Background reduction in top-down antibody analysis

Abstract

Methods and apparatus for performing a top-down analysis of an antibody are described that involve generating a plurality of ions from a sample containing at least one intact antibody with an ion source. Further, the plurality of ions are transmitted through a quadrupole rod set while applying RF signals to the quadrupole rod set and in the absence of a resolving DC voltage so as to preferentially transmit precursor ions having m / z values greater than a low mass cutoff of about 1500 m / z from the quadrupole rod set to an ECD cell. The methods and apparatus can also perform an ECD reaction on the precursor ions in the ECD cell and can also detect reaction products from the ECD reaction.

Description

[0001] Related applications

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 847,130, filed May 13, 2019, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to a method and apparatus for using electron capture dissociation (ECD) in the mass spectrometry analysis of intact antibodies. Background Technology

[0004] Mass spectrometry (MS) is an analytical technique used to determine the elemental composition of a test substance, with both quantitative and qualitative applications. For example, MS can be used to identify unknown compounds and / or determine their structure by observing the fragmentation of specific compounds. Recently, MS has played an increasingly important role in proteomics due to its speed, specificity, and sensitivity in characterizing and identifying peptides and proteins.

[0005] One strategy for characterizing proteins in MS-based proteomics is the "bottom-up" approach, in which the protein (one or more) of interest undergoes enzymatic digestion (e.g., by trypsin, LysC, etc.) and the peptide fragments are then subjected to MS analysis (MS). 1 ) or tandem MS / MS analysis (MS 2 (e.g., multidimensional LC) This process involves one or more separations prior to the initial separation. In a "bottom-up" MS... 2 In the workflow, collision-induced dissociation (CID) is typically used to further dissociate the precursor peptide fragment selected in the first MS stage into product ion fragments. The amino acid sequence of the precursor peptide ion can then be deduced from the quality of the product ion fragment. In CID, high-energy collisions between the ionized precursor ion and inert neutral gas and / or nitrogen molecules vibrate and ultimately dissociate (cleave) the backbone amide bond, producing b-type (N-terminal) and y-type (C-terminal) product ions. By identifying several product ion peptides, the original protein can be determined (e.g., by referencing known sequences in protein or genome databases). However, because CID reactions typically occur only at the weakest peptide amide bonds, incomplete fragmentation along the peptide backbone makes complete reconstruction of the peptide sequence difficult. Another key limitation of using CID in proteomics is the loss of post-translational modifications (PTMs) during dissociation. PTMs that are typically only weakly bound to the peptide backbone (e.g., phosphorylated or sulfated functional groups) are stripped from the peptide during fragmentation, preventing their formation in MS. 2 Detection and characterization of PTM in the spectrum.

[0006] In contrast to the aforementioned "bottom-up" approach, alternative MS-based proteomics strategies utilize a "top-down" analysis, where the intact protein undergoes dissociation in the mass spectrometer using, for example, ion-ion interactions. While conventional CID often dissociates too few sites to provide complete information to characterize the entire amino acid sequence of a whole protein, ion-ion interactions can be more efficient for "top-down" sequencing of intact proteins due to the more complete fragmentation of the peptide backbone. However, a potential obstacle to the "top-down" approach is the wide distribution of mass among various multi-charged precursors after ionization and the limitations of MS. 2 The complexity of the spectrum. Furthermore, real-world applications involve complex, unknown samples, which further increases the complexity of the MS. 2 The ambiguity of spectral analysis.

[0007] Therefore, there is still a need for improved methods and apparatus for mass spectrometry analysis of antibodies using a top-down approach. Summary of the Invention

[0008] Based on various aspects of this teaching, methods and systems are provided herein for performing top-down MS-based analysis of antibodies. In some aspects, the method includes generating multiple ions from a sample containing at least one intact antibody using an ion source, and transmitting the multiple ions through a quadrupole assembly while an RF signal is applied and in the absence of a resolving DC voltage, so as to preferentially transmit precursor ions having m / z values ​​larger than a low-mass cutoff of about 1500 m / z from the quadrupole assembly to an ECD unit. An ECD reaction is then performed on the precursor ions in the ECD unit, and the reaction products from the ECD reaction are detected. In various embodiments, the quadrupole assembly includes Q1 in a triple quadrupole. In some aspects, the amplitude and / or frequency of the RF signal is adjusted such that the low-mass cutoff is about 1700 m / z. Alternatively, the amplitude and / or frequency of the RF signal is adjusted such that the low-mass cutoff is about 2000 m / z. In some aspects, at least a portion of the reaction products has an m / z value below the low-mass cutoff.

[0009] In some implementations, performing an ECD reaction includes introducing electrons into the ECD unit while transporting the precursor ions through the ECD unit.

[0010] In some aspects, multiple precursor antibody ions include multiple charge states. For example, in some aspects, multiple precursor antibody ions may include antibodies of the same species but exhibiting at least two different m / z values ​​in the range of about 2000 to about 4000.

[0011] In various aspects of this teaching, a mass spectrometer system is provided, comprising: an ion source for generating multiple ions from a sample containing intact antibodies; a quadrupole assembly extending along a central longitudinal axis and configured to receive the multiple ions from the ion source through an inlet end and transmit at least a portion of the ions through an outlet end, the quadrupole assembly including a first pair of extension rods and a second pair of extension rods disposed around and parallel to the central longitudinal axis; an ECD unit for receiving ions transmitted from the quadrupole assembly, and the ions received from the quadrupole assembly reacting with electrons within the ECD unit to generate product ions therefrom; and a detector for detecting the product ions. A power system electrically coupled to the quadrupole assembly is configured to provide an RF signal to the quadrupole assembly in the absence of a resolving DC voltage, so as to substantially prevent ions having an m / z value smaller than a low mass cutoff of about 1500 m / z from the quadrupole assembly to the ECD unit. In some embodiments, the low mass cutoff may be higher than 1500 m / z. For example, in some embodiments, the power system is configured to provide an RF signal to the quadrupole assembly in the absence of a distinguishing DC voltage, so as to substantially prevent ions with m / z values ​​smaller than the low-mass cutoff of about 1700 m / z from traveling from the quadrupole assembly to the ECD unit. In other embodiments, the power system is configured to provide an RF signal to the quadrupole assembly in the absence of a distinguishing DC voltage, so as to substantially prevent ions with m / z values ​​smaller than the low-mass cutoff of about 2000 m / z from traveling from the quadrupole assembly to the ECD unit. Furthermore, the system may include a controller to adjust the low-mass cutoff. For example, in some embodiments, the controller is configured to increase the amplitude of the RF signal to increase the low-mass cutoff. In various embodiments, the amplitude of the RF signal is in the range of about 0.1 kV. p-p Up to approximately 10kV p-p Within the range.

[0012] In some embodiments, the quadrupole assembly includes Q1. Additionally, in some aspects, the quadrupole assembly is housed at a temperature maintained at less than about 1 × 10⁻⁶. -4 The chamber at the pressure point of the Torr. The system includes an electron source for introducing electrons into the ECD unit.

[0013] In various respects, the intact antibody can have an m / z in the range of about 2000 to about 4000. Additionally, in some embodiments, at least a portion of the product ions has an m / z below the low mass cutoff.

[0014] Each of the above features can be paired and / or combined and / or removed in various ways with the specific embodiments described to obtain alternative embodiments that should be considered within the scope of this teaching.

[0015] These and other characteristics of the applicant's education are described in this article. Attached Figure Description

[0016] The foregoing and other objects and advantages of the invention will be more fully understood from the following further description, with reference to the accompanying drawings. Those skilled in the art will understand that the drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of the applicant's teachings in any way.

[0017] Figure 1 An exemplary ECD-mass spectrometer system is schematically depicted based on various aspects of the applicant's teachings.

[0018] Figure 2 The illustration depicts various aspects of this teaching suitable for use in Figure 1 An example quadrupole used in the system.

[0019] Figure 3 The illustration schematically depicts various aspects of this teaching operating in RF-only mode. Figure 2 An example of a quadrupole.

[0020] Figure 4 A flowchart is provided illustrating an exemplary method for performing top-down ECD-based analysis of antibodies, according to various aspects of this teaching.

[0021] Figures 5A-5C Example property spectra of antibody-containing samples to be analyzed according to this teaching are depicted. Detailed Implementation

[0022] It will be understood that, for clarity, the following discussion will illustrate various aspects of embodiments of the applicant's teachings, while certain specific details are omitted wherever convenient or appropriate. For example, the discussion of similar or analogous features in alternative embodiments may be simplified. For brevity, well-known ideas or concepts may also not be discussed in detail. Those skilled in the art will recognize that some embodiments of the applicant's teachings may not require certain details specifically described in each implementation, which are set forth herein merely to provide a thorough understanding of the embodiments. Similarly, it will be clear that the described embodiments can be readily changed or varied based on common general knowledge without departing from the scope of this disclosure. The following detailed description of the embodiments should not be construed as limiting the scope of the applicant's teachings in any way. As used herein, the terms "about" and "substantially equal" refer to variations in numerical quantities that may occur, for example, by: real-world measurements or processes; negligence or errors in these processes; differences in the manufacture, origin, or purity of the composition or reagent, etc. Generally, the terms "about" and "substantially" as used herein mean greater than or less than 1 / 10 of the value or range of values, for example, ±10%. For example, a concentration value of approximately 30% or substantially equal to 30% can represent a concentration between 27% and 33%. The term also refers to equivalent variations that those skilled in the art will recognize, provided such variations do not include values ​​known in prior art practice.

[0023] In various aspects, this paper provides methods and systems for analyzing ions to enable top-down ECD-based analysis of intact antibodies. Conventional MS-based proteomics methods can lead to complex and / or inaccurate data due to interfering species and the presence of multi-charged precursor ions with different charge states, while the teachings of this paper result in less complex mass spectrometry while utilizing the high dissociation efficiency of ECDs to produce the complete sequence of antibody ions. As discussed in detail below, various aspects of the methods and systems disclosed herein selectively prevent ions exhibiting m / z below the low mass cutoff (LMCO) from entering the ECD unit and allow the transport of ions formed from intact antibodies, regardless of their charge state.

[0024] Although the systems, apparatus, and methods described herein can be used in conjunction with many different mass spectrometer systems having fewer, more, or different components than those depicted, Figure 1 The example property spectrometer system 100 used according to this teaching is schematically illustrated. Figure 1As illustrated in the exemplary embodiments, the mass spectrometer system 100 typically includes: a sample ion source 102 for ionizing a sample containing or suspected of containing one or more antibodies of interest, thereby generating a variety of precursor ions; and a quadrupole assembly 104 for receiving the generated ions from the ion source 102 and then transferring a portion of the generated ions to an ECD reaction unit 110. The ECD unit 110 includes an interaction region in which the precursor cation interacts with electrons generated by an electron source 106 to dissociate the precursor ions into a variety of product ions detected by a detector 140. Figure 1 As shown, the exemplary mass spectrometer system 100 further includes one or more power supplies (e.g., DC power supply 105 and RF power supply 107), controlled by a controller 108 to apply potentials having RF, AC, and / or DC components to the electrodes of various components, thereby configuring the elements of the mass spectrometer system 100 in a coordinated manner and / or for various different operating modes. As discussed in detail below, in an exemplary embodiment of this teaching, the electrical signal applied to the quadrupole assembly 104 is set and / or adjusted to preferentially transport precursor ions with m / z values ​​greater than LMCO (e.g., approximately 1500 m / z) to the ECD unit 110. In a particular embodiment, an RF signal exhibiting amplitude and frequency is applied to the individual bars of the quadrupole assembly, and in the absence of a DC resolution voltage on these bars, this amplitude and frequency are selected to substantially prevent ions with m / z values ​​below LMCO from being transported to downstream elements, thereby eliminating these low m / z ions to prevent interference with the MS generated by detecting product ions after the antibody precursor ions react in the ECD unit 110. 2 Subsequent analysis and / or interpretation of the spectrum.

[0025] Ion source 102 can have various configurations, but is generally configured to generate ions (e.g., cations) from antibodies contained in a sample. In some embodiments, a suitable sample source used according to these teachings is configured to contain a sample (e.g., a solution containing or suspected of containing an antibody of interest) and / or introduce the sample into ion source 102, for example, via fluid coupling to transfer a liquid sample to ion source 102 through one or more conduits, channels, tubes, pipes, capillaries, etc. As a non-limiting example, the sample source includes a reservoir of the sample to be analyzed or an input port through which the sample is injected. In some aspects, for example, the sample source includes an infusion pump (e.g., a syringe pump) for continuously flowing the sample into ion source 102. Alternatively, also as a non-limiting example, the liquid sample to be analyzed is in the form of eluent from an online liquid chromatography column, but in some aspects, one or more sample preparation steps (e.g., multidimensional LC separation, electrophoresis, disulfide bond reduction, etc.) can be performed offline.

[0026] In some exemplary aspects of this teaching, ion source 102 includes a conduit in direct or indirect fluid communication with a sample source, terminating at an outlet end that extends at least partially into an ionization chamber. As a liquid sample is discharged from the outlet end into the ionization chamber (e.g., as multiple droplets), antibodies (and other interfering analytes) contained within the droplets are ionized (i.e., charged) by ion source 102. As the liquid (e.g., solvent) within the droplets evaporates, ions are released and drawn toward and through orifices to be transported to quadrupole assembly 104 and ECD unit 110. It will be understood that a variety of different devices known in the art and modified according to the teachings herein can be used as ion source 102. As a non-limiting example, ion source 102 can be an electrospray ionization device, a nebulizer-assisted electrospray device, a chemical ionization device, a nebulizer-assisted atomization device, a photoionization device, a laser ionization device, a thermal spray ionization device, or an acoustic spray ionization device.

[0027] like Figure 1 As shown in the embodiment, system 100 includes an ECD unit 110, in which precursor ions transported by quadrupole assembly 104 can undergo an ECD reaction. ECD reactions typically involve the interaction of a multiprotonated molecule M with free electrons to form a radical species with an odd number of electrons.

[0028] [M+nH] n+ +e - →[M+nH] (n-1)+· →ECD fragment

[0029] An electron release binding energy is added to the incomplete molecular orbitals of the precursor antibody cation. If this binding energy is sufficient to exceed the dissociation threshold, it will cause fragmentation of the electron acceptor ion, as is known in the art. Once the precursor ion has reacted within the ECD unit 110, the fragment or product ion can be transferred to one or more mass analyzers for further analysis before detection by the detector 110. For example, the mass analyzer positioned between the ECD unit 110 and the detector 140 may include any suitable mass spectrometry module, including but not limited to, time-of-flight (TOF) mass spectrometry modules, quadrupole mass spectrometry modules, linear ion trap (LIT) modules, etc., for scanning the product ion therefrom. As a non-limiting example, the ECD reaction can be performed in the ECD device described in PCT Publication WO2014191821 entitled “Inline Ion Reaction Device Cell and Method of Operation,” the teachings of which are incorporated herein by reference in their entirety. To meet the need to provide electrons to the ECD unit 110 for the ECD reaction, the system 100 further includes an electron source 106. Those skilled in the art will understand that any electron source suitable for a mass spectrometer system to provide electrons for ion-ion reactions and modified in accordance with this teaching can be used in system 100. As a non-limiting example, electrons can be generated by a filament (e.g., tungsten, thorium-tungsten, etc.) or another electron emitter (e.g., a Y₂O₃ cathode). In exemplary operation, a current of 1 to 3 A can be applied to heat the electron source, which generates 1 to 10 W of thermal power to generate electrons. It will be understood that, in some aspects, the electron source 106 can be additionally associated with a magnetic field generator (e.g., a permanent neodymium magnet or an electromagnet, not shown) to control the path of electrons within the ECD unit 110, and with a cooling mechanism (e.g., a heat sink, active cooling) to keep the temperature of the magnet (if present) below its Curie temperature (at which permanent magnets lose magnetization). Other known methods for cooling the magnet can also be utilized.

[0030] As shown in the figure, system 100 includes detector 140 (e.g., time-of-flight mass analyzer, ion trap mass analyzer, Faraday cup, or other ion current measurement device) to efficiently detect precursor and / or product ions transmitted from ECD unit 110. The detected ion data can be stored in memory and analyzed by a computer or computer software.

[0031] As those skilled in the art will understand, system 100 may additionally include any number of additional mass analyzer elements or ion optics disposed upstream or downstream of quadrupole assembly 104 and ECD unit 110 for further ion processing, manipulation, and / or mass analysis. For example, ions may be transported through one or more additional differential pumping vacuum stages (e.g., a first stage maintained at approximately 2.3 Torr, a second stage maintained at approximately 6 millitors, and a third stage maintained at approximately 10...). -5 The third stage at the pressure of the Torr, wherein the third unit contains a detector 140 and two or more quadrupole mass analyzers, and an ECD unit 110 is located therebetween. For example, in one embodiment, the quadrupole group 104 represents Q1 (maintained at less than about 1 × 10⁻⁶). -4 (The pressure of the Torr) and ECD unit 110 represents or replaces Q2 in the QqQ triple quadrupole mass spectrometer (see, for example, Baba et al., “Electron Capture Dissociation in a Radio Frequency IonTrap,” Anal. Chem. 1 Aug. 2004; 76(15):4263-6, and PCT Publication No. WO2014191821 entitled “Inline Ion Reaction Device Cell and Method of Operation,” the teachings of each of these exemplary references describing ECD devices are incorporated herein by reference in their entirety).

[0032] As shown in the figure, the depicted system 100 further includes a controller 108 operatively coupled to one or more of the elements of the system 100 to control its operation. For example, the controller 108 may include a processor for processing information, a data storage device for storing mass spectrometry data and instructions to be executed. As discussed in detail below and as is known in the art and modified according to this teaching, for example, the controller 108 may control the sample ion source 102 to generate ions and the electron source 106 to generate electrons and / or control the movement of ions into and through the quadrupole assembly 104 and the ECD unit 110 via applying one or more RF / DC voltages to the electrodes of the quadrupole assembly 104 and the ECD unit 110. It will be understood that although the controller 108 is depicted as a single component, one or more controllers (whether local or remote) may be configured to operate the mass spectrometer system 100 according to any of the methods described herein. Furthermore, in some embodiments, controller 108 may be operatively associated with an output device such as a display (e.g., a cathode ray tube (CRT) or liquid crystal display (LCD) for displaying information to a computer user) and / or an input device including alphanumeric and other keys and / or cursor control to transmit information and command selection to a processor. Consistent with some embodiments of this teaching, controller 108 executes one or more sequences of one or more instructions, such as those contained in a data storage device or read into memory from another computer-readable medium such as a storage device (e.g., a disk). One or more controllers may take the form of hardware or software; for example, controller 108 may take the form of a suitably programmed computer in which a computer program is stored, which is executed to operate the mass spectrometer system 100 as otherwise described herein, but embodiments of this teaching are not limited to any particular combination of hardware circuitry and software. For example, various software modules associated with controller 108 may execute programmable instructions to perform the following references. Figure 4 The exemplary method described.

[0033] As described above, the exemplary mass spectrometer system 100 includes one or more power supplies controlled by a controller 108 to apply potentials having RF, AC, and / or DC components to the electrodes of various components to configure the elements of the mass spectrometer system 100 in a coordinated manner and / or for various different operating modes, as discussed elsewhere herein. Referring now to... Figure 2 An exemplary quadrupole assembly 104 includes four rods 104a-104d arranged around and parallel to a central longitudinal axis (Z) extending from an inlet end (e.g., toward the ion source) to an outlet end (e.g., toward the ECD cell). Rods 104a-104d include cylindrical shapes (i.e., circular cross-sections with radius r) arranged equidistantly from the central axis (Z). Figure 2The rods (shown) are identical in size and shape to each other. The minimum distance between each rod of 104a-104d and the central axis (Z) is defined by a distance r0 such that the innermost surface of each primary rod 104a-104d and the innermost surface of the other rod in its pair are separated by a minimum distance 2r0 across the central longitudinal axis (Z). In some exemplary embodiments, r0 of the rod assembly 104 ranges from about 3 mm to about 10 mm. It will be understood that although the rods 104a-104d are depicted as cylindrical, the cross-sectional shape, size, and / or relative spacing of the rods 104a-104d can vary as is known in the art. For example, in some aspects, the rods 104a-104d can be formulated according to the formula... It exhibits a radially inner hyperbolic surface, where r0 (field radius) is the radius of the inscribed circle between the electrodes in order to generate a quadrupole field.

[0034] Rods 104a-104d are conductive (i.e., they can be made of any conductive material such as metal or alloy) and can be coupled to an electrical system (including...). Figure 1 One or more power supplies 105, 107) are provided to enable one or more electrical signals to be applied individually or in combination to each rod 104a-104d. Specifically, rods 104a-104d typically comprise two pairs of rods (e.g., a first pair comprising rods 104a and 104c and a second pair comprising rods 104b and 104d), wherein each pair of rods is positioned on opposite sides of the central axis (Z) and the same electrical signal can be applied to each pair of rods. For example, in... Figure 2 In some aspects shown, the power system may include: a power supply 106a electrically coupled to the first pair of rods 104a, 104c to apply the same potential to the first pair of rods 104a, 104c; and a power supply 106b electrically coupled to the second pair of rods 104b, 104d to apply different electrical signals to the second pair of rods 104b, 104d. Figure 2As shown, in some embodiments, the exemplary power system can apply a potential of [U-VcosΩt] to the first pair of poles 104a, 104c, where U is the magnitude of the DC electrical signal, V is the zero-to-peak amplitude of the AC or RF signal, Ω is the frequency of the AC or RF signal, and t is time. Similarly, the exemplary power system can apply a potential of -[U-VcosΩt] to the second pair of poles 104b, 104d. In this exemplary configuration, the electrical signals applied to the first pair of poles 104a, 104c and the electrical signals applied to the second pair of poles 104b, 104d differ in the polarity of the DC signal (i.e., the sign of U), while the RF portions of the electrical signals are 180° out of phase with each other. Therefore, those skilled in the art will understand that the quadrupole assembly 104 can be considered, in some respects, as a quadrupole mass filter capable of selectively transporting ions in a selected m / z range by a suitable selection of the DC / RF ratio. For example, consider DC electrical signals (i.e., ±U) applied individually to the four primary rods 104a-104d, such as Figure 2 The cations injected into the quadrupole assembly 104 will experience stabilizing forces in the XZ plane (towards the central axis Z) based on the application of a positive DC voltage to the first pair of electrodes 104a, 104c, while simultaneously experiencing destabilizing forces in the YZ plane based on the application of a negative DC voltage to the second pair of electrodes 104b, 104d. Considering the effect of the RF signal alone, as the RF signal applied to the pole pairs changes over time, the cations will be attracted and repelled sequentially by the respective pole pairs 104a, 104c and 104b, 104d. Because low m / z cations are more likely to follow the alternating components of the field, they will tend to remain more in phase with the RF signal, gain energy from the field, and oscillate with increasingly larger amplitudes until they encounter one of the poles 104a-104d and discharge. Now, considering the combined effects of the DC and RF signals, it will be understood that the field in the XZ plane will act as a high-pass mass filter, as only high m / z ions will be transported to the other end of the quadrupole without impacting the first pair of electrodes 104a, 104c. On the other hand, in the YZ plane, high m / z cations will be unstable due to the defocusing / attraction effect of the negative DC voltage. However, if the amplitude of the RF component is set to correct the trajectory whenever the cation deviation increases, some lower m / z ions can be stabilized by the RF component. Therefore, it can be said that the field in the YZ plane acts as a low-pass mass filter, because only lower m / z ions will be transported to the other end of the quadrupole assembly 104 without impacting the second pair of rods 104b, 104d.

[0035] By appropriately selecting the RF / DC ratio of the electrical signal applied to the quadrupole assembly 104, the two effects described above, in both the XZ and YZ planes, provide a mass filter capable of resolving individual atomic masses, as illustrated in the exemplary interpolation Mathieu stability plots of the following parameters:

[0036]

[0037]

[0038] Where e is the charge on the electron, U is the amplitude of the DC voltage, V is the applied zero-to-peak RF voltage, m is the mass of the ion, r0 is the effective radius between 10⁴a and 10⁴d, and Ω is the applied RF frequency. It should be noted that parameters a and q are proportional to the DC voltage U and the RF voltage V, respectively, and q = 0.908 at the stability boundary of the Mathieu stability plot.

[0039] As described above, the exemplary mass spectrometer system 100 includes one or more power supplies controlled by a controller 108 to apply potentials having RF, AC, and / or DC components to the electrodes of various components to configure the elements of the mass spectrometer system 100 in a coordinated manner and / or for various different operating modes, as discussed elsewhere herein. Although Figure 2 The text describes the application of both a DC signal (i.e., ±U) and an RF signal (i.e., ±[U-VcosΩt]) to the bars 104a-104d of the quadrupole assembly 104 during mass filter implementation (e.g., by ramping U and V). However, this teaching provides a system and method in which electrical signals are applied to the individual bars of the quadrupole assembly 104 without a DC-resolving voltage during periods of preferential transport of ions at and above LMCO m / z. Referring now to... Figure 3 The quadrupole assembly 104 is shown in such an RF-only configuration that those ions with m / z greater than LMCO are preferentially transported to the ECD unit 110 for further processing, as discussed elsewhere herein. That is, as Figure 3 As shown, the DC signal (U) is set to 0V, making parameter a in equation (1) zero. Under these conditions, where only the RF signal exhibiting peak-to-peak amplitude (V) and frequency (Ω) is applied to the individual bars 104a-104d, such as... Figure 3 As shown in the interpolation Mathieu stability plot, the mass scan line becomes horizontal, allowing entry into quadrupole group 104 and at q max Ions stable at or below 0.908 are selectively transported to ECD110. In other words, by rearranging the above formula (2) and replacing q with 0.908, according to the following formula m / e LMCOThe lowest mass-to-charge ratio ion transported by the quadrupole assembly 104:

[0040]

[0041] By appropriately selecting the RF amplitude (V) and frequency (Ω) applied to the quadrupole assembly 104 according to various aspects of this teaching, the LMCO can be set and / or adjusted to substantially prevent the transport of low m / z ions to downstream elements, thereby eliminating interference with subsequent analysis and / or interpretation of the spectrum generated by the detection of product ions after the reaction with higher m / z antibody precursor ions in the ECD unit 110. In some aspects, for example, the amplitude and frequency of the RF signal can be set such that ions exhibiting m / z greater than 1500, greater than 1700, or greater than 2000 can be selectively transported by the quadrupole assembly 104. Exemplary combinations of V and Ω include those from about 0.1 kV. p-p Approximately 10kV p-p The RF amplitude and / or RF frequency in the range from about 0.8 MHz to about 3 MHz, wherein the values ​​are selected, for example, according to the above formula (3), based on the effective field radius of the quadrupole and the desired LMCO. In an exemplary embodiment, to obtain an LMCO equal to about 1500 m / z, the field radius (r0) may be 0.4 mm, the RF amplitude may be 6.4 kV, and the RF frequency may be about 1.2 MHz. Those skilled in the art will understand that such values ​​of RF amplitude according to this teaching can be significantly larger than those values ​​typically used by quadrupole ion traps operating in narrow-bandpass mass filter mode (e.g., as...). Figure 2 (as shown in the image).

[0042] Now refer to Figure 4 It describes the various aspects of operation based on this teaching. Figure 1 An exemplary method 400 for a mass spectrometer system. As shown in step 401, method 400 may begin by delivering a sample containing an antibody to an ion source 104, whereby molecules within the sample are ionized. Once the molecules within the sample (including the antibody) are ionized, these ions are delivered, for example, through one or more ion lenses, a mass analyzer, and / or a differential pump to a vacuum stage (e.g., Q0 or Q10). jet ) and are transported to quadrupole assembly 104, as shown in step 402. When ions are transported into and through quadrupole assembly 104, as Figure 3An RF signal is applied to each of the bars 104a-104d, such that a combination of RF amplitude (V) and frequency (Ω) destabilizes and discharges ions with an LMCO lower than that associated with the RF signal, while allowing those ions with an LMCO higher (including antibody ions with relatively high m / z) to be transported to the ECD unit (step 403). According to this teaching, it will be understood that operating the quadrupole assembly 104 in this RF-only mode allows the transport of antibody ions of interest to the ECD unit 110 even when the antibody ions exhibit different charge states (e.g., antibodies of the same species, where one molecule carries a single charge and another carries a double charge). In step 404, these precursor antibody ions transported to the ECD unit 110 then undergo an ECD reaction (e.g., via interaction with electrons captured within or transported through the ECD unit 110 in step 405), resulting in the formation of product ions. In step 406, the ECD product ions are then detected by detector 110 to generate an ECD product ion spectrum, which can be analyzed as is known in the art to reconstruct the antibody structure in a “top-down” analysis.

[0043] Refer to the following examples and Figures 5A-5C The data presented can provide a more comprehensive understanding of the applicant's teaching and offer... Figures 5A-5C This is for illustrative purposes only and does not limit the teachings. As described below, the methods and systems disclosed herein selectively block the transport of ions exhibiting m / z below the low mass cutoff (LMCO) to downstream ECD cells.

[0044] Example 1

[0045] Samples containing humanized monoclonal IgG antibodies (NIST-mAb) obtained from NIST were prepared. Samples were desalted and separated by LC using a desalted LC column (water), followed by electrospray ionization and mass spectrometry analysis using a research-grade quadrupole-TOF system (SCIEX). This SCIEX system was modified to include an ECD unit as described in the article “Electron Capture Dissociation in a Branched Radio-Frequency Ion Trap” published in Anal. Chem. 87(1):785-792 (incorporated in its entirety by reference). The ECD unit was mounted between Q1 and Q2 of the mass spectrometer system. A typical electron beam irradiation time was 10 ms, with the electron beam intensity adjusted to achieve appropriate dissociation efficiency. The TOF-MS had a mass resolution of 35,000–47,000, resolving isotopic modes up to Z–30+ fragments. Figure 5A The spectrum produced by the ionized sample was depicted, where the RF amplitude was selected at 400 V.p-p This allows virtually all ion transport to pass through. Figure 5A The spectrum includes a significant signal from ions exhibiting m / z values ​​less than 500, as well as several prominent peaks at approximately m / z 1520. Now refer to... Figure 5B The amplitude of the RF signal applied to the quadrupole assembly is increased to 5kV. p-p (The DC voltage was set to 0V), and the ions transported by Q1 were detected again (without ECD reaction). This will be compared... Figure 5A and Figure 5B It was observed that by increasing the amplitude of the RF signal, Q1 blocked the transport of ions exhibiting m / z values ​​less than approximately 1700. Figure 7 depicts the effect of... Figure 5B The ions detected after ECD of precursor ions transported from Q1 under the specified conditions. The low-intensity peaks (m / z less than approximately 2000) represent product ion fragments after ECD. Comparison Figure 5A and 5C It will be understood that if low m / z ions are not removed according to this teaching, then in Figure 5C Many of the peaks in the product ion peaks observed will be confused, making top-down analysis of antibodies more challenging.

[0046] Those skilled in the art will know or be able to determine many equivalents of the embodiments and practices described herein using only conventional experiments. For example, the dimensions of the various components and the exact values ​​of the specific electrical signals applied to the various components (e.g., amplitude, frequency, etc.) are merely exemplary and not intended to limit the scope of this teaching. Therefore, it will be understood that the invention is not limited to the embodiments disclosed herein, but should be understood from the following claims, which should be interpreted broadly to the extent permitted by law.

[0047] The chapter headings used herein are for organizational purposes only and should not be construed as limiting. While the applicant's teachings have been described in conjunction with various embodiments, it is not intended that the applicant's teachings be limited to these embodiments. Rather, as those skilled in the art will understand, the applicant's teachings encompass various alternatives, modifications, and equivalents.

Claims

1. A method for performing top-down analysis of antibodies, comprising: Multiple ions are generated from a sample containing at least one intact antibody using an ion source. While applying an RF signal to the quadrupole assembly that operates as a mass filter and without distinguishing DC voltage, the various ions are transported through the quadrupole assembly in order to preferentially transport precursor ions with m / z values ​​larger than the low mass cutoff of 1500 m / z from the quadrupole assembly to the ECD unit. The precursor ions undergo an ECD reaction in the ECD unit; The reaction products from the ECD reaction are detected.

2. The method according to claim 1, wherein the quadrupole assembly includes Q1 in a triple quadrupole configuration.

3. The method according to claim 1 or 2, further comprising adjusting at least one of the amplitude and frequency of the RF signal such that the low-quality cutoff is 1700 m / z.

4. The method according to claim 1 or 2, further comprising adjusting at least one of the amplitude and frequency of the RF signal such that the low-quality cutoff is 2000 m / z.

5. The method according to claim 1 or 2, wherein the low-quality cutoff is 1700 m / z.

6. The method according to claim 1 or 2, wherein the low-quality cutoff is 2000 m / z.

7. The method according to claim 1 or 2, wherein at least a portion of the reaction product has an m / z value lower than the low mass cutoff.

8. The method of claim 1 or 2, wherein performing the ECD reaction includes introducing electrons into the ECD unit while transmitting the precursor ions through the ECD unit.

9. The method according to claim 1 or 2, wherein the precursor ion comprises an ion of the antibody in multiple charge states.

10. The method of claim 9, wherein the ions of the antibody in multiple charge states exhibit at least two m / z values ​​in the range of 2000 to 4000.

11. A mass spectrometer system, comprising: An ion source is used to generate multiple ions from a sample containing intact antibodies; As a mass filter, the quadrupole assembly extends along a central longitudinal axis and is configured to receive the plurality of ions from the ion source through an inlet end and transmit at least a portion of the ions through an outlet end, the quadrupole assembly including a first pair of extension rods and a second pair of extension rods arranged around and parallel to the central longitudinal axis. An ECD unit is used to receive ions transported from the quadrupole assembly, and the ions received from the quadrupole assembly react with electrons in the ECD unit to produce product ions therefrom; A detector is used to detect the product ions; A power system, electrically coupled to the quadrupole assembly, is configured to provide an RF signal to the quadrupole assembly in the absence of a distinguishing DC voltage, so as to substantially prevent ions with m / z values ​​smaller than the low mass cutoff of 1500 m / z from the quadrupole assembly to the ECD unit.

12. The system of claim 11, wherein the quadrupole assembly comprises Q1.

13. The system of claim 11 or 12, wherein the quadrupole assembly is housed in a state maintained at less than 1 × 10⁻⁶. -4 The chamber at the pressure point of the torrent.

14. The system of claim 11 or 12, wherein the power system is configured to provide an RF signal to the quadrupole assembly in the absence of a distinguishing DC voltage in order to substantially prevent ions with an m / z value smaller than the low mass cutoff of 1700 m / z from traveling from the quadrupole assembly to the ECD unit.

15. The system of claim 11 or 12, wherein the power system is configured to provide an RF signal to the quadrupole assembly in the absence of a distinguishing DC voltage in order to substantially prevent ions with an m / z value smaller than a low mass cutoff of 2000 m / z from traveling from the quadrupole assembly to the ECD unit.

16. The system of claim 11 or 12 further includes a controller configured to increase the amplitude of the RF signal to increase the low-quality cutoff.

17. The system of claim 11 or 12, wherein at least a portion of the product ions has an m / z value lower than the low mass cutoff.

18. The system of claim 11 or 12, wherein the intact antibody has an m / z in the range of 2000 to 4000.

19. The system of claim 11 or 12 further includes an electron source for introducing electrons into the ECD unit.

20. The system according to claim 11 or 12, wherein the amplitude of the RF signal is from 0.1 kV. p-p Up to 10kV p-p Within the range.

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