 mass filter device 318 of the embodiment selects the enhanced precursor ion having an m / z value of 1413.82 and removes Figure 7 The remaining precursor ions with different charge states and high m / z contaminant ions generated from impurities are shown in FIG.

[0080] Figure 8 It is assumed that according to various embodiments Figure 3An exemplary hypothetical plot 800 of a pure myoglobin precursor ion selected and transmitted by a Q1 mass filter device. Figure 8 Shows Figure 3 The Q1 mass filter device 318 acts as a bandpass m / z extraction filter. Only the Figure 8 The precursor ion shown in FIG. 5 has an m / z value of 1413.82. Figure 3 The Q1 mass filter device 318 effectively removes all other precursor ions having m / z values ​​above the threshold m / z.

[0081] return Figure 3 , the Q1 mass filter device 318 now transmits the selected precursor ions downstream to other components of the mass spectrometer 310 for mass analysis or fragmentation. For example, the accumulated and isolated precursor ions enable sensitive ExD analysis. For example, the mass spectrometer 310 includes a second Q2 chimera device 319, which can be used to apply ExD to the precursor ions selected by the Q1 mass filter device 318. Alternatively, the mass spectrometer 310 includes a Q2 CID collision cell 320, which can be used to apply CID to the precursor ions selected by the Q1 mass filter device 318. The product ions are then mass analyzed by a mass analyzer device 321.

[0082] In a further alternative embodiment, the accumulated and isolated precursor ions may simply be mass analyzed. In this case, the precursor ions selected by the Q1 mass filter device 318 are simply transmitted through the Q2 chimera device 319 and the Q2 CID collision cell 320 to the mass analyzer device 321.

[0083] Although Q0 ion guide apparatus 315 filters and charge reduces a continuous flow of precursor ions from ion source apparatus 311, in various embodiments, ions of Q0 ion guide apparatus 315 are periodically refreshed or discharged to empty Q0 ion guide apparatus 315. For example, periodically emptying Q0 ion guide apparatus 315 prevents accumulation of contaminant ions.

[0084] Fig. 9 According to various embodiments Figure 3 Schematic diagram 900 of an apparatus in which a Q0 ion guide device that simultaneously receives sample ions and reactants through different ports is replaced by a Q0 ion guide device that separately and sequentially receives sample ions and reactants through the same port. Specifically, Figure 3 The chimera Q0 ion guide device 315 is Fig. 9 The multipole Q0 ion guide device 915 in the embodiment of the present invention is replaced. The multipole Q0 ion guide device 915 can be, but is not limited to, a quadrupole, a hexapole, or an octopole.

[0085] At this time, the ion source device 311 and the reactant source device 312 transmit their two or more precursor ions and reactants to the Q0 ion guide device 915 through a single inlet port of the Q0 ion guide device 915, respectively. For example, the two or more precursor ions and reactants are transmitted through the orifice and skimmer 313 and the ion guide 314. Since the two or more precursor ions and reactants use the same ion path, they need to be transmitted separately and sequentially. For example, the two or more precursor ions are first transmitted to the Q0 ion guide device 915. Then, the ion source device 311 is stopped and the reactant source device 312 is turned on to transmit the charge reduction reactant to the Q0 ion guide device 915. In various embodiments, when negative chemical ionization is used at atmospheric pressure, the charge reduction reactant is introduced by the reactant source device 312 through the orifice and skimmer 313 and the ion guide 314.

[0086] Pseudopotential trapping and charge reduction devices

[0087] return Figure 3 The mass spectrometer 310 includes a device for reducing the charge of precursor ions of the same compound with different m / z values ​​so as to continuously accumulate and transmit precursor ions of a single m / z value. The device includes an ion source device 311, a reactant source device 312, a Q1 mass filter device 318, and a Q0 ion guide device 315.

[0088] Ion source device 311 ionizes the compound of sample. This produces two or more precursor ions with different m / z values ​​of the compound. Ion source device 311 can be but not limited to electrospray ion source (ESI) device, electron impact source and fast atom bombardment source device, chemical ionization (CI) source device such as atmospheric pressure chemical ionization source (APCI) device, atmospheric pressure photoionization (APPI) source device or matrix-assisted laser desorption source (MALDI) device. In a preferred embodiment, ion source device 311 is ESI device.

[0089] Reactant source device 312 supplies charge reducing reactants. The charge reducing reactants can be neutral molecules or charged ions.

[0090] The Q1 mass filter device 318 is shown as a quadrupole. However, the Q1 mass filter device 318 may be any type of mass filter, such as a magnetic sector mass spectrometer.

[0091] The Q0 ion guide device 315 is positioned between both the ion source device 311 and the reactant source device 312 and the Q1 mass filter device 318. The Q0 ion guide device 315 receives two or more precursor ions from the ion source device 311 and receives a charge-reducing reactant from the reactant source device 312. The Q0 ion guide device 315 applies an AC voltage to one or more electrodes of the Q0 ion guide device 315 that creates a pseudopotential to trap the received two or more precursor ions having m / z values ​​below a threshold m / z in the Q0 ion guide device 315. The AC voltage in turn causes the trapped two or more precursor ions to be charge-reduced due to the received charge-reducing reactant, so that the m / z values ​​of the two or more precursor ions are increased to a single m / z value above the threshold m / z. The Q0 ion guide device 315 applies a DC voltage to one or more electrodes of the Q0 ion guide device 315 relative to the DC voltage applied to the electrodes of the mass filter device 318 so that two or more precursor ions having m / z values ​​increasing to the single m / z value are continuously transmitted to the mass filter device 318.

[0092] In various embodiments, the charge reducing reactant supplied by reactant source device 312 may be a neutral charge scavenger reactant. The neutral charge scavenger reactant may include, but is not limited to, ammonia or acetone.

[0093] In various alternative embodiments, the reactant source device 312 is a PTR reactant source device. The charge-reduced reactants include PTR reactant ions. Additionally, the ion guide device 315 applies an AC voltage to mutually trap both the received PTR reactant ions having an m / z value below a threshold m / z and the received two or more precursor ions.

[0094] In various embodiments, the one or more electrodes of the Q0 ion guide device 315 are rods of the Q0 ion guide device 315. In various alternative embodiments, the one or more electrodes of the Q0 ion guide device 315 include an exit aperture of the Q0 ion guide device 315 or an IQ1 lens 316.

[0095] return Fig. 9In various embodiments, two or more precursor ions from the ion source device 311 and the charge-reduced reactants from the reactant source device 312 are received separately and sequentially by the same entrance of the Q0 ion guide device 915. The two or more precursor ions from the ion source device 311 and the charge-reduced reactants from the reactant source device 312 are introduced separately and sequentially into the same entrance of the Q0 ion guide device 915 through the orifice and skimmer 313 and the ion guide 314. The Q0 ion guide device 915 is, for example, a multipole ion guide. The Q0 ion guide device 915 can be, but is not limited to, a quadrupole, a hexapole, or an octopole ion guide device.

[0096] return Figure 3 In various embodiments, two or more precursor ions from the ion source device 311 and the charge reduced reactant from the reactant source device 312 are received sequentially and simultaneously at different entrances of the Q0 ion guide device 315 .

[0097] In various embodiments, the Q0 ion guide device 315 is a chimera device. The device includes eight L-shaped electrodes, thereby providing four branches. A pair of aligned branches receives two or more precursor ions from the ion source device 311. At the same time, another pair of aligned branches receives charge reduction reactants from the reactant source device 312.

[0098] In various embodiments, the second ST1 ion guide device 317 is positioned between the Q0 ion guide device 315 and the Q1 mass filter device 318. The Q0 ion guide device 315 applies a DC voltage to one or more electrodes of the Q0 ion guide device 315 relative to a DC voltage applied to electrodes of the second ST1 ion guide device 317 and relative to a DC voltage applied to electrodes of the Q1 mass filter device 318. The DC voltage applied to one or more electrodes of the Q0 ion guide device 315 causes two or more precursor ions having m / z values ​​that increase to a single m / z value to be sequentially transmitted through the second ST1 ion guide device 317 and to the Q1 mass filter device 318.

[0099] In various embodiments, the ExD device is positioned after the Q1 mass filter device 318. The ExD device is, for example, a second Q2 chimera device 319. The Q1 mass filter device 318 selects two or more precursor ions having m / z values ​​increased to a single m / z value and transmits the two or more precursor ions having m / z values ​​increased to a single m / z value to the ExD device. The ExD device fragments the two or more precursor ions having m / z values ​​increased to a single m / z value.

[0100] In various embodiments, the processor 330 is used to control or provide instructions to the ion source device 311, the reactant source device 312, the Q1 mass filter device 318, and the Q0 ion guide device 315 and analyze the collected data. The processor 330 controls or provides instructions by controlling one or more voltage, current, or pressure sources (not shown), for example. The processor 330 may be, for example, Figure 3 , or may be a processor or controller of one or more devices of mass spectrometer 310. Processor 330 may be, but is not limited to, a controller, a computer, a microprocessor, Figure 1 A computer system or any device capable of sending and receiving control signals and data.

[0101] Methods for pseudopotential trapping and charge reduction

[0102] Fig.10 is a flow chart illustrating a method 1000 for reducing the charge of precursor ions of the same compound having different m / z values ​​in order to continuously accumulate and transmit precursor ions of a single m / z value, according to various embodiments.

[0103] In step 1010 of method 1000 , a processor is used to instruct an ion source device to ionize a compound of a sample, thereby generating two or more precursor ions of the compound having different m / z values.

[0104] In step 1020, a processor is used to instruct a reactant source device to supply a charge reducing reactant.

[0105] In step 1030, using a processor, an ion guide apparatus positioned between both the ion source apparatus and the reactant source apparatus and the mass filter apparatus is directed to receive two or more precursor ions from the ion source apparatus and a charge-reduced reactant from the reactant source apparatus.

[0106] In step 1040, the processor is used to instruct the ion guide device to apply an AC voltage and a DC voltage to one or more electrodes of the ion guide device that create a pseudopotential to trap two or more precursor ions received having m / z values ​​below a threshold m / z in the ion guide device. The AC voltage in turn causes the trapped two or more precursor ions to be charge-reduced due to the received charge-reducing reactant, such that the m / z values ​​of the two or more precursor ions are increased to a single m / z value above the threshold m / z.

[0107] In step 1050, the processor is used to instruct the ion guide device to apply a DC voltage to one or more electrodes relative to the DC voltage applied to the electrodes of the mass filter device. The DC voltage applied to the one or more electrodes of the ion guide device causes two or more precursor ions having m / z values ​​increased to a single m / z value to be sequentially transmitted to the mass filter device.

[0108] Computer program product for pseudopotential trapping and charge reduction

[0109] In various embodiments, a computer program product includes a tangible computer-readable storage medium, the contents of which include a program with instructions executed on a processor to perform a method for reducing the charge of precursor ions of the same compound having different m / z values ​​so as to continuously accumulate and transmit precursor ions of a single m / z value. The method is performed by a system including one or more different software modules.

[0110] Fig.11 1 is a schematic diagram of a system 1100 including one or more different software modules that implement a method for reducing the charge of precursor ions of the same compound having different m / z values ​​so as to continuously accumulate and transmit precursor ions of a single m / z value according to various embodiments. The system 1100 includes a control module 1110.

[0111] The control module 1110 instructs the ion source device to ionize the compound of the sample, thereby generating two or more precursor ions of the compound having different m / z values. The control module 1110 instructs the reactant source device to supply a charge reduction reactant. The control module 1110 instructs the ion guide device positioned between the ion source device and the reactant source device and the mass filter device to receive the two or more precursor ions from the ion source device and receive the charge reduction reactant from the reactant source device.

[0112] The control module 1110 instructs the ion guide device to apply an AC voltage and a DC voltage to one or more electrodes of the ion guide device that create a pseudopotential to trap two or more precursor ions received with m / z values ​​below a threshold m / z in the ion guide device. The AC voltage in turn causes the trapped two or more precursor ions to be charge-reduced due to the received charge-reducing reactant, so that the m / z values ​​of the two or more precursor ions are increased to a single m / z value above the threshold m / z.

[0113] The control module 1110 instructs the ion guide device to apply a DC voltage to one or more electrodes relative to the DC voltage applied to the electrodes of the mass filter device. The DC voltage applied to the one or more electrodes of the ion guide device causes two or more precursor ions having m / z values ​​increased to a single m / z value to be sequentially transmitted to the mass filter device.

[0114] Although the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, as will be appreciated by those skilled in the art, the present teachings encompass various alternatives, modifications, and equivalents.

[0115] In addition, when describing various embodiments, this specification may have proposed methods and / or processes as specific sequences of steps. However, insofar as the method or process does not rely on the specific order of the steps set forth herein, the method or process should not be limited to the specific sequence of steps described. As will be appreciated by those of ordinary skill in the art, other sequences of steps may be possible. Therefore, the specific order of the steps set forth in the specification should not be interpreted as a limitation on the claims. In addition, claims for methods and / or processes should not be limited to performing their steps in the order written, and those of ordinary skill in the art can readily appreciate that the sequence can vary and still remain within the spirit and scope of the various embodiments.

Claims

1. A device for reducing the charge of precursor ions of the same compound having different mass-to-charge ratios m / z values ​​so as to continuously accumulate and transmit precursor ions of a single m / z value, include: an ion source device for ionizing a compound of the sample to generate two or more precursor ions of the compound having different m / z values; a reactant source device that supplies a charge reducing reactant; mass filter equipment; as well as An ion guide device, the ion guide device is positioned between the ion source device and the reactant source device and the mass filter device, the ion guide device receives the two or more precursor ions from the ion source device and receives the charge reduction reactant from the reactant source device, the ion guide device applies an alternating current (AC) voltage and a direct current (DC) voltage to one or more electrodes of the ion guide device to create a pseudopotential to capture the received two or more precursor ions with m / z values ​​below a threshold m / z in the ion guide device and thereby reduce the charge of the captured two or more precursor ions due to the received charge reduction reactant so that the m / z values ​​of the two or more precursor ions are increased to a single m / z value above the threshold m / z, and the ion guide device applies a DC voltage to the one or more electrodes relative to the DC voltage applied to the electrodes of the mass filter device so that the two or more precursor ions with m / z values ​​increased to the single m / z value are continuously transmitted to the mass filter device.

2. The device according to claim 1, in, The charge reducing reactant includes a neutral charge scavenger reactant.

3. The device according to claim 2, in, The neutral charge scavenger reactant includes ammonia or acetone.

4. The device according to claim 1, in, The charge reduction reactant source device includes a proton transfer reaction (PTR) reactant source device, the charge reduction reactant includes PTR reactant ions, and the ion guide device applies an AC voltage to the one or more electrodes of the ion guide device that creates a pseudopotential to mutually capture both the received PTR reactant ions having an m / z value below the threshold m / z and the received two or more precursor ions.

5. The device according to claim 1, in, The one or more electrodes of the ion guide device include rods of the ion guide device.

6. The device according to claim 1, in, The one or more electrodes of the ion guide device comprise electrodes of an exit aperture or lens of the ion guide device.

7. The device according to claim 1, in, The two or more precursor ions from the ion source apparatus and the charge-reduced reactant from the reactant source apparatus are received separately and sequentially through a same inlet of the ion guide apparatus.

8. The device according to claim 7, in, The two or more precursor ions from the ion source apparatus and the charge-reduced reactant from the reactant source apparatus are separately and sequentially introduced into the same inlet of the ion guide apparatus through an orifice and an ion guide.

9. The device according to claim 7, in, The ion guide apparatus comprises a quadrupole, hexapole or octopole ion guide apparatus.

10. The device according to claim 1, in, The two or more precursor ions from the ion source apparatus and the charge-reduced reactant from the reactant source apparatus are received sequentially and simultaneously at different inlets of the ion guide apparatus.

11. The device according to claim 10, in, The ion guide apparatus comprises a chimera apparatus comprising eight L-shaped electrodes providing four branches, wherein a pair of aligned branches receives the two or more precursor ions from the ion source apparatus and simultaneously another pair of aligned branches receives the charge reduced reactants from the reactant source apparatus.

12. The apparatus of claim 10, further comprising a second ion guide device positioned between the ion guide device and the mass filter device, in, The ion guide device applies a DC voltage to the one or more electrodes of the ion guide device relative to the DC voltage applied to the electrodes of the second ion guide device and relative to the DC voltage applied to the electrodes of the mass filter device so that the two or more precursor ions having m / z values ​​increasing to the single m / z value are continuously transmitted through the second ion guide device and to the mass filter device.

13. The apparatus of claim 10, further comprising an ExD device positioned after the mass filter device, in, The mass filter device selects the two or more precursor ions having m / z values ​​that increase to the single m / z value and transmits the two or more precursor ions having m / z values ​​that increase to the single m / z value to the ExD device, and wherein the ExD device fragments the two or more precursor ions having m / z values ​​that increase to the single m / z value.

14. A method for reducing the charge of precursor ions of the same compound having different mass-to-charge ratios m / z values ​​so as to successively accumulate and transmit precursor ions of a single m / z value, include: instructing the ion source device to ionize a compound of the sample using a processor to generate two or more precursor ions of the compound having different m / z values; instructing a reactant source device to supply a charge reducing reactant using the processor; instructing, using the processor, an ion guide device positioned between both the ion source device and the reactant source device and a mass filter device to receive the two or more precursor ions from the ion source device and to receive the charge-reduced reactant from the reactant source device; instructing the ion guide device to apply, using the processor, an alternating current (AC) voltage and a direct current (DC) voltage to one or more electrodes of the ion guide device that create a pseudopotential to trap two or more precursor ions received having m / z values ​​below a threshold m / z in the ion guide device and thereby cause the trapped two or more precursor ions to be charge-reduced due to the received charge-reducing reactant so that the m / z values ​​of the two or more precursor ions are increased to a single m / z value above the threshold m / z; as well as Using the processor, instructing the ion guide device to apply a DC voltage to the one or more electrodes relative to a DC voltage applied to electrodes of the mass filter device that causes the two or more precursor ions having m / z values ​​increasing to the single m / z value to be sequentially transmitted to the mass filter device.

15. A computer program product comprising a non-transitory and tangible computer-readable storage medium, the contents of which include a program having instructions to be executed on a processor to perform a method for reducing the charge of precursor ions of the same compound having different mass-to-charge ratios m / z values ​​so as to continuously accumulate and transmit precursor ions of a single m / z value, the method include: Providing a system, wherein the system comprises one or more different software modules, and wherein the different software modules comprise a control module; instructing the ion source device to ionize a compound of the sample using the control module, thereby generating two or more precursor ions of the compound having different m / z values; instructing a reactant source device to supply a charge reducing reactant using the control module; instructing, using the control module, an ion guide device positioned between both the ion source device and the reactant source device and a mass filter device to receive the two or more precursor ions from the ion source device and the charge-reduced reactant from the reactant source device; Instructing the ion guide device to apply an alternating current (AC) voltage and a direct current (DC) voltage to one or more electrodes of the ion guide device to create a pseudopotential to trap two or more precursor ions received with m / z values ​​below a threshold m / z in the ion guide device and thereby cause the trapped two or more precursor ions to be charge-reduced due to the received charge-reducing reactant so that the m / z values ​​of the two or more precursor ions are increased to a single m / z value above the threshold m / z using the control module; and Using the control module, instructing the ion guide device to apply a DC voltage to the one or more electrodes relative to a DC voltage applied to electrodes of the mass filter device that causes the two or more precursor ions having m / z values ​​increasing to the single m / z value to be sequentially transmitted to the mass filter device.

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