Background reduction in top-down antibody analysis

Abstract

A method and device for performing top-down analysis of antibodies is described, which involves using an ion source to generate a plurality of ions from a sample containing at least one intact antibody.Furthermore, while applying an RF signal thereto, in the absence of a resolving DC voltage, the plurality of ions are transmitted through a quadrupole rod set so as to transmit precursor ions having m / z values ​​above a low mass cutoff value of about 1,500 m / z from the quadrupole rod set to an ECD cell.The method and device can also perform an ECD reaction on the precursor ions in the ECD cell, and detect reaction products from the ECD reaction.

Description

[Technical field]

[0001] 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.

[0002] The present invention relates to methods and apparatus that utilize electron capture dissociation (ECD) in the mass spectrometric analysis of the mass of intact antibodies. [Background technology]

[0003] Mass spectrometry (MS) is an analytical technique for determining the elemental composition of a test substance that has both quantitative and qualitative applications. For example, MS can be used to identify unknown compounds and / or to determine the structure of a particular compound by observing its fragmentation. Recently, MS has played an increasingly important role in proteomics due to the speed, specificity, and sensitivity of MS strategies in characterizing and identifying peptides and proteins.

[0004] One strategy for characterizing proteins in MS-based proteomics is to analyze peptide fragments using MS (MS 1 ) or tandem MS / MS analysis (MS 2 A "bottom-up" approach is where the protein of interest is subjected to enzymatic digestion (e.g., via trypsin, LysC, etc.) and one or more rounds of separation (e.g., multidimensional LC) prior to exposure to "bottom-up" MS. 2In the workflow, collision-induced dissociation (CID) is traditionally utilized to further dissociate the precursor peptide fragments selected in the first MS stage into product ion fragments. The amino acid sequence of the precursor peptide ion can then be inferred from the masses of the product ion fragments. In CID, energetic collisions between the ionized precursor ion and an inert neutral gas and / or nitrogen molecule vibrate the backbone amide bond and ultimately dissociate (cleave) it, thereby resulting in b-type (N-terminal) and y-type (C-terminal) product ions. By identifying some of the product ion peptides (e.g., by referencing known sequences in proteins or genome databases), the original protein can be determined. However, because the CID reaction generally occurs only at the weakest peptide amide bonds, incomplete fragmentation along the peptide backbone can make complete reconstruction of the peptide sequence difficult. Another important limitation to the use of CID in proteomics is the loss of post-translational modifications (PTMs) during dissociation. PTMs, which are often only weakly attached to the peptide backbone (e.g., phosphorylation or sulfation functionalities), are stripped from the peptide during fragmentation, thereby reducing the number of PTMs that can be bound to the peptide backbone. 2 hindering the detection and characterization of PTMs in the spectrum.

[0005] In contrast to the "bottom-up" approach described above, an alternative MS-based proteomics strategy instead utilizes "top-down" analysis, where intact proteins undergo dissociation in a mass spectrometer, for example, using ion-ion interactions. Conventional CID generally provides too few dissociation sites to provide complete information and characterize the entire amino acid sequence of an intact protein, whereas ion-ion interactions can be effective for "top-down" sequencing of intact proteins due to more complete fragmentation of the peptide backbone. However, one possible obstacle to the "top-down" approach is the wide distribution of masses of the various multiply charged precursors following ionization, and the large number of fragments that can be generated by the MS. 2 Moreover, real-world applications involve complex unknown samples, which can be difficult to measure using MS2 This may add further ambiguity in the analysis of the spectrum.

[0006] Thus, there remains a need for improved methods and devices for mass spectrometric analysis of antibodies utilizing a top-down approach. Summary of the Invention [Means for solving the problem]

[0007] According to various aspects of the present teachings, methods and systems for performing top-down MS-based analysis of antibodies are provided herein. In one aspect, the method includes utilizing an ion source to generate a plurality of ions from a sample containing at least one intact antibody, and transmitting the plurality of ions through a quadrupole rod set while applying an RF signal to the quadrupole rod set in the absence of a resolving DC voltage to preferentially transmit precursor ions having m / z values ​​above a low mass cutoff value of about 1,500 m / z from the quadrupole rod set to an ECD cell. An ECD reaction is then performed on the precursor ions in the ECD cell, and reaction products from the ECD reaction are detected. In various implementations, the quadrupole rod set comprises Q1 in a triple quadrupole. In one aspect, the amplitude and / or frequency of the RF signal is adjusted such that the low mass cutoff value is about 1,700 m / z. Alternatively, the amplitude and / or frequency of the RF signal is adjusted such that the low mass cutoff value is about 2,000 m / z. In some aspects, at least a portion of the reaction products have an m / z below a low mass cutoff value.

[0008] In some implementations, performing an ECD reaction includes introducing electrons into the ECD cell while transmitting precursor ions through the ECD cell.

[0009] In one aspect, the plurality of precursor antibody ions comprises a plurality of charge states. For example, in one aspect, the plurality of precursor antibody ions can comprise antibodies of the same species of antibody, but exhibiting at least two different m / z within the range of about 2,000 to about 4,000.

[0010] In various aspects of the present teachings, a mass spectrometer system is provided that includes an ion source for generating a plurality of ions from a sample containing intact antibodies; a quadrupole rod set extending along a central longitudinal axis, the quadrupole rod set configured to receive the plurality of ions from the ion source through an entrance end and transmit at least a portion of the ions through an exit end, the quadrupole rod set including a first pair of elongate rods and a second pair of elongate rods disposed about and parallel to the central longitudinal axis; an ECD cell for receiving the ions transmitted from the quadrupole rod set, in which the ions received from the quadrupole rod set are reacted with electrons to generate product ions from the ECD cell; and a detector for detecting the product ions. A power system electrically coupled to the quadrupole rod set is configured to provide an RF signal to the quadrupole rod set in the absence of a resolving DC voltage to substantially prevent ions having m / z values ​​less than a low mass cutoff value of about 1,500 m / z from being transmitted from the quadrupole rod set to the ECD cell. In some implementations, the low mass cutoff value can be higher than 1,500 m / z. For example, in some implementations, the power system is configured to provide an RF signal to the quadrupole rod set in the absence of a resolving DC voltage to substantially prevent ions having m / z values ​​less than a low mass cutoff value of about 1,700 m / z from being transmitted from the quadrupole rod set to the ECD cell. In other implementations, the power system is configured to provide an RF signal to the quadrupole rod set in the absence of a resolving DC voltage to substantially prevent ions having m / z values ​​less than a low mass cutoff value of about 2,000 m / z from being transmitted from the quadrupole rod set to the ECD cell. In addition, the system can include a controller for adjusting the low mass cutoff value. For example, the controller may be configured in some embodiments to increase the amplitude of the RF signal and increase the low mass cutoff value. In various implementations, the amplitude of the RF signal is about 0.1 kV. p-p ~about 10kV p-p is within the range.

[0011] In some implementations, the quadrupole rod set comprises Q1. Additionally, in some aspects, the quadrupole rod set comprises about 1×10 -4 The ECD cell is housed in a chamber that is maintained at a pressure below Torr. An electron source is included in the system for introducing electrons into the ECD cell.

[0012] In various aspects, the intact antibody can have an m / z in the range of about 2,000 to about 4,000. Additionally, at least a portion of the product ions, in some implementations, have an m / z below a low mass cutoff value.

[0013] Each of the features described above can be combined and / or combined in various ways with the specific embodiments described, and / or removed to result in alternative embodiments that are to be considered within the scope of the present teachings.

[0014] These and other features of the applicants' teachings are described herein. [Brief description of the drawings]

[0015] The foregoing and other objects and advantages of the present invention will be more fully understood from the following further description taken in conjunction with the accompanying drawings, in which: Those skilled in the art will appreciate that the drawings described below are for illustrative purposes only and are not intended to limit the scope of applicant's teachings in any way.

[0016] [Figure 1] FIG. 1 diagrammatically depicts an exemplary ECD mass spectrometer system in accordance with various aspects of the applicant's teachings.

[0017] [Diagram 2] FIG. 2 diagrammatically depicts one exemplary quadrupole suitable for use in the system of FIG. 1 in accordance with various aspects of the present teachings.

[0018] [Diagram 3]FIG. 3 diagrammatically depicts the example quadrupole of FIG. 2 operated in an RF-only mode in accordance with various aspects of the present teachings.

[0019] [Figure 4] FIG. 4 depicts a flow chart of one exemplary method for performing ECD-based top-down analysis of antibodies according to various aspects of the present teachings.

[0020] [Diagram 5] 5A-C depict exemplary mass spectra of a sample containing an antibody to be analyzed in accordance with the present teachings. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] For clarity, it should be understood that the following discussion details various aspects of embodiments of the applicant's teachings while omitting certain specific details whenever it is convenient or appropriate to do so. For example, discussion of like or similar features in alternative embodiments may be somewhat abbreviated. Well-known ideas or concepts may also not be discussed in further detail for brevity. Those skilled in the art will recognize that some embodiments of the applicant's teachings may not require some of the specifically described details set forth herein solely to provide a complete understanding of the embodiment in all implementations. Similarly, it will be apparent that the described embodiments may be subject to modification or variation in accordance with common general knowledge without departing from the scope of the present disclosure. The following detailed description of the embodiments should not be considered in any way as limiting the scope of the applicant's teachings. As used herein, the terms "about" and "substantially equal" refer to variations in numerical quantities that may occur, for example, through real-world measurement or handling procedures, through inadvertent errors in these procedures, through differences in the manufacture, source or purity of compositions or reagents, and through equivalents. Typically, as used herein, the terms "about" and "substantially" refer to 1 / 10 of the stated value, e.g., ±10%, a value or range greater or less than the stated value. For example, a concentration value of about 30% or substantially equal to 30% can refer to a concentration of 27% to 33%. The terms also refer to such variations that are recognized by those skilled in the art as equivalents, unless the variations encompass known values ​​practiced by the prior art.

[0022] In various aspects, methods and systems are provided herein for analyzing ions to enable ECD-based top-down analysis of intact antibodies.While conventional methods of MS-based proteomics can result in cluttered and / or inaccurate data due to the presence of interfering species and multiply charged precursor ions of different charge states, the present teachings can take advantage of the high dissociation efficiency of ECD to generate less cluttered mass spectra while still generating complete sequences for antibody ions.As discussed in detail below, various aspects of the methods and systems disclosed herein selectively prevent ions exhibiting m / z below a low mass cutoff value (LMCO) from entering the ECD cell, allowing transmission of ions formed from intact antibodies regardless of their charge state.

[0023] Although the systems, devices, and methods described herein may be used with many different mass spectrometer systems with fewer, more, or different components than those depicted, one exemplary mass spectrometer system 100 for use in accordance with the present teachings is illustrated diagrammatically in Figure 1. As shown in the exemplary embodiment depicted in Figure 1, the mass spectrometer system 100 generally comprises a sample ion source 102 for ionizing a sample containing or suspected of containing one or more antibodies of interest to generate a plurality of precursor ions therefrom, and a quadrupole rod set 104 for receiving the generated ions from the ion source 102 and then transmitting a portion of the generated ions to an ECD reaction cell 110. The ECD cell 110 includes an interaction region in which precursor cations interact with electrons generated by an electron source 106 to dissociate the precursor ions into a plurality of product ions that are detected by a detector 140. 1, the exemplary mass spectrometer system 100 additionally includes one or more power supplies (e.g., DC power supply 105 and RF power supply 107) controlled by a controller 108 to apply electrical potentials having RF, AC, and / or DC components to the electrodes of the various components to configure the elements of the mass spectrometer system 100 in a coordinated manner and / or for a variety of different operating modes. As discussed in detail below, in an exemplary implementation of the present teachings, the electrical signal applied to the quadrupole rod set 104 is preferably set and / or adjusted to transmit those precursor ions having m / z values ​​above LMCO (e.g., about 1,500 m / z) to the ECD cell 110. In a particular implementation, RF signals exhibiting an amplitude and frequency are applied to various rods of the quadrupole rod set in the absence of a DC resolving voltage to those rods, the amplitude and frequency being such that ions having m / z values ​​below the LMCO are substantially prevented from being transmitted into downstream elements, thereby preventing MS signaling generated by reaction of antibody precursor ions in the ECD cell 110 followed by detection of product ions. 2 These low m / z ions are selected to eliminate interference with subsequent analysis and / or interpretation of the spectra.

[0024] The ion source 102 may have a variety of configurations, but is generally configured to generate ions (e.g., cations) from antibodies contained in a sample. In certain implementations, a suitable sample source for use in accordance with the present teachings contains a sample (e.g., a solution containing or suspected of containing an antibody of interest) and / or is configured to introduce the sample to the ion source 102, e.g., via a fluid coupling, to transmit the liquid sample to the ion source 102 through one or more conduits, channels, tubing, pipes, capillary tubes, etc. As a non-limiting example, the sample source comprises 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 comprises an injection pump (e.g., a syringe pump) for continuously flowing the sample into the ion source 102. Alternatively, and by way of non-limiting example, the liquid sample to be analyzed is in the form of an eluate from an online liquid chromatographic column, although in some aspects one or more sample preparation steps (e.g., multidimensional LC separation, electrophoresis, disulfide bond reduction, etc.) may be performed offline.

[0025] In some exemplary aspects of the present teachings, the ion source 102 includes a conduit in direct or indirect fluid communication with the sample source, the conduit terminating in an outlet end that extends at least partially into the ionization chamber. When the liquid sample is released from the outlet end into the ionization chamber (e.g., as a plurality of microdroplets), the antibodies (and other interfering analytes) contained in the microdroplets are ionized (i.e., charged) by the ion source 102. When the liquid (e.g., solvent) in the droplets evaporates, the ions are released and drawn toward and through an opening for transmission to the quadrupole rod set 104 and the ECD cell 110. It should be understood that a number of different devices known in the art and modified according to the teachings herein can be utilized as the ion source 102. As non-limiting examples, the ion source 102 can be an electrospray ionization device, a nebulizer-assisted electrospray device, a chemical ionization device, a nebulizer-assisted powder ionization device, a photoionization device, a laser ionization device, a thermospray ionization device, an ultrasonic spray ionization device.

[0026] As shown in the implementation of FIG. 1, system 100 includes an ECD cell 110 in which precursor ions transmitted by quadrupole rod set 104 can undergo an ECD reaction. An ECD reaction typically involves a multiply protonated molecule M interacting with a free electron to form a radical species having an odd number of electrons. [M+nH] n+ +e - →[M+nH] (n-1)+· →ECD fragment Adding an electron to the incomplete molecular orbital of the precursor antibody cation releases binding energy, which, if sufficient to exceed the dissociation threshold, causes fragmentation of the electron acceptor ion, as known in the art. Once the precursor ions are reacted in the ECD cell 110, the fragment or product ions can be transported to one or more mass analyzers for further analysis prior to detection of the ECD product ions by the detector 110. By way of example, the mass analyzer disposed between the ECD cell 110 and the detector 140 can comprise any suitable mass analyzer module, including, but not limited to, a time-of-flight (TOF) mass spectrometry module, a quadrupole mass spectrometry module, a linear ion trap (LIT) module, and the like, for example, to scan the product ions therefrom. As a non-limiting example, the ECD reaction can be carried out in an ECD device described in PCT Publication No. WO2014191821, entitled "Inline Ion Reaction Device Cell and Method of Operation," the teachings of which are incorporated by reference in their entirety. Consistent with the need to provide electrons to the ECD cell 110 for the ECD reaction, the system 100 additionally includes an electron source 106. Those skilled in the art will appreciate that any electron source suitable for use in a mass spectrometer system to provide electrons for ion-ion reactions and modified according to the present teachings may be utilized in the system 100. As a non-limiting example, the electrons may be generated by a filament (e.g., tungsten, thoriated tungsten, and others) or another electron emitter such as a Y2O3 cathode. In one exemplary operation, a current of 1-3 A may be applied to heat the electron source, which generates a thermal power of 1-10 W to generate electrons. It should be appreciated that the electron source 106, in some aspects, may additionally be associated with a magnetic field generator (e.g., a permanent neodymium magnet or electromagnet not shown) to control the path of electrons within the ECD cell 110 and a cooling mechanism (e.g., a heat sink, active cooling) to maintain the temperature of the magnet, if present, below its Curie temperature at which the permanent magnet loses magnetization.Other known methods of cooling a magnet may also be utilized.

[0027] As shown, system 100 includes a detector 140 (e.g., a time-of-flight mass analyzer, an ion trap mass analyzer, a Faraday cup, or other ion current measuring device) effective to detect precursor and / or product ions transmitted from the ECD cell 110. The detected ion data may be stored in memory and analyzed by a computer or computer software.

[0028] As will be appreciated by one of ordinary skill in the art, system 100 may additionally include any number of additional mass analyzer or ion optical elements disposed upstream or downstream of quadrupole rod set 104 and ECD cell 110 for further ion processing, manipulation, and / or mass analysis. By way of example, ions may be pumped through one or more additional differentially pumped vacuum stages (e.g., a first stage maintained at a pressure of about 2.3 Torr, a second stage maintained at a pressure of about 6 mTorr, and a third stage maintained at a pressure of about 10 mTorr). -5 A third stage, maintained at a pressure of 1000 MPa (1.0 MPa), may be transferred through the third cell, which contains a detector 140 and two or more quadrupole mass analyzers with the ECD cell 110 located therebetween, maintained at a pressure of 100 MPa (1.0 MPa). For example, in one embodiment, the quadrupole rod set 104 is connected to Q1 (approximately 1×10 -4 Torr) and ECD cell 110 represents or replaces Q2 in a QqQ triple quadrupole mass spectrometer (see, e.g., Baba et al., "Electron Capture Dissociation in a Radio Frequency Ion Trap," Anal. Chem. 2004, Aug. 1;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 by reference in their entirety)).

[0029] As shown, the depicted system 100 additionally includes a controller 108 operably coupled to one or more of the elements of the system 100 to control the operation of the system 100. By way of example, the controller 108 may include a processor for processing information, a data storage device for storing mass spectral data, and instructions to be executed. As generally known in the art and modified in accordance with the present teachings, as discussed in detail below, the controller 108 may, by way of example, control the generation of ions by the sample ion source 102 and the generation of electrons by the electron source 106, and / or control the movement of ions into and through the quadrupole rod set 104 and the ECD cell 110 via application of one or more RF / DC voltages to their electrodes. Although the controller 108 is depicted as a single component, it should be understood that 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. Additionally, in some implementations, the 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 controls for communicating information and command selections to the processor. Consistent with certain implementations of the present teachings, the controller 108 executes one or more sequences of one or more instructions, for example, contained in a data storage device or read into memory from another computer-readable medium, such as a storage device (e.g., disk). The one or more controller(s) may take the form of hardware or software, e.g., the controller 108 may take the form of a suitably programmed computer having a computer program stored therein that is executed to cause the mass spectrometer system 100 to operate as otherwise described herein, although implementations of the present teachings are not limited to any specific combination of hardware circuitry and software.For example, various software modules associated with the controller 108 may execute programmable instructions to implement the example methodology described below with reference to FIG.

[0030] As noted 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 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 otherwise discussed herein. Referring now to FIG. 2, the exemplary quadrupole rod set 104 includes four rods 104a-d arranged around and parallel to a central longitudinal axis (Z) that extends from an entrance end (e.g., ion source side) to an exit end (e.g., ECD cell side). The rods 104a-d comprise rods having a cylindrical shape (i.e., a circular cross section of radius r, as shown in FIG. 2) that are arranged equidistant from the central axis (Z), and each of the rods 104a-d are uniform in size and shape to one another. The minimum distance between each of the rods 104a-d and the central axis (Z) is defined by a distance r0 such that the innermost surface of each primary rod 104a-d is separated from the innermost surface of the other rod in that rod pair across the central longitudinal axis (Z) by a minimum distance of 2r0. In some example implementations, r0 of the rod set 104 is in the range of about 3 mm to about 10 mm. Although the rods 104a-d are depicted as cylindrical, it should be understood that the cross-sectional shape, size, and / or relative spacing of the rods 104a-d may be varied as known in the art. For example, in some aspects, the rods 104a-d are oriented in a circular arc, as defined by the equation x 2 -y 2 =r0 2 where r0 (field radius) is the radius of the inscribed circle between the electrodes for generating the quadrupole field.

[0031] The rods 104a-d can be electrically conductive (i.e., they can be made of any conductive material, such as a metal or alloy) and coupled to a power system (comprising one or more power supplies 105, 107 of FIG. 1) such that one or more electrical signals can be applied, alone or in combination, to each rod 104a-d. In particular, the rods 104a-d generally comprise two pairs of rods (e.g., a first pair comprising rods 104a and 104c, and a second pair comprising rods 104b and 104d), with the rods of each pair disposed on opposite sides of a central axis (Z) to which the same electrical signal can be applied. For example, in some aspects as illustrated in FIG. 2, a power system can include a power supply 106a electrically coupled to the first pair of rods 104a,c to apply the same electrical potential to the first pair of rods 104a,c, and a power supply 106b electrically coupled to the second pair of rods 104b,d to apply a different electrical signal to the second pair of rods 104b,d. As illustrated in FIG. 2, in some implementations, an example power system can apply an electrical potential of [U-VcosΩt] to the first pair of rods 104a,c, 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, an example power system can apply an electrical potential of -[U-VcosΩt] to the second pair of rods 104b,d. In this exemplary configuration, the electrical signals applied to the first pair of rods 104a,c and the second pair of rods 104b,d may differ in polarity (i.e., the sign of U) of the DC signal, while the RF portions of the electrical signals may be 180° out of phase with each other. Thus, it will be appreciated by those skilled in the art that in some aspects the quadrupole rod set 104 may be viewed as a quadrupole mass filter that may selectively transmit ions in a selected m / z range by a suitable choice of DC / RF ratio.For example, considering only the DC electrical signals (i.e., ±U) applied to the four primary rods 104a-d, cations injected into a quadrupole rod set 104 as shown in FIG. 2 may experience a stabilizing force 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,c, while the cations may experience a destabilizing force in the YZ plane based on the application of a negative DC voltage to the second pair of electrodes 104b,d. Considering only the effect of the RF signal, the cations may be attracted and repelled by the various rod pairs 104a,c and 104b,d in succession as the RF signal applied to the rod pairs changes over time. Because low m / z cations can more easily follow the AC component of the field, they may tend to stay more in phase with the RF signal, gain energy from the field, and oscillate with increasingly larger amplitudes until they encounter one of the rods 104a-d and are released. Considering now the effect of the combined DC and RF signals, it should be understood that the field in the XZ plane can act as a high-pass mass filter in that only ions with high m / z are transmitted to the other end of the quadrupole without striking the first pair of electrodes 104a,c. On the other hand, in the YZ plane, high m / z cations become unstable due to the diverging / attracting effect of the negative DC voltage, but some ions with lower m / z can be stabilized by the RF component if the amplitude of the RF component is set to correct the trajectory whenever the deviation of the cations increases. Thus, it can be said that the field in the YZ plane acts as a low-pass mass filter in that only ions with lower m / z are transmitted to the other end of the quadrupole rod set 104 without striking the second pair of rods 104b,d.

[0032] By suitable choice of the RF / DC ratio of the electrical signal applied to the quadrupole rod set 104, the two effects described above in the XZ and YZ planes together provide a mass filter capable of resolving individual atomic masses, as depicted in the exemplary inset Mathieu stabilization diagram of the following parameters:

number

number

[0033] As noted 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 the various components to configure the elements of the mass spectrometer system 100 in a coordinated manner and / or for a variety of different operating modes as otherwise discussed herein. Although Figure 2 depicts both DC signals (i.e., ±U) and RF signals (i.e., ±[U-VcosΩt]) being applied to the rods 104a-d of the quadrupole rod set 104 during mass filter implementation (e.g., by ramping U and V), the present teachings provide systems and methods in which electrical signals are applied to the various rods of the quadrupole rod set 104 without DC resolving voltages during the period of establishing the LMCO and preferential transmission of ions above the LMCO m / z. 3, the quadrupole rod set 104 is shown in an RF-only configuration such that those ions having m / z above LMCO are preferentially transmitted to the ECD cell 110 for further processing as otherwise discussed herein. That is, as depicted in FIG. 3, the DC signal (U) is set to 0V such that the parameters from equation (1) are zero. Under these conditions, with RF-only signals exhibiting peak-to-peak amplitudes (V) and frequencies (Ω) applied to the various rods 104a-d, the mass scan line is q max3 inset, Mathieu Stability Diagram, such that ions entering quadrupole rod set 104 that are stable below m / e = 0.908 are selectively transmitted to ECD 110. In other words, rearranging equation (2) above and substituting 0.908 for q results in m / e = 0.908 according to the formula LMCO represents the lowest mass / charge ratio of ions that will be transmitted by the quadrupole rod set 104.

number

[0034] By suitable selection of the RF amplitude (V) and frequency (Ω) applied to the quadrupole rod set 104 according to various aspects of the present teachings, the LMCO can be set and / or adjusted to substantially prevent low m / z ions from being transmitted into downstream elements, thereby eliminating interference with subsequent analysis and / or interpretation of the spectrum generated by detection of product ions following reaction of higher m / z antibody precursor ions in the ECD cell 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 1,500, greater than 1,700, or greater than 2,000 can be selectively transmitted by the quadrupole rod set 104. Exemplary combinations of V and Ω are about 0.1 kV p-p ~about 10kV p-p and / or an RF frequency in the range of about 0.8 MHz to about 3 MHz, with values ​​selected based on the effective field radius of the quadrupole and the desired LMCO, for example, according to equation (3) above. In one exemplary implementation for obtaining an LMCO equal to about 1,500 m / z, the field radius (r0) can be 0.4 mm, the RF amplitude can be 6.4 kV, and the RF frequency can be about 1.2 MHz. It will be appreciated by those skilled in the art that such values ​​for the RF amplitude according to the present teachings can be substantially greater than those in which a quadrupole ion trap conventionally operates in a narrow bandpass mass filter mode (e.g., as shown in FIG. 2).

[0035] 4, an exemplary method 400 for operating the mass spectrometer system of FIG. 1 in accordance with various aspects of the present teachings is depicted. As shown in step 401, the method 400 may begin with delivering a sample containing antibodies to the ion source 104, whereby molecules in the sample are ionized. Once the molecules in the sample (including the antibodies) are ionized, these ions may be passed through, for example, one or more ion lenses, a mass analyzer, and / or a differential pumping vacuum stage (e.g., Q0 or Q2), as shown in step 402. jet ) into the quadrupole rod set 104. As the ions are transmitted into and through the quadrupole rod set 104, the various rods 104a-d have an RF signal applied thereto as shown in FIG. 3, whereby a combination of RF amplitude (V) and frequency (Ω) destabilizes and ejects ions below the LMCO associated with the RF signal, while allowing those ions above the LMCO (including relatively high m / z antibody ions) to be transmitted into the ECD cell (step 403). It should be appreciated that, in accordance with the present teachings, operating the quadrupole rod set 104 in this RF-only mode can allow transmission of ions of an antibody of interest into the ECD cell 110 even when the antibody ions exhibit different charge states (e.g., antibodies of the same species where one molecule is singly charged and the other is doubly charged). In step 404, these precursor antibody ions transmitted to ECD cell 110 then undergo an ECD reaction (e.g., via interaction with electrons captured in or transmitted through ECD cell 110 in step 405), thereby 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 known in the art to reconstruct the antibody structure in a "top-down" analysis.

[0036] Applicant's teachings may be understood even more fully with reference to the following examples and the data presented in Figures 5A-C, which are provided to demonstrate but not limit the present teachings. As explained herein below, the disclosed methods and systems selectively prevent ions exhibiting m / z below a low mass cutoff value (LMCO) from being transmitted to a downstream ECD cell. EXAMPLES

[0037] A sample was prepared containing a humanized monoclonal IgG antibody (NIST-mAb) obtained from NIST. The sample was subjected to desalting and LC separation utilizing a desalted LC column (water), then electrospray ionization and mass spectrometry using a research grade quadrupole TOF system (SCIEX) modified to include an ECD cell as described in the paper entitled "Electron Capture Dissociation in a Branched Radio-Frequency Ion Trap" (published in Anal. Chem. 87(1):785-792), which is incorporated by reference in its entirety. The ECD cell was disposed between Q1 and Q2 of the mass spectrometer system. The typical electron beam exposure time was 10 ms, and the electron beam intensity was adjusted to obtain appropriate dissociation efficiency. The mass resolution of the TOF-MS was 35,000-47,000, which resolved the isotopic patterns of fragments up to Z~30+. FIG. 5A depicts the spectrum generated from an ionized sample, where the RF amplitude was 400 V. p-p The amplitude of the RF signal applied to the quadrupole rod set is selected to allow substantially all ions to be transmitted therethrough. The spectrum of Figure 5A contains significant signals for ions exhibiting m / z less than 500, and several significant peaks at about m / z 1,520. Referring now to Figure 5B, the amplitude of the RF signal applied to the quadrupole rod set is 5 kV p-p(the resolving DC voltage was set to 0 V) ​​and ions transmitted by Q1 were again detected (no ECD reaction was performed). As can be seen by comparing Figures 5A and 5B, by increasing the amplitude of the RF signal, Q1 prevented the transmission of those ions exhibiting m / z less than about 1,700. Figure 7 depicts ions detected when performing ECD on precursor ions transmitted from Q1 under the conditions of Figure 5B. The low intensity peaks exhibiting m / z less than about 2,000 represent product ion fragments following ECD. Comparing Figures 5A and 5C, it can be seen that many of the product ion peaks observed in those Figures 5C would be obscured if the low m / z ions were not removed in accordance with the present teachings, thereby making top-down analysis of antibodies more difficult.

[0038] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the embodiments and practices described herein. By way of example, the dimensions of the various components and the explicit values ​​(e.g., amplitudes, frequencies, etc.) for the particular electrical signals applied to the various components are merely illustrative and are not intended to limit the scope of the present teachings. It is therefore to be understood that the present invention is not limited to the embodiments disclosed herein, but should be understood from the following claims, which should be interpreted as broadly as permitted under law.

[0039] The headings used herein are for organizational purposes only and should not be construed as limiting. While the applicant's teachings are described in conjunction with various embodiments, it is not intended that the applicant's teachings be limited to such embodiments. To the contrary, the applicant's teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those skilled in the art.

Claims

1. 1. A method for performing a top-down analysis of an antibody, comprising: generating a plurality of ions from a sample containing at least one intact antibody using an ion source; transmitting the plurality of ions through the quadrupole rod set while applying an RF signal to the quadrupole rod set in the absence of a resolving DC voltage so as to preferentially transmit precursor ions having m / z values ​​above a low mass cutoff value of about 1,500 m / z from the quadrupole rod set to an ECD cell; performing an ECD reaction on the precursor ions in the ECD cell; detecting a reaction product from the ECD reaction; A method comprising:

2. The method of claim 1 , wherein the quadrupole rod set comprises Q1 in a triple quadrupole.

3. 3. The method of claim 1 or 2, further comprising adjusting at least one of the amplitude and frequency of the RF signal such that the low mass cutoff is about 1,700 m / z.

4. 3. The method of claim 1 or 2, further comprising adjusting at least one of the amplitude and frequency of the RF signal such that the low mass cutoff is about 2,000 m / z.

5. The method of claim 1 or 2, wherein the low mass cutoff is about 1,700 m / z.

6. The method of claim 1 or 2, wherein the low mass cutoff is about 2,000 m / z.

7. The method of any one of claims 1 to 6, wherein at least a portion of the reaction products have an m / z below the low mass cutoff value.

8. The method of any one of claims 1 to 7, wherein performing an ECD reaction comprises introducing electrons into the ECD cell while transmitting the precursor ions through the ECD cell.

9. The method of any one of claims 1 to 8, wherein the precursor ions comprise ions of the antibody in multiple charge states.

10. 10. The method of claim 9, wherein the ions of the antibody in multiple charge states exhibit at least two m / z in the range of about 2,000 to about 4,000.

11. 1. A mass spectrometer system comprising: an ion source for generating a plurality of ions from a sample containing an intact antibody; a quadrupole rod set extending along a central longitudinal axis, the quadrupole rod set configured to receive the plurality of ions from the ion source through an entrance end and transmit at least a portion of the ions through an exit end, the quadrupole rod set comprising a first pair of elongate rods and a second pair of elongate rods disposed about and parallel to the central longitudinal axis; an ECD cell for receiving ions transmitted from the quadrupole rod set, in which the ions received from the quadrupole rod set are reacted with electrons to generate product ions from the ECD cell; a detector for detecting the product ions; a power system electrically coupled to the quadrupole rod set, the power system configured to provide an RF signal to the quadrupole rod set in the absence of a resolving DC voltage so as to substantially prevent ions having m / z values ​​less than a low mass cutoff value of about 1,500 m / z from being transmitted from the quadrupole rod set to the ECD cell; A system comprising:

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

13. The quadrupole rod set has a diameter of about 1×10 －４ 13. The system of claim 11 or 12, housed in a chamber maintained at a pressure below Torr.

14. 14. The system of claim 11, wherein the power system is configured to provide an RF signal to the quadrupole rod set in the absence of a resolving DC voltage to substantially prevent ions having m / z values ​​below a low mass cutoff value of about 1,700 m / z from being transmitted from the quadrupole rod set to the ECD cell.

15. 14. The system of claim 11, wherein the power system is configured to provide an RF signal to the quadrupole rod set in the absence of a resolving DC voltage to substantially prevent ions having m / z values ​​below a low mass cutoff value of about 2,000 m / z from being transmitted from the quadrupole rod set to the ECD cell.

16. The system of any one of claims 11 to 15, further comprising a controller configured to increase the amplitude of the RF signal and to increase the low mass cutoff value.

17. The system of any one of claims 11 to 16, wherein at least some of the product ions have an m / z below the low mass cutoff value.

18. The system of any one of claims 11 to 17, wherein the intact antibody has an m / z in the range of about 2,000 to about 4,000.

19. The system of any one of claims 11 to 18, further comprising an electron source for introducing electrons into the ECD cell.

20. The amplitude of the RF signal is about 0.1 kV. ｐ－ｐ ~about 10kV ｐ－ｐ The system according to any one of claims 11 to 19, wherein

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