Systems and methods for operating mass spectrometers with improved robustness

Abstract

A method of mass spectrometry comprises selecting an operational mode of a mass spectrometer from two operational modes a high-ion-energy mode and a low-ion-energy mode, wherein the high-ion-energy mode comprises directing a plurality of high-energy ions into a mass filter, wherein the plurality of high-energy ions are characterized by having, in the mass filter, kinetic energies that are greater than a high energy level; the low-ion-energy mode comprises directing a plurality of low-energy ions into the mass filter, wherein the plurality of low-energy ions are characterized by having, in the mass filter, kinetic energies that are smaller than a low energy level; and the low energy level is not larger than the high energy level; and performing mass spectrometry in the selected operational mode.

Description

SYSTEMS AND METHODS FOR OPERATING MASS SPECTROMETERS WITH IMPROVED ROBUSTNESSRELATED APPLICATION

[0001] The present application claims priority to provisional application number 63 / 788,862, filed on April 15, 2025, which is herein incorporated by reference in its entirety.TECHNICAL FIELD

[0002] The present disclosure relates generally to mass spectrometry and more specifically improving robustness in mass spectrometry.BACKGROUND

[0003] Mass spectrometry (MS) is an analytical technique for determining the elemental composition of a substance. Specifically, MS measures a mass-to-charge ratio (m / z) of ions generated from a test substance. MS can be used to identify unknown compounds, to determine isotopic composition of elements in a molecule, to determine the structure of a particular compound by observing its fragmentation, and to quantify the amount of a particular compound in a sample.

[0004] A mass spectrometer may include an ion source, a mass analyzer, and a detector. The ion source may convert a test sample into ions and direct those ions into the mass analyzer. The mass analyzer may in turn separate (or mass analyze) the ions based on their m / z ratios and send a subset of those ions into the detector. The detector may detect the ions that it receives.

[0005] In general, it is desired to have more robust instruments that are able to process larger quantities of samples before requiring cleaning or servicing. In MS proteomics applications, for example, there is an increased need for the instrument robustness owing to more sensitive modem instruments and the necessity of maintaining the instrument performance during the analysis of large cohorts of patients / samples. One of the factors that may reduce the robustness of a mass spectrometer is contamination of different parts with increased use. Therefore,increasing the stability of the mass spectrometer against such contaminations by, for example, maintaining its accuracy during use may improve the robustness of the mass spectrometer.

[0006] The importance of robustness arises, for example, for mass spectrometers that employ ionization techniques with improved ionization efficiency or more sensitive mass spectrometers that may have larger intake orifice than prior instruments. These techniques result in an increased rate of ionization and introduction of the ions into the mass analyzer.

[0007] Therefore, increasing the robustness of the MS systems will improve the efficiency of the MS technology.SUMMARY

[0008] Some embodiments relate to a mass spectrometer, including: an ion source configured to generate sample ions in response to receiving a sample; and an analytical mass filter positioned downstream of the ion source to receive the sample ions and configured to provide an ion transmission window to allow passage of precursor ions with m / z ratios within an m / z range of the ion transmission window; wherein: the mass spectrometer is configured to selectively operate in any of the following operational modes: a first operational mode characterized by a first m / z range of the ion transmission window and a first ion energy imparted to the precursor ions entering mass filter, and a second operational mode characterized by a second m / z range of the ion transmission window and a second ion energy imparted to the precursor ions entering mass filter, the second m / z range is greater than the first m / z range, and the second ion energy is greater than the first ion energy. By way of example, the analytical mass filter can be an RF / DC quadrupole mass filter. The analytical mass filter defines a final mass window for specific target ions, i.e. precursor ions, to be transmitted for analysis within a narrow range of m / z values.

[0009] Some embodiments relate to a mass spectrometer, wherein said second operational mode is further characterized by the mass filter operating in a Data Independent Acquisition (DIA) mode.

[0010] Some embodiments relate to a mass spectrometer, wherein the first ion energy is in a range of about 0.5 eV to about 4 eV and the second ion energy is in a range of about 5 eV to about 15 eV.

[0011] Some embodiments relate to a mass spectrometer, wherein said transmission window of the mass filter is scanned across m / z dimension in said DIA mode.

[0012] Some embodiments relate to a mass spectrometer, wherein said transmission window of the mass filter is discretely varied across said m / z dimension in said DIA mode.

[0013] Some embodiments relate to a mass spectrometer, further including a controller, wherein the controller is configured to control operation of the mass spectrometer in the first and second operational modes.

[0014] Some embodiments relate to a mass spectrometer, further including: an ion guide positioned upstream of the mass filter; an ion lens separating the ion guide from the mass filter; and a DC voltage source operating under controller of the controller and configured to apply a DC voltage to the ion lens.

[0015] Some embodiments relate to a mass spectrometer, wherein the controller is configured to set the DC voltage so as to maintain a DC offset voltage between said ion lens and said ion guide within a target range predetermined for the second operational mode.

[0016] Some embodiments relate to a mass spectrometer, wherein: the target range is a first target range; the controller is further configured to set the DC voltage so as to maintain the DC offset voltage between said ion lens and said ion guide within a second target range predetermined for the first operational mode.

[0017] Some embodiments relate to a mass spectrometer, wherein the 1st m / z range is less than or equal to ITh and the 2nd m / z range is greater than 2 Th.

[0018] Some embodiments relate to a method of configuring an operational mode of a mass spectrometer, the method including: directing a plurality of ions into the mass spectrometer; measuring a parameter indicative of a level of contamination of at least one ion optic of the mass spectrometer; and in response to a value of the measured parameter, selecting an energy level for imparting to the ions.

[0019] Some embodiments relate to a method, further including: selecting a robust mode for the mass spectrometer when the value of the measured parameter exceeds a threshold, wherein the robust mode is related to the selected energy level;

[0020] Some embodiments relate to a method, further including operating the mass spectrometer in a high-energy mode related to the selected energy level.

[0021] Some embodiments relate to a method, wherein the parameter is an electrical current measured at the at least one ion optic.

[0022] Some embodiments relate to a method, wherein the selected energy level corresponds to a high-energy mode.

[0023] Some embodiments relate to a method, wherein the method further includes determining an acceptable range for a voltage applied to the at least one ion optic in the high-energy mode.

[0024] Some embodiments relate to a method, wherein determining the acceptable range includes: acquiring a plurality of intensities for the ions passing the mass filter to derive an intensity function as a function of the voltage applied to the component; determining a maximum intensity for the intensity function acquired at an optimal focusing voltage; determining a lower limit for the acceptable range that is lower than or equal to the optimal focusing voltage and atwhich the intensity function is equal to a predetermined fraction of the maximum intensity; determining a higher limit for the acceptable range that is higher than or equal to the lower limit and is further lower than or equal to the optimal focusing voltage.

[0025] Some embodiments relate to a method, determining the higher limit includes: determining a degradation voltage larger than the optimal focusing voltage at which the intensity function reaches a degraded intensity that is lower than the maximum intensity; determining a 1st distance between the optimal focusing voltage and the degradation voltage; determining a 2nd distance that is equal to a factor multiplied by the 1st distance, wherein the factor is nonnegative; and determining the higher limit to be equal to the optimal focusing voltage minus the 2nd distance.

[0026] Some embodiments relate to a method, wherein: measuring the parameter is performed in a low-energy mode selected for operating the mass spectrometer; and the low-energy mode is different from the high-energy mode.

[0027] Some embodiments relate to a mass spectrometer including: an ion source; and a mass filter, wherein: the mass spectrometer is configured to operate two operational modes: a high-ion-energy mode and a low-ion-energy mode; in the high-ion-energy mode: the ion source is configured to direct a plurality of high-energy ions into the mass filter, wherein the plurality of high-energy ions are characterized by having, in the mass filter, kinetic energies that are greater than a high energy level; in the low-ion-energy mode: the ion source is configured to direct a plurality of low-energy ions into the mass filter, wherein the plurality of low-energy ions are characterized by having, in the mass filter, kinetic energies that are smaller than a low energy level; and the low energy level is not larger than the high energy level.

[0028] Some embodiments relate to a method of mass spectrometry including: selecting an operational mode of a mass spectrometer from two operational modes: a high-ion-energy modeand a low-ion-energy mode, wherein: the high-ion-energy mode includes: directing a plurality of high-energy ions into a mass filter, wherein the plurality of high-energy ions are characterized by having, in the mass filter, kinetic energies that are greater than a high energy level; the low-ion-energy mode includes: directing a plurality of low-energy ions into the mass filter, wherein the plurality of low-energy ions are characterized by having, in the mass filter, kinetic energies that are smaller than a low energy level; and the low energy level is not larger than the high energy level; and performing mass spectrometry in the selected operational mode.

[0029] Some embodiments relate to a method, wherein operating the mass spectrometer in the high-ion-energy mode includes directing a plurality of high-energy ions into a mass filter of the mass spectrometer, wherein the plurality of high-energy ions are characterized by having, in the mass filter, kinetic energies that are greater than a high energy level.

[0030] In some embodiments, a mass spectrometer may be operative to activate a high-ion-energy mode when a data independent acquisition (DIA) is executed by the mass spectrometer in which a precursor mass filter is set to allow transmission of ions wherein a width of the precursor mass filter is set to greater than unit resolution. The mass spectrometer further operative to activate a low-ion-energy mode when the precursor mass filter is set to transmit ions within a unit resolution mass filter width.

[0031] Some embodiments relate to a method, wherein: measuring the parameter is performed in a low-energy mode selected for operating the mass spectrometer; and operating the mass spectrometer in the low-energy mode includes: directing a plurality of low-energy ions into the mass filter, wherein the plurality of low-energy ions are characterized by having, in the mass filter, kinetic energies that are smaller than a low energy level; and the low energy level is not larger than the high energy level

[0032] Some embodiments relate to a method, wherein in the high-ion-energy mode: a mass filter of the mass spectrometer is configured to operate with a wide bandpass window for m / z selection so as to allow passage of a subset of the plurality of high-energy ions having m / z ratios within the wide bandpass window through the mass filter; and the wide bandpass window is greater than an upper bandpass threshold.

[0033] Some embodiments relate to a method, wherein: in the high-energy mode: the mass filter is configured to operate with a wide bandpass window for m / z selection so as to allow passage of a subset of the plurality of high-energy ions having m / z ratios within the wide bandpass window through the mass filter; and the wide bandpass window is greater than an upper bandpass threshold; and in the low-ion-energy mode: the mass filter is configured to operate with a narrow bandpass window for m / z selection so as to allow passage of a subset of the plurality of low-energy ions having m / z ratios within the narrow bandpass window through the mass filter; and the narrow bandpass window is smaller than a lower bandpass threshold.

[0034] Some embodiments relate to a method, wherein the upper bandpass threshold is substantially equal to the lower bandpass threshold.

[0035] Some embodiments relate to a method, wherein the high energy level is substantially equal to the low energy level.

[0036] Some embodiments relate to a method, wherein: the high energy level is larger than n times the low energy level; and n is equal to 2, 3, or 4.

[0037] Some embodiments relate to a method, wherein the low-energy mode provides unit resolution.

[0038] Some embodiments relate to a method, wherein the unit resolution is configured to detect mass spectrometry peaks with widths around 0.7 Th.

[0039] Some embodiments relate to a method, wherein the low-energy mode is configured for isolation with a narrow bandpass and the high -energy mode is configured for isolation with a wide bandpass.

[0040] Some embodiments relate to a method, wherein the narrow bandpass is <1 Th.

[0041] Some embodiments relate to a method, wherein the wide bandpass is >2Th.

[0042] Some embodiments relate to a method, wherein the high -ion-energy mode is utilized for a proteomics workflow.

[0043] Some embodiments relate to a method of mass spectrometry including: selecting an operational mode of a mass spectrometer from two operational modes: a high-ion-energy mode and a low-ion-energy mode, wherein: the high-ion-energy mode includes: directing a plurality of high-energy ions into a mass filter, wherein the plurality of high-energy ions are characterized by having, in the mass filter, kinetic energies that are greater than a high energy level; the low-ion-energy mode includes: directing a plurality of low-energy ions into the mass filter, wherein the plurality of low-energy ions are characterized by having, in the mass filter, kinetic energies that are smaller than a low energy level; and the low energy level is not larger than the high energy level; and performing mass spectrometry in the selected operational mode.

[0044] Some embodiments relate to a method, wherein the operational mode is a first operational mode selected to be the high-ion-energy mode, the method further including: selecting the low-ion-energy mode as a second operational mode; and performing mass spectrometry in the second operational mode.

[0045] Some embodiments relate to a method, wherein performing mass spectrometry in the high-ion-energy mode is followed by performing mass spectrometry in the low-ion-energy mode.

[0046] Some embodiments relate to a method, wherein performing mass spectrometry in the low-ion-energy mode is followed by performing mass spectrometry in the high-ion-energy mode.

[0047] Some embodiments relate to a method, wherein: in the high-ion-energy mode: the mass filter is configured to operate with a wide bandpass window for m / z selection so as to allow passage of a subset of the plurality of high-energy ions having m / z ratios within the wide bandpass window through the mass filter; and the wide bandpass window is greater than an upper bandpass threshold; and in the low-ion-energy mode: the mass filter is configured to operate with a narrow bandpass window for m / z selection so as to allow passage of a subset of the plurality of low-energy ions having m / z ratios within the narrow bandpass window through the mass filter; and the narrow bandpass window is smaller than a lower bandpass threshold.

[0048] Some embodiments relate to a method, wherein the upper bandpass threshold is substantially equal to the lower bandpass threshold.

[0049] Some embodiments relate to a method, wherein the high energy level is substantially equal to the low energy level.

[0050] Some embodiments relate to a method, wherein the high energy level is larger than n times the low energy level, wherein n is equal to 2, 3, or 4.

[0051] Some embodiments relate to a method, wherein the low energy level is determined to provide unit resolution.

[0052] Some embodiments relate to a method, wherein the unit resolution is configured to detect mass spectrometry peaks with widths around 0.7 Th.

[0053] Some embodiments relate to a method, wherein the low energy level is determined based on a minimal attainable peak width one or more physical parameters of the mass filter.

[0054] Some embodiments relate to a method, wherein: the mass filter includes an RF + DC quadrupole; and the low energy level is determined based on an equation, in which, is the low energy level, is an RF frequency of the quadrupole, is a length of the quadrupole, and is a desired peak width.

[0055] Some embodiments relate to a method, wherein the desired peak width is calculated based on the desired resolution.

[0056] Some embodiments relate to a method, wherein the low-ion-energy mode is configured for isolation with a narrow bandpass and the high-ion-energy mode is configured for isolation with a wide bandpass.

[0057] Some embodiments relate to a method, wherein the narrow bandpass is <1 Th.

[0058] Some embodiments relate to a method, wherein the wide bandpass is >2Th.

[0059] Some embodiments relate to a method, wherein: the high energy level is around 10 eV and the upper bandpass threshold is around 5 Th., and the low energy level is around 1 eV and the lower bandpass threshold is around 2 Th..

[0060] Some embodiments relate to a method, wherein the mass filter includes a plurality of rods arranged in a multipole configuration so as to provide an ion passageway for transit of ions through the mass filter.

[0061] Some embodiments relate to a method, wherein the multipole configuration includes a quadrupole configuration.

[0062] Some embodiments relate to a method, wherein: the ion passageway has an axial length L; in the high-ion-energy mode the mass filter is operated at a radiofrequency RFH and the high-energy ions have an average axial velocity VH, based on which a high-energy oscillation number (NH) is defined as NH; in the low-ion-energy mode the mass filter is operated at a radiofrequency RFL and the low-energy ions have an average axial velocity VL, based on which a low-energy oscillation number (NL) is defined as NL; and the NL is at least 1.5 times the NH.

[0063] Some embodiments relate to a method, wherein the high -ion-energy mode is utilized for a proteomics workflow.

[0064] Some embodiments relate to a mass spectrometer including: an ion source; and a mass filter, wherein: the mass spectrometer is configured to operate two operational modes: a high-ion-energy mode and a low-ion-energy mode; in the high-ion-energy mode: the ion source is configured to direct a plurality of high-energy ions into the mass filter, wherein the plurality of high-energy ions are characterized by having, in the mass filter, kinetic energies that are greater than a high energy level; in the low-ion-energy mode: the ion source is configured to direct a plurality of low-energy ions into the mass filter, wherein the plurality of low-energy ions are characterized by having, in the mass filter, kinetic energies that are smaller than a low energy level; and the low energy level is not larger than the high energy level.

[0065] Further understanding of various aspects of the embodiments may be obtained by reference to the following detailed description in conjunction with the associated drawings, which are described briefly below.BRIEF DESCRIPTION OF THE DRAWINGS

[0066] The drawings are not necessarily to scale or exhaustive. Instead, emphasis is generally placed upon illustrating the principles of the embodiments described herein. The accompanying drawings, which are incorporated in this specification and constitute a part of it, illustrate several embodiments consistent with the disclosure. Together with the description, the drawings serve to explain the principles of the disclosure.

[0067] In the drawings:

[0068] Fig. 1 A is a schematic of a mass spectrometer according to some embodiments.

[0069] FIGS. IB and 1C are potential diagrams for the low energy mode and the high energy mode in some embodiments.

[0070] FIG. 2 is a sensitivity graph set according to some embodiments.

[0071] FIG. 3 is another sensitivity graph set according to some embodiments.

[0072] Figs. 4A and 4B illustrate the effect of using different energy modes on the resolution of the data according to some embodiments.

[0073] FIG. 5 is a flowchart for a MS operation method according to some embodiments.

[0074] FIG. 6 shows a table depicting results of five operations according to some embodiments.

[0075] Fig. 7A shows a transmission rate curve for a high resolution experiment according to an embodiment.

[0076] Fig. 7B, shows an intensity curve for a low resolution or wide bandpass acquisition according to an embodiment.

[0077] Fig. 8A illustrates one example of the change in the optimum focusing settings due to contamination according to an embodiment.

[0078] Figs. 8B and 8C also show a comparison of the shift in the stopping potential in the two operational modes.

[0079] FIG. 9 schematically depicts two differentially-pumped chambers and of a mass spectrometer, according to some embodiments.

[0080] FIG. 10A depicts two profiles of an ion current measured according to an embodiment.

[0081] FIG. 10B shows two current profiles operated in a robust operational mode according to an embodiment.

[0082] Figs. 11A-11C show data related to charges deposited on ion optics under different conditions according to various embodiments.

[0083] Figs. 12A and 12B illustrate the process of determining the optimal ion energy for the robust mode according to some embodiments.

[0084] Fig. 12C shows a flowchart for a configuration method according to someembodiments.

[0085] Fig. 13 A includes a graph showing the ion transition as the function of IQ1 offset according to an embodiment.

[0086] Figs. 13B and 13C, on the other hand, illustrate determining an optimum IQ1 offset range in a high energy mode.

[0087] FIG. 14 schematically depicts an example of an implementation of a module according to some embodiments.DETAILED DESCRIPTION

[0088] It will be appreciated that for clarity, the following discussion will explicate various aspects of embodiments of the applicant’s teachings, while omitting certain specific details wherever convenient or appropriate to do so. For example, discussion of like or analogous features in alternative embodiments may be somewhat abbreviated. Well-known ideas or concepts may also for brevity not be discussed in any great detail. The skilled person will recognize that some embodiments of the applicant’s teachings may not require certain of the specifically described details in every implementation, which are set forth herein only to provide a thorough understanding of the embodiments. Similarly, it will be apparent that the described embodiments may be susceptible to alteration or variation according to common general knowledge without departing from the scope of the disclosure. The following detailed description of embodiments is not to be regarded as limiting the scope of the applicant’s teachings in any manner.

[0089] The following detailed description refers to the accompanying drawings. The same or similar reference numbers may have been used in the drawings or in the description to refer to the same or similar parts. Also, similarly named elements may perform similar functions and may be similarly designed, unless specified otherwise. Details are set forth to provide an understanding of the exemplary embodiments. Embodiments, e.g., alternative embodiments, maybe practiced without some of these details. In other instances, well known techniques, procedures, and components have not been described in detail to avoid obscuring the described embodiments.

[0090] Throughout this disclosure, the robust mode refers to an operational mode in which at least one selected operating parameter is set to reduce rate of contamination of at least one ion optic, which may result from incidence of ions on at least one surface of the ion optic. The rate is reduced relative to a respective rate of contamination in a normal operational mode (alternatively called default mode). In various embodiments, the normal operational mode and the robust operational mode may differ by the setting of one or more operating parameters that include the selected operating parameter. In some embodiments, the set of selected operating parameters includes an energy level of the ions such that the energy level is higher for the robust mode as compared to the normal mode.

[0091] Some embodiments improve the robustness of spectrometry analyses by adjusting various parameters such as the ion energies or the bandpass windows, as further described below.

[0092] The terms parameter or operating parameter may be used interchangeably throughout this disclosure.

[0093] In particular, in some embodiments, the acquisition mode of the mass analyzer may take advantage of the present methods in order to provide additional robustness. For example, data independent acquisition (DIA) methods, which utilize relatively wider bandpass windows (a.k.a. isolation windows) with m / z ranges greater than unit resolution, e.g. 2Th or greater, in which additional selectivity is provided by analysing fragment ions, may take advantage of the present methods, whereas information dependent acquisition (IDA) methods or parallel reaction monitoring methods, for which the bandpass window is often narrower and of the order of lessthan 2 Th, and often unit resolution (e.g. ITh or less), may provide better performance at low-ion-energy levels.

[0094] Such DIA techniques may be utilized in, for example, proteomics studies. In this approach, inherently chimeric spectra may be matched against spectral libraries that are either acquired experimentally or generated in silico often employing machine learning approaches. Over the last decade these new proteomics search engines experienced a tremendous progress in matching sensitivity and accuracy for processing of increasingly more complex spectra.Importantly such search engines are rather tolerant toward imprecise quadrupole isolation windows.

[0095] Some embodiments may leverage the relationship between robustness, on the one hand, and factors such as ion energies and bandpass windows, on the other hand, to improve the robustness of the system. For example, in some embodiments, the mass spectrometer may be operated in a special regime where deleterious effects of instrument contamination are delayed to accommodate a higher number of sample injections as compared to conventional techniques.

[0096] Some embodiments also include techniques for improving the robustness of the precursor isolation in RF+DC quadrupole. Such an improvement is in particular beneficial to many of the conventional systems in which the contamination starts in the isolation quadrupole and adjacent ion optics, parts that will be further described below.

[0097] Fig. 1 A is a schematic of a mass spectrometer 100 according to some embodiments. Mass spectrometer 100 includes, among other parts, an ion source 110, an ion guide 120 (sometimes labeled as Q0), an ion lens 130 (sometimes labeled as IQ1, standing for inter quad lens 1), a mass filter 140 (sometimes labeled as QI), a collision cell 150 (sometimes labeled as Q2), and a mass analyzer 160. In some embodiments, such as the one shown in FIG. 1 A, mass spectrometer 100 may further include an additional ion guide 115, located between ion source110 and ion guide 120, or include an Electron-Capture Dissociation cell 170 (a.k.a. ECD cell), located between mass filter 140 and collision cell 150. Mass spectrometer 100 may further include a controller module 180 in communication with one or more parts of the mass spectrometer.

[0098] Ion source 110 may generate ions from a sample and send those ions to ion guide 120. In various embodiments, ion source 110 may be an electrospray ion source, and APCI ion source, a MALDI ion source, etc. or other known ion source.

[0099] Ion guide 120 may receive ions from ion source 110 and direct a subset of those ions through ion lens 130 into mass filter 140. In various embodiments, ion guide 120 may be a multipole ion guide, such as a quadrupole, a hexapole, or an octupole. Alternatively, in various embodiments, ion guide 120 may be a stack ring ion guide or another type of RF ion guide. In some embodiments ion guide 120 may be operated as a trapping device and may be configured as an ion mobility device, such as a trapped ion mobility device.

[0100] In various embodiments, a particular operational mode of the mass spectrometer may expose ion lens 130 (e.g. IQ 1) to a high rate of contamination by ions or render it sensitive to such contamination. More specifically, in some embodiments, ion lens 130 may include a narrow orifice to enable mass filter 140 to operate at a pressure that is lower than the pressure in ion guide 120. For example, the gas pressure in ion guide 120 may be between 2 and 20mTorr while the gas pressure in mass filter 140 may be below 0.5mTorr and preferably below 0.05mTorr. Therefore, ion lens 130 may be in the form of a narrow restricting orifice such that the pressure difference on its two sides may be maintained. In various embodiments, the size of this orifice may be around l-3mm. Moreover, in some embodiments the mass spectrometer is operated with a “bright” ion beam, in which the ion beam radial size may increase due to space charge.Therefore, due to the narrow size of its aperture, IQ1 may be collided by a high number of ions.

[0101] Moreover, in various embodiments, ion lens 130 (IQ 1 ) also acts as a focusing lens either alone or in a cascade of focusing lenses preceding the QI mass filter. In such embodiments, the potential applied to IQ1 may be adjusted to achieve better focusing of the beam at QI entrance. In particular, IQ1 may be configured to operate at a small potential difference with respect to its adjacent lenses, such as Q0. Therefore, with such a small potential difference, ion lens 130 may experience a high relative rate of change in electrical potential resulting from a relatively small amount of ion contamination. Therefore, ion lens 130 may show a high sensitivity to contamination by ions and its contamination may affect the performance of the mass spectrometer.

[0102] In some embodiments, mass filter 140 (QI) may include a plurality of rods that are arranged in a multipole configuration and connected to one or more sources of electrical voltage. In particular, mass filter 140 may be an isolation quadrupole, including four rods, one or more of which are connected to one or more sources of DC (direct current) voltage and also one or more of which are connected to one or more sources of RF (radio frequency) voltage. Different parameters of the quadrupole may be tuned such that QI would allow a first subset of the ions that enter the mass filter to pass through and reach collision cell 150 but would divert a second subset of those ions toward the periphery of the quadrupole. The tuned parameters may include one or more of the amplitude or frequency of the RF voltage, or the amplitude of the DC voltage.

[0103] The mass filter may select the first subset or the second subset of ions based on their mass to charge ratios (m / z). More specifically, mass filter 140 may be a bandpass mass filter, thus selecting the first subset of ions from ions that have m / z values within a bandpass window, and the second subset of ions from ions that have m / z values outside the bandpass window. The bandpass window may be controlled by setting the values of some tuned parameters.

[0104] In various embodiments, the second subset of the ions may collide with, or be absorbed by, different parts of the periphery of the quadrupole including the rods. Such absorptions may further reduce the robustness of the mass spectrometer by contaminating the rods during the operation of the mass spectrometer.

[0105] In various embodiments, collision cell 150 may be a cell that receives ions and causes those ions to dissociate into charged fragments. The fragmentation may result from, for example, collisions of the received ions with the gas molecules inside the collision cell. Collision cell 150 may be also operated to collisionally focus ions and condition them for further mass analysis in mass analyzer 160.

[0106] In various embodiments, mass analyzer 160 may receive the ionized fragments from collision cell 150 and analyze them by, for example, determining their m / z. Mass analyzer 160 may utilize different technologies such as time-of-flight (TOF) analysis; ion traps, such as linear ion traps, q traps, and Paul ion traps; or Fourier based mass analyzers, such as ion cyclotron resonance (ICR) or Orbitrap mass analyzers.

[0107] Various embodiments may further include one or more additional stages of differential pumping comprising appropriate RF ion guides such as ion guide 115. In some embodiments, an additional ion guide 115, identified without limitation as QJet in FIG. 1 A, may be included to assist in transitioning ions from atmosphere to lower pressure stages of the mass spectrometer. In some embodiments, the additional ion guide 115 may include other multipole or stacked ring ion guides such as an ion funnel, a Diet, a travelling wave stack ring ion guide, etc. to similarly assist with transitioning ions from atmosphere to lower pressure stages of the mass spectrometer.

[0108] In some embodiments, ECD cell 170 may be placed between mass filter 140 and collision cell 150. In such cases, ECD cell 170 may receive ions from mass filter 140 andfragment some of those ions via irradiating them with an electron beam. ECD cell 170 may direct some or all of the remaining ions or the resulting ion fragments toward collision cell 150.

[0109] Additional mass filters, such as quadrupole mass filters, may be provided upstream or downstream of the QI and Q2 regions identified in FIG. 1A.

[0110] In some embodiments, controller module 180 may be a module that controls different aspects of the mass spectrometer operation, such as setting parameters for different parts or scheduling the operation in different modes at different times. In some embodiments, controller module 180 may include a computer implemented module in communication with one or more of the other parts of the mass spectrometer listed earlier. Some implementations of modules such as controller module 180 are described further below.

[0111] In some embodiments the term ion optic may be used for one or more components or component assemblies of the mass spectrometer such as ion lenses, rods, to which RF or DC voltages may be applied.

[0112] Some embodiments improve the robustness of the mass spectrometer by reducing the rate of contamination in some parts of the instrument or by reducing the impact of the contamination on the instrument performance. In particular, those parts may include electrodes in sections such as ion lens 130 or the rods in mass filter 140. The degradation of the mass spectrometer performance due to contamination may relate to the contamination of some parts of the mass spectrometer due to absorption of ions. More specifically, the ions that land on some of the surfaces may act similar to a dielectric layer. This dielectric layer may get charged with further exposure to the ion current, resulting to a change in the electric field around the contaminated surfaces. Such a change in the electrical field or the corresponding electrical potential may alter the ion transmission, resulting in a decrease in the efficiency of the instrument and, in some cases, rendering the instrument inoperable.

[0113] Some embodiments may reduce the unwanted effects of contamination by increasing one or more of the voltages utilized by the instrument. Such increases may reduce those unwanted effects for different reasons. As a first reason, for example, increasing the voltages may be beneficial by reducing AV / V, i.e., the ratio of the undesired contamination-induced variation in the electrical potential (AV) to an electrical potential difference utilized by the instrument in the vicinity of the contaminated parts (V). For example, V may be a potential difference between a pair of ion optics elements, such as an IQ1 lens and the Q0 ion guide, or a potential difference between the mass filter QI and the ion guide Q0.

[0114] As a numerical example, if in some part of the instrument, the contamination induces a potential variation of AV = 0.5eV, the relative effect of this contamination would be different between a first case where in that part the operational potential difference is set to V = leV, and a second case where that operational potential difference is set to V = lOeV. More specifically, in the first case, the contamination changes the operational potential difference by 50% while in the second case the contamination changes the operational potential difference by a much less relative amount of 5%. Accordingly, in some applications, the 50% variation due to the contamination may significantly alter the outcome, while the 5% variation may be negligible. Such operating conditions may, for example, be used to reduce or suppress the contamination effects on the lens and on the quadrupole mass filter.

[0115] Moreover, as a second reason, increasing some of the potential differences may increase the energies of the ions causing the contamination to spread over a larger area and thus reducing the effect of the contamination. Consequently, tuning some of the voltages may result in a more spread distribution of the contamination layer across surfaces such as ion optics surfaces or quadrupole surfaces. For example, when quadrupole mass filter is operated in resolving DC mode, larger incoming ion energies may cause the contamination to be spread over a larger area.Alternatively, operating at different voltages and operating potentials can shift the unwanted deposition from one of the susceptible ion optic elements to another susceptible ion optic element. This can be beneficial when the susceptible element receiving lesser amount of deposition is located closer to the ion beam and hence affecting the ion motion stronger when the potential is altered by ΔV induced by contamination.

[0116] On the other hand, some applications may prefer lower ion energies. For example, in some embodiments, the required quadrupole mass filter resolution may have a direct relation to the number of RF cycles that the ions experience within the quadrupole in order to be separated. In some embodiments, where the mass filter is a quadrupole mass filter, the mass filter resolution may be defined as a ratio of the bandpass window size to the m / z value corresponding to the center of the band pass window. Because the quadrupole may have a fixed and finite length, the number of RF cycles may be inversely proportional to the speed of the ion and thus inversely related to the energy of the ion. Thus, to achieve higher resolutions the energy of the ion may need to be set to the lower values.

[0117] In some cases, when due to the accumulation of contamination the mass spectrometer is rendered inoperable for the required resolution, the MS operation needs to be halted and the contamination to be removed from some of the affected surfaces before the operation is resumed. Such interruptions reduce the efficiency of the mass spectrometer. Some embodiments improve the efficiency of the mass spectrometer by increasing the duration of uninterrupted operation before such interruption is needed.

[0118] Some embodiments achieve such an increased efficiency and longer uninterrupted operation by selectively operating the mass spectrometer in two different operational modes at different times. In various embodiments, those two operational modes may be termed as a high-ion-energy mode (also referred to as a “robust mode”) and a low-ion-energy mode (also referred to as a “normal mode”),which can also be abbreviated to the high-energy mode and low-energy mode, respectively. In some embodiments, these two modes are differentiated by various characteristics including the kinetic energies of the ions entering different parts of the mass spectrometer or the width of the bandpass window when operating the mass filter in the corresponding mode, as further detailed below.

[0119] Some embodiments control the kinetic energies of the ions in different parts by adjusting the electromagnetic forces exerted on the ions in some parts or equivalently by adjusting the electrical potential level in some parts. FIGS. IB and 1C are potential diagrams that schematically depict, respectively, for the low energy mode and the high energy mode, the electrical potential energy levels U and the corresponding ion energies in different parts of the mass spectrometer in some embodiments. In these figures, the electrical potential energy level U at each point may be interpreted to be equal to z x e x f, in which z x e is the charge of the ion and V is the electrical potential at that point. In some embodiments, the ion may be assumed to be singly charged (z = 1) and, by adopting a unit system in which the unit of charge is e, the schematic electrical potential levels in FIGS. IB and 1C may indicate the corresponding ion energy levels.

[0120] More specifically, FIG. IB shows a potential diagram 102 that schematically depicts, for the low energy mode, potential levels of some of the parts of mass spectrometer 100 and some corresponding ion kinetic energies, according to some embodiments. In particular, according to potential diagram 102, in the low energy mode, ion guide 120 is at a potential level 122, ion lens 130 is at a potential level 132, mass filter 140 is at a potential level 142, and collision cell 150 is at a potential level 152.

[0121] The relative differences between the potential levels in different parts may approximately determine the relative increase or decrease of the kinetic energies of the ions in those parts. In describing potential diagrams 102 and 104, the potential energy and the kinetic energy at ion guide 120 (Q0) may be set to zero without loss of generality, thus measuring the potential energy or the kinetic energy inother parts relative to those energies in QO. For example, in ion lens 130 (IQ 1), the kinetic energy of the ions may be assumed to be around a first energy difference 133 (sometimes labeled as IQ1 energy or alternatively just IQ 1, if unambiguous in the context), which is the difference between potential levels 122 and 132. Similarly, the kinetic energies of the ions in mass filter 140 (QI) may be assumed to be around a second energy 143 (sometimes labeled as IE1 energy), which is the difference between potential levels 122 and 142. Moreover, the kinetic energy of the ions in collision cell 150 (Q2) may be assumed to be around a third energy 153 (sometimes labeled as CE, standing for collision energy) which is the difference between potential levels 122 and 152. In some embodiments, for example, in the low-energy mode, the values of IQ1 energy, IE1, and CE can respectively be set to 2eV, leV, and 40eV. Some embodiments may use different values for these energy as shown by some examples below.

[0122] In a similar manner, FIG. 1C shows, for the high-energy mode, a potential diagram 104 that schematically depicts potential levels of, and the kinetic energies of the ions in, the same parts of mass spectrometer 100 according to some embodiments. More specifically, potential diagram 104 shows that, in the high-energy mode, ion guide 120, ion lens 130, mass filter 140, and collision cell 150 are respectively at potential levels 124, 134, 144, and 154. Further, in the high-energy mode, the kinetic energies of the ions in ion lens 130, mass filter 140, and collision cell 150, each exceed the kinetic energies of the ions in ion guide 120 by amounts that are respectively around values schematically shown by first, second, and third energy differences 135, 145, and 155. In some embodiments, for example, in the high-energy mode, the values of IQ1, IE1, and CE energies may be respectively set to lleV, lOeV, and 40eV.

[0123] Diagrams 102 and 104 illustrate some of the mechanisms utilized in the high-energy mode enabling the higher robustness of the high-energy mode compared to the low energy mode according to some embodiments. In particular, these two diagrams depict that in various parts of the mass spectrometer such as in ion lens 130 or in mass filter 140, the electrical potential differences with the ionguide 120, and thus the kinetic energies of the ions, are higher in the high-energy mode. These differences may cause higher robustness for the reasons described above.

[0124] In various embodiments, therefore, the high-energy mode may be utilized to prolong the uninterrupted operation of the MS before an intermption is required to decontaminate the mass spectrometer. FIG. 2 is a sensitivity graph set 200, illustrating the higher longevity of a mass spectrometer operated in the high-energy mode according to some embodiments. More specifically, sensitivity graph set 200 depicts that utilizing the high-energy mode may reduce the speed of onset of contamination effects and thus delay the intermption required for decontamination. The y-axis in graph set 200 shows the value of the signal intensity in each measurement divided by the signal intensity at the start of the operation (at which time the contamination is assumed to be the lowest) and the x-axis shows the cumulative amount of the sample infused into the mass spectrometer from the start of the operation measured in milligrams.

[0125] Graph set 200 includes two graphs, a low-energy sensitivity graph 210 and a high-energy sensitivity graph 220, that respectively show measurements of the change in the sensitivity of a mass spectrometer during continuous operation in the low-energy mode and high-energy mode. To derive each data point in each graph, two steps were taken, a one-hour contamination step followed by a one-minute measurement step.

[0126] In the contamination step, the mass spectrometer was continuously operated for one hour while being directly infused with a K562 cell line tryptic digest. Further, in the contamination step, a DIA acquisition mode of a SCIEX research time-of-flight (ToF) mass spectrometer, known as SWATH (Sequential Windowed Acquisition of all Theoretical fragment ions), was utilized, which contained a single TOF MS scan and a number of TOF MS / MS scans with wide isolation windows (6 Th-15Th).

[0127] In the measurement step, the mass spectrometer was infused with a Sciex calibration mixture containing an ALILTLVS peptide ion at concentration of Ipmol / u. Then the mass spectrometer wasoperated in the MS / MS mode, in which a singly charged ALILTLVS peptide ion (m / z = 829.53) was isolated in the mass filter QI, the isolated ions were fragmented in the collision cell Q2, and the intensity of six most intense fragments of the peptide ion were measured by the mass analyzer.

[0128] In FIG. 2, the two different operations that result in the two graphs 210 and 220 differ as follows: for low-energy sensitivity graph 210, the first and second energy differences (i.e., the IQ1 and IE1 energies) were respectively set to leV and 2eV, while for high-energy sensitivity graph 220, the first and second energy differences were respectively set to 10 eV and 11 eV. Graph set 200 shows that in the high energy mode of high-energy sensitivity graph 220, as compared to the low-energy mode of low-energy sensitivity graph 210, the mass spectrometer was able to operate for more than five times longer before losing 50% of signal intensity. More specifically, in low-energy sensitivity graph 210, the sensitivity reaches 50% after infusion of about 2 mg of the sample, while in high-energy sensitivity graph 220, that deterioration does not happen until infusion of about 11 mg of the sample.

[0129] FIG. 3 is another sensitivity graph set 300 depicting that utilizing the high-energy mode may reduce the speed of onset of contamination effects in some embodiments. Graph set 300, similar to graph set 200, includes a low-energy sensitivity graph 310 and a high-energy sensitivity graph 320. Moreover, graph set 300 is also similar to graph set 200 in some other respects such as the utilized sample, the potential levels of different parts, and the details of the contamination step. For graph set 300, however, instead of using the data from the measurement step, the data from the contamination step was used for assessment of sensitivity degradation. In particular, the contamination step contained an MS experiment, in which the ions were analyzed unisolated and unfragmented, and the mass filter QI was utilized as an ion guide. Further, the collision energy was set at lOeV to reduce fragmentation events, not for isolation. Accordingly, the signal intensity was measured to select unfragmented ions with m / z below 300 amu. In this case, graph set 300 shows that in the low-energy mode depicted by low-energy sensitivity graph 310 the sensitivity deteriorates to about 50% after infusion of around 1 mg of the sample. In the high-energymode, depicted by high-energy sensitivity graph 320, on the other hand, the sensitivity does not reach 50% even after infusion of around 13 mg of the sample. Combining graph sets 200 and 300 may indicate that, in this setup, if the mass spectrometer is utilized for both the MS mode and the MS / MS mode, the high-energy mode may be utilized for at least five times longer periods as compared to the low-energy mode.

[0130] As explained above, operating in different energy modes may affect the resolution of the mass spectral data. Figs. 4A and 4B illustrate the effect of using different energy modes on the resolution of the data according to some embodiments. More specifically, Fig. 4A includes a low-energy MS plot 400 of ion fragments of a sample derived by a mass spectrometer operating in a low-energy mode. Fig. 4B, on the other hand, includes a high-energy MS plot 402 of the same ion fragments of the same sample derived by the same mass spectrometer operating in a high-energy mode. Both plots 400 and 402 show the signal intensities as a function of the ion mass, for ions with masses between limits that are around 827 Da and 832 Da. Each plot in FIG. 4 may be a section of a larger MS plot that covers a larger mass range. For deriving low-energy MS plot 400, the mass spectrometer was operated at a low-energy mode by setting the second energy difference IE1 to leV. On the other hand, for deriving high-energy MS plot 402 the mass spectrometer was operated at a high-energy mode by setting the second energy difference IE1 to lOeV.

[0131] Figs. 4A and 4B show that in some embodiments the low-energy mode provides a higher resolution. More specifically, low-energy MS plot 400 shows three clearly pronounced peaks 410, 420, and 430 corresponding to three ions with ion mass values that are respectively around 829.5 Da, 830.5 Da, and 831.5 Da. These ions, therefore, may be resolved when utilizing a unit resolution operational setting, that is, an isolation window with the width of roughly 1 Th, illustrated by a rectangle 405.

[0132] High-energy MS plot 402, on the other hand, does not show those three peaks in a clear manner. In particular, in high-energy MS plot 402, while a first peak 460 may be detected at an ion massvalue around 829.5 Da, a second peak 470 at around 830.5 Da is not easily discernible or distinguishable from first peak 460, and the third peak at around 831.5 Da is almost completely missing. Therefore, when utilizing a unit resolution such as an isolation window 455, the first ion corresponding to first peak 460 may be detected, but the second and third ions may be missed. More specifically, in low-energy MS plot 400 the width of peak 410 corresponding to the first ion is around 0.7 Th, while in high-energy MS plot 402 the width of peak 460, also corresponding to the first ion, is around 1.5 Th.

[0133] In some embodiments, plots similar to low-energy MS plot 400 or high-energy MS plot 402 may be utilized to tune the mass spectrometer for unit resolution. Accordingly, a mass spectrum for a known set of ions (such as the singly charged ions of ALILTLVS peptide in this case) may be acquired. Then, the mass spectrometer setting may be sequentially optimized to get an approximately 0.7Da peak width at full width half maximum. In some embodiments, such a tuning may be possible for a low-energy mode operation, but not possible for a high-energy mode operation.

[0134] While, therefore, operation in the high-energy mode may increase the robustness against contamination, the low-energy mode may be more suitable for some operations. For example, as seen in the above embodiments, the low-energy mode may provide a higher resolution. Therefore, the low-energy mode may be more suitable for narrow bandpass spectrometry.

[0135] As a result, various embodiments may alternate operating the mass spectrometer between the two modes based on the type of sample or needed spectrum. For example, the high-energy mode may be utilized for wide bandpass spectrometry while the low-energy mode may be utilized for narrow bandpass spectrometry. Therefore, in some embodiments, the two modes may also differ in the width of the bandpass setting of the mass filter. More specifically, the mass filter bandpass in the high-energy mode may be tuned to be wider as compared to the bandpass in the low-energy mode. Further, some embodiments may optimize the division of the operation between the two modes by balancing the need for higher resolutions in some cases and an increase in the longevity of the mass spectrometer before adecontamination procedure is required. For example, in various embodiments, a high-energy mode may be utilized when a Data Independent Acquisition (DIA) acquisition modality is employed.

[0136] FIG. 5 is a flowchart for a MS operation method 500 that corresponds to operating a mass spectrometer in different modes according to some embodiments. More specifically, MS operation method 500 includes steps for operating the mass spectrometer in different modes at different times, as detailed below. In some embodiments, some or all steps of operations such as MS operation method 500 may be performed or controlled by a controller module such as controller module 180.

[0137] At step 502, an operational mode may be selected. In some embodiments, the choices for the operational mode may include a high-energy mode and a low-energy mode.

[0138] At step 504, the operating parameters of the mass spectrometer may be set in accordance with the selected mode. In some embodiments, the operating parameters may include one or more of the energies of the ions in different parts of the mass spectrometer, the bandpass of the mass filter, the magnitude of the resolving DC voltage in the mass filter, and the amplitude or the frequency of the RF voltage in the mass filter. The energies of the ions may be set by, for example, setting the values of electrical potentials at different parts.

[0139] In various embodiments, for the high-energy mode, the kinetic energy of the ions in the mass filter may be set to be greater than a high-energy level, and for the low-energy mode the kinetic energy of the ions in the mass filter may be set to be smaller than a low-energy level. The low-energy level may be set to be smaller than or equal to the high-energy level. In particular, the low-energy level may be set to be in the range 0.5-5 eV, and the high-energy level may be set in the range 2-15 eV. The specific limits may vary depending on a particular geometric and operational implementation of the mass filter, including rod dimensions, spacing, operating frequency (RF) and DC voltages. While for an exemplar mass filter the low energy range will constitute a value from 0.5-1,5eV and high energy range 2-15eV for the mass filter with different operational parameters the selected low energy ranges can be forexample l-5eV for low energy and 5-15 for high energy. In various embodiments, these energy levels may be set to other values. In some embodiments, the high-energy level may be n times the low-energy level, where n may be an integer between 2-5, or higher. In other embodiments, n may adopt a noninteger value.

[0140] In some embodiments, for the high-energy mode, the mass filter may be set to operate with a wide bandpass window for m / z selection of the ions directed to the collision cell. For the low-energy mode, on the other hand, the mass filter may be set to operate with a narrow bandpass window for that m / z selection. In some embodiments, the wide bandpass window is selected to have a width greater than an upper bandpass threshold while the narrow bandpass window is selected to have a width smaller than a lower bandpass threshold. The lower bandpass threshold may be selected to be smaller than the upper bandpass threshold. In some embodiments, the upper bandpass threshold may be selected to be between 3-200 Th while the lower bandpass threshold may be selected to be between 1 -3 Th. Alternatively or in addition, the narrow bandpass window may be selected to be between 1-3 Th while the wide bandpass window may be selected to be between 5-200 Th.

[0141] In some embodiments, various parameters may be set based on physical relationships between the RF frequency, the ion energies, the selected bandpass, or the desired resolution. For example, some embodiments may utilize the relationship in Eq. (1):in which (M / Z) / D(M / Z) is the desired resolution (M / Z being the mass to charge of the ion and A(M / Z) being the measured peak width of the ions corresponding to a select analyte ion) and N is the number of RF cycles experienced by each ion while passing through the mass filter, such as the quadrupole. Moreover, K is a dimensionless proportionality factor and n is an integer power, both of which may be determined empirically. The number of RF cycles, N, may bedetermined as f x t, where / is the frequency of the RF voltage and t is the duration of each ion’s passage through the mass filter. Therefore, N may be calculated by Eq. (2):in which ZeVzis the kinetic energy of an ion with a charge Ze inside the mass filter, and m is the mass of the ion, based on which the quantity in the parentheses would be the inverse of the ion’s velocity inside the mass filter, and thus the outcome of the brackets would be the time t. In some embodiments, based on experimental data, the quantities K and n in Eq. (1) are determined to be 20 and 2, respectively. Inserting Eq. (2) in Eq. (1) as well as the values for K, n, and e, and solving for A(M / z) results in Eq. (3):or equivalently Eq. (4):7Z= 2.5 x 10-10 2L2A(M / Z) (4).

[0142] In some embodiments, Eq. (4) may be utilized for determining the maximum electrical potential level difference between different parts of the mass spectrometer, such as the ion guide(M\—J. For example, in some embodiments,Mfor which f=940kHz, L=0.127m, for achieving a “unit” resolution or better ( A(— ) < 0.7), Eq. (4)results in Vz< 2.57, setting an upper boundary on the kinetic energy of the ions or equivalently the electrical potential difference between the mass filter and the ion guide to achieve unit resolution. In various embodiments, setting the mass filter to unit mass filtration resolution is utilized to isolate singly charged isotopes of an ion, which would be separated by m / z differencesthat are multiples of ITh. Various embodiments may achieve higher resolutions by selecting smaller peak widths.

[0143] Returning to MS operation method 500, at step 506, the mass spectrometer is operated and data are acquired for one or more samples.

[0144] In some embodiments, the operation in the selected mode may continue for a predetermined time or based on some criteria. The operation may be stopped when the analysis of one or more samples is complete. Alternatively, or in addition, the operation may be stopped to decontaminate the mass spectrometer because, for example, the sensitivity or accuracy of the operation has reached a minimum level.

[0145] In some embodiments, the selected operational mode may be the same or different compared to the previously selected operational mode. For example, MS operation method 500 may switch from the high-energy mode to the low-energy mode if a higher resolution or a smaller bandpass window is required for a new sample or for the same sample. Alternatively, or inversely, MS operation method 500 may switch from the low-energy mode to the high-energy mode if, instead of a higher resolution, a larger bandpass or a longer operation before decontamination is desired.

[0146] After performing one or more rounds of operations in different modes according to MS operation method 500, the decision may be made to stop the operation at which time the method proceeds to an ending step 508.

[0147] In some embodiments, the controller module of the mass spectrometer may provide a selector for switching between the high-energy mode and the low-energy mode. The selector may be operated via an automated program, a logic module, or an operator of the mass spectrometer, which may, for example, make the selection at step 502, set the operating parameters for different parts of the mass spectrometer at step 504, or make the decision atdecision step 508. For example, at step 504, the controller module may set the voltage level at one or more parts of the mass spectrometer, described above. In some embodiments, the controller module may also set the parameters related to the desired resolution or the bandpass window.

[0148] In various embodiments, the controller module may set one or more of the parameters in accordance with the selected mode or in accordance with the selection of the other parameters. For example, the controller module may set the voltage levels such that in the high-energy mode the ions in the mass filter would have kinetic energies greater than a preset high energy level; and in low-energy mode the ions in the mass filter would have kinetic energies lower than a preset low-energy level. For example, the low-energy level may be set to values between 0.5eV-4eV and the high-energy level may be set to values between 5eV-15eV. Further, the high energy level may be set to be larger than n times the low-energy level, where the integer number n may be selected to be between 2-10.

[0149] Further, in various embodiments, the controller module may set the bandpass window to a wide bandpass in the high-energy mode and a narrow bandpass in the low-energy mode.Moreover, the narrow bandpass may be selected to be smaller than a preset lower bandpass threshold and the wide bandpass may be selected to be greater than a preset upper bandpass threshold. In various embodiments, the lower bandpass threshold may be selected to be equal to, or smaller than, the upper bandpass threshold. The lower bandpass threshold may, for example, have a value below 2Th, and the upper bandpass threshold may, for example, have a value between 5-200Th. In various embodiments, the preset values may be determined by the operator of the mass spectrometer or may be set automatically, based on results of some previous operations of the mass spectrometer, such as calibration operations.

[0150] Some embodiments may utilize adjusting the potential level, or equivalently the energy level, at the ion lens (IQ 1) in addition to the potential or energy level in the mass filter (IE1) for obtaining optimum results. FIG. 6 shows a table 600 depicting the effects of changing IQ1 and IE1 according to some embodiments. Table 600 includes results of operating a mass spectrometer under five different conditions. All operations corresponded to identifying proteins using the DIA method. In each operation, 10ng of K562 mixture was injected and analyzed using 5min gradient, microflow conditions. Further, in each operation, a 13 Th bandpass window was used, and experiments were performed in triplicate for shown experimental conditions. Moreover, the results were searched using the DIANN 1.8.1 automated software suite for DIA proteomics data analysis.

[0151] Table 600 includes four columns. The first column lists the energy settings for each of the five operations. The next three columns list the results of the operations, that is, respectively the number of proteins identified, the number of proteins quantified with coefficient of variation (CV) <20%, and the number of proteins quantified with CV <10%.

[0152] As shown in the first column of table 600, in rows 2-6, for the first operation IE1 was set to 2eV, and was set to 10eV for the rest of operations. Therefore, the first operation may be considered to be a low-energy mode operation while the remainder of the operations may be considered to be high-energy mode operations. Moreover, for the 1stto 5thoperations, IQ1 voltage was respectively set to values 2 V, 11 V, 8 V, 6 V, and 4 V. Thus, the results may indicate the effect of changing IQ1 in different modes.

[0153] The data in table 600 show that, for these operations, the accuracy of the results does not change significantly between the low-energy mode and the high-energy mode, or upon changing the values of IQ 1. Nevertheless, the outcome of the first operation at the low-energy mode seems to be marginally better than some outcomes at high-energy mode operations and essentially similar to the outcomes of some other high-energy mode operations, e.g. IEl=10V, IQ1=4V. Therefore, based on these results for this type of operation, the high-energy mode may be chosen for higher robustness in some cases if theslight decrease in the number of identified proteins is acceptable. Otherwise, the low-energy mode may be chosen or an additional optimization step may be performed to identify the high-energy robustness mode with minimal loss of accuracy of the results. Such optimization may be performed based on a group of instruments and then the resulting setting may be used as default acquisition parameters for high energy mode. Alternatively, for one or more of instruments, the optimization may be performed individually and the optimal settings determined for each instrument.

[0154] In some embodiments involving MS / MS operations, some of the most contamination prone areas may include the precursor isolation sections such as the interface between the ion guide (Q0) and the mass filter (QI), and in particular in the ion lens (IQ 1 ) depicted in Fig. 1 A. Some embodiments may also employ other types of lenses, e.g., a Brubacker lens (STI) or and RF free DC lens for improved ion transmission. Such ion optic elements may be among elements that are the most susceptible to contamination, in particular depending on the type of analysis. For example, when the analysis is performed at a low bandwidth (e.g., at unit resolution), small changes in resolving potential or axial potential may lead to a shift of one of the edges of the bandpass filter. As a result, the center of the bandpass may shift away from the ion of interest and drastically reduce transmission of that compound.

[0155] Similarly, in some cases, the contamination of the ion optic element may lead to some performance degradations. For instance, in proteomics experiments performed with wide isolation windows (>2Th), the disclosed techniques may permit slight shifts of the isolation window edges and therefore illustrate more robustness against the contamination of the RF+DC filter. Some systems, however, may still be susceptible to deleterious effects primarily caused by the contamination of other optic elements, such as the IQ1 lens. In some embodiments, the IQ1 lens may serve two distinct purposes, first it focuses the ion beam into narrow acceptance area of resolving QI mass filter. In this case ions hitting the acceptance area are successfullytransmitted, while the ions outside of this area are rejected.. Second, the IQ1 separates the upstream higher pressure region of QO, where collisional cooling is performed, from the lower pressure region of QI, where RF+DC isolation is performed. Because of the focusing property of IQ1, there are advantageous optimal tuning values. Such tuning values may particularly be advantageous when data is acquired at a higher resolution, such as the unit resolution.

[0156] In various embodiments, contamination of ion optic elements may show different effects in a high resolution experiment versus a low resolution experiment, or alternatively in a high energy mode versus a low energy mode.

[0157] Fig. 7A shows an intensity curve 700 for a high resolution experiment according to an embodiment. Intensity curve 700 shows the measured intensity of a transmitted ion of interest, in this example, a calibrant ion with m / z of 829.5, as a function of various IQ1 electrical potentials (aka IQ1 offset or just offset for brevity). In this setup, the quadrupole was set at a unit resolution (around 0.7 Da) and the value of the ion energy derived from IE1 was set to IV (similar to the situation depicted in Fig. IB). As seen, in this case the maximum intensity is found around the maximum point 702 with an offset of about -2.0 V, indicating the settings for optical focusing effect of IQ1. Moreover, intensity curve 700 has a steep incline before maximum point 702 and a steep decline after that point. Substantially positive voltages are blocking the ions instead of letting them pass through. Therefore, the available range for maximum transition is narrowly located around maximum point 702.

[0158] Fig. 7B, shows an intensity curve 750 for a low resolution or wide bandpass acquisition according to an embodiment. The setup in this experiment was similar to that of Fig. 7A except that the ion energy or IE1 was set to 10V and the mass filter width was set to 10 Da (similar to the situation depicted in Fig. 1C). As seen, in this case the maximum intensity is observed around the maximum point 752, with an offset of about 0V. But, contrary to the high-energyhigh-resolution case of transmission rate curve 700 in Fig. 7A, intensity curve 750 has a flatter profile before maximum point 752, therefore providing a wider available range of tunable offset (located before the maximum point) for achieving transitions close to maximum transition.

[0159] In various systems, when the IQ1 lens gets contaminated, it may become charged more easily and may cause the settings for the optimum focusing to change. Fig. 8A illustrates one example of the change in the optimum focusing settings due to contamination according to an embodiment. In particular, Fig. 8 A includes a graph set 800 that includes two intensity plots 810 and 820. Each of intensity graphs 810 and 820 shows a measured transmission rate expressed as a percentage of maximum expected intensity of a detected target ion, as a function of the offset recorded in volts. In this embodiment, the instrument was exposed to K562 LC injections of a reference K562 cell line tryptic digest using nanoflow chromatography. Mass spectrometry runs were interspersed with contamination check experiments, where the IQ1 tuning ramp was acquired using tuning solutions. In particular, intensity plot 810 was acquired at the start of the experiment when the instrument was clean. Intensity plot 820, on the other hand, was acquired after 15 days of injection of K562 to simulate performance after repeated use.

[0160] As illustrated, the optimal IQ1 offset shifted between plots 822 and 812 such that the optimal DC offset for 812 yields poor transmission performance for the later acquisition illustrated in plot 822. As a result, the later acquisition run would require an adjustment in the IQ1 offset value for optimal performance.

[0161] As can be seen in graph set 800, as the contamination increases (i.e., upon transitioning from intensity graphs 810 to intensity graphs 820), the optimal IQ1 offset value, shifts to a different value and in some cases the required shift due to contamination may prevent useful transmission over time as contamination increases.

[0162] Therefore, in some embodiments, the shift in the optimal IQ1 offset may indicate the extent of contamination of the ion optic elements. To that end, Fig. 8B illustrates a method of determining the extent of contamination based on the shift in the IQ1 offset value according to some embodiments. In various embodiments, the shift may be measured as a shift in the optimal IQ1 offset (i.e. the offset for the highest intensity). Alternatively, and in some embodiments, the shift may be measured as a shift in the measured IQ1 offset value to a “stopping potential” or “stopping offset” where less than maximum transmission is observed below some threshold, such as 70% of maximum, 50% maximum, etc. In some aspects, the shift may be measured as a shift in the measured IQ1 offset value where no transmission is observed. As will be appreciated, in many cases a drop in measured intensity to the 70% or the 50% level of expected intensity may commonly be indicative of an instrument that requires cleaning due to contamination.

[0163] Figs. 8B and 8C also show a comparison of the shift in the stopping potential in the two operational modes. Fig. 8B includes a graph set 830 and Fig. 8C includes a graph set 840, corresponding to two different target ions (with m / z= 118 for graph set 830 and m / z= 829 for graph set 840). Each graph set includes two graphs corresponding to the two operational modes for the same ion. The value of the y-component of each dot in each of the graphs is the value of the stopping offset (measured in volts). The value of the x-component, on the other hand, is the length of the duration of time from the start of the experiment, at which time there is no contamination (measured in days).

[0164] Graph set 830 includes a low-energy graph 832 and the high-energy graph 834. Low-energy graph 832 shows that the stopping offset for the low-energy experiment drops from around -0.7 V at the beginning (data point 832-0) to -1.8 V after 7 days (data point 832-7). High-energy graph 834, on the other hand, shows that the stopping offset for the high-energy experiment drops from -0.6 V at the beginning (data point 834-0) to around -1.3 V after 14 days(data point 834-14). That is, the stopping offset is consistently higher and varies less for the high-energy experiment as compared to the low-energy experiment after the same duration of time and drops more slowly indicating less contamination (as expected).

[0165] Such experiments may be conducted as tuning experiments to approximate the duration of the up time between consecutive cleanings of the instrument in each mode. For example, if an acceptable amount of contamination is taken to be equivalent to a stopping offset of around -1.4 V, this threshold is reached after around 3 days in the low-energy mode (data point 832-3) while, for the high-energy mode, the threshold is not reached after more than 14 days (data point 834-14).

[0166] Similar behavior is observed in graph set 840. This graph set includes a low-energy graph 842 and a high-energy graph 844. Low-energy graph 842 shows that, in the low-energy operational mode, the stopping offset drops from -0.5 V at the start to -1.6 V after 7 days. High-energy graph 844, on the other hand, drops from around -0.4 V at the start to -1.3 V after 14 days, indicating a slower rate of contamination in the high-energy.

[0167] FIG. 9 schematically depicts two differentially-pumped chambers 902 and 904 of a mass spectrometer, according to some embodiments. The pressure inside upstream chamber 902 may generally be higher relative to a respective pressure in the downstream chamber 904. An ion optic 905 in the form of an electrically conductive plate having an aperture 905a separates chamber 902 from chamber 904. Aperture 905a provides gas flow restriction between the two chambers to facilitate maintaining a desired pressure differential between them. In some embodiments, in addition to providing a pressure-regulating partition, the application of a DC voltage to the ion optic 905 allows it to function as an ion lens to focus an ion beam 908 passing through chamber 902 to reach chamber 904.

[0168] A variety of ion optics may be housed in the two chambers. By way of example, in this embodiment, a set of rods 906 arranged in a quadrupole arrangement is housed in chamber 902, two of which are illustrated as rods 906a and 906b. In various implementations, the application of RF voltages to rods 906 in a manner known in the art can generate an electromagnetic field in a space between the rods that can provide radial confinement of ion beam 908 as it passes through that space.

[0169] Further, the mass spectrometer includes an RF ion lens 910 formed of four rods arranged in a quadrupole arrangement (two of which 912a and 912b are illustrated). RF voltages maybe applied to the four rods to provide additional focusing of the ions received by chamber 904 into a mass filter 914. Mass filter 914 includes four rods arranged in a quadrupole arrangement (two of which 916a and 916b are illustrated). The application of RF voltages and DC discriminating voltages to these rods in a manner known in the art allows radial confinement of the ion beam as it passes through the filter. The voltages further establish an ion transmission window characterized by an m / z range for passage of ions through the filter.

[0170] As shown in FIG. 9, the minimum distance between ion beam 908 and ion optic 905 (designated as d2) is less than the minimum distance between the ion beam and other components, namely, rods sets 908, 912 and 916 (designated, as dl, d3, and d4, respectively). In various embodiments, this is the result of a small size of aperture 905a, which is typically needed to maintain a desired pressure differential between the two chambers. As a result, in this example, ion optic 905 is more susceptible to contamination via incidence of a portion of ions from the ion beam.

[0171] As discussed above, in various embodiments, the energy of the ions can be adjusted to reduce the rate of contamination of an ion optic of a mass spectrometer, such as ion optic 905 discussed above. By way of illustration, FIG. 10A depicts two profiles of an ioncurrent measured at an electrically conductive ion optic having an aperture that separates a chamber in which a mass filter is positioned from an upstream chamber, such as ion optic 905 described above. In this example, the ions were generated by injecting 300 nanograms of K562 tryptic digest on LC column operated at nanoflow (300nl / min) regime.

[0172] For acquiring the data associated with profile (a), the mass spectrometer was operated in a normal mode with an ion energy of 1eV and the mass filter was configured to provide a mass transmission window having an m / z range of about 10Da. Further, an RF ion lens, such as RF ion lens 910 discussed above, was configured to block passage of ions and return the ions passing through an aperture of the ion optic back to the ion optic. Consequently, profile (a) corresponds to a current profile induced in the conductive ion optic by substantially all ions arriving at the ion optics. In contrast, profile (b) was measured by configuring the RF ion lens to allow passage of ions passing through the aperture of the ion optic to the mass filter, such as mass filter 914 described above. Consequently, profile (b) corresponds to a faction of ions received at the ion optic, i.e., those that do not pass through the ion optic’s aperture and hence can lead to contamination of the ion optic.

[0173] FIG. 10B shows two current profiles (a) and (b), which were obtained in a manner similar to that discussed above, but with the mass spectrometer operated in a robust operational mode characterized by a higher ion energy of 5eV. Similar to FIG. 10A, profile (a) corresponds to a current profile induced in the ion optic when the RF lens blocks the passage of ions so that the ion optic receives substantially all of the ions while profile (b) corresponds to a current profile induced in the ion optic when the RF lens allows the passage of ions passing through the ion optic’s aperture to the downstream ion filter.

[0174] As state above, the specific energy levels are architecture dependent, but generally the “robust” operational mode will impart an energy level that is two or more timesgreater (e.g., n times greater) than the “normal” mode of operation to ensure extended operational performance. As will be appreciated, for simplicity n may be an integer value or may adopt a decimal value as may be convenient.

[0175] A comparison of current profile (b) in FIG. 10A with current profile (b) in FIG.10B shows that operating the mass spectrometer in the robust operational mode results in a lower induced current in the ion lens, thus reducing its rate of contamination.

[0176] FIG. 11 A shows total charge deposited on an ion optic, such as above ion optic 905, during an entire elution period of a sample loaded onto a LC column of an LC / MS spectrometer as a function of different amounts of the sample for operating the spectrometer in a normal and in a robust operational mode, when the RF lens was set to block the passage of ions and hence substantially all ions arriving at the ion optic were collected by the ion optic. The data shows that as the sample load increases, so as the ion current induced in the ion optic by the ions striking the ion optic. Further, for each sample load, the induced ion current is the same for the normal and the robust mode, when the ion optics configured to block all the ion current.

[0177] In contrast, FIG. 1 IB shows data corresponding to the induced current in the ion optic in both the normal and the operational mode as a function of the sample load when the RF lens allows passage of the ions to the mass filter. The data indicates that in both the normal and the robust modes, the ion current induced in the ion optic due to ions striking the body of the ion optic rather than passing through its aperture increases as the sample load increases. However, at all measured sample loads, the ion current measured at the ion optic is lower in the robust mode relative to that measured in the normal mode. In other words, for each sample load, less ions strike the body of the ion optic in the robust mode than in the normal mode and hence contribute less to the contamination of the ion optic. Further, the data shows a lower rate of increase of the ion current as the sample load increases for the robust mode relative to the normal mode.

[0178] By way of further illustration, FIG. 11C presents data corresponding to a fraction of ion charge deposited on the ion optic over an entire LC elution period of the same sample for normal and robust operational modes of the LC-MS spectrometer for different sample loads. The data indicates that for each sample load, the fraction of charge deposited on the ion optic is less in the robust operational mode than in the normal mode, and hence there is less contamination due to ion deposition on the ion optic in the robust mode.

[0179] In some embodiments, the behavior of the contamination as a function of different parameters, such as the ion energy or the IQ1 offset may be used for configuring the mass spectrometer for optimal contamination rate. For example, determining the changes of the contamination rate (based on the IQ1 ion current) as a function of the ion energy may determine optimal values for the ion energy in a robust mode. Figs. 12A and 12B illustrate the process of determining the optimal ion energy for the robust mode according to some embodiments. Figs.12A and 12B show graph sets 1200 and 1250, respectively corresponding to a mass spectrometer right after cleaning (i.e., a clean MS) and a mass spectrometer after substantial contamination (i.e., a contaminated MS). The y-axis in each graph set shows the value of the IQ1 ion current in Amperes (indicating the rate of contamination of IQ1) and the y-axis shows the value of the IQ1 offset in volts. Both graph sets 1200 and 1250 include 3 graphs corresponding to setting the ion energy at a specific value and determining the changes in the IQ1 as a function of the IQ1 offset for that ion energy.

[0180] More specifically, graph set 1200 includes a graph 1210 corresponding to an ion energy of 1 eV, a graph 1220 corresponding to an ion energy of 5eV, and a graph 1230 corresponding to an ion energy of lOeV. In all 3 graphs, the section for IQ1 offset values below -0.5 V is considered the operational regime and the section for IQ1 offset values above -0.5 V is considered the nonoperational regime.

[0181] Further, in the operational regime, the IQ1 ion current generally decreases with increasing IQ1 offset up to about an optimum offset 1240 (around -0.5 V). The value of the IQ1 ion current at the minimum IQ1 offset value, however, is not the same for the 3 graphs. In particular, the IQ1 ion current for graph 1210 at optimum offset 1240 is around 2.5x10-10A, while the IQ1 ion currents for graphs 1220 and 1230 at minimum offset 1240 are almost the same and less than 0.5x10-10A. These results show that while the rate of contamination (indicated by the value of the IQ1 current) is relatively high for the low-energy mode of 1eV, that rate reduces significantly for the high-energy mode of 5eV and does not change much for higher energies at least up to 10eV. In other words, while the rate of contamination for the low-energy mode (a.k.a. default mode) is high, the rate of contamination in a range of high-energy modes (a.k.a. robust mode) reaches a minimum plateau and almost does not change.

[0182] Similar conclusions may be made from graph set 1250 for the operation of the mass spectrometer after some contamination. More specifically graph set 1250 includes 3 graphs 1260, 1270, and 1280 corresponding to the same 3 ion energies depicted in graph set 1200. In this case, as compared to the clean MS in graph set 1200, the nonoperational mode starts at a lower IQ1 offset value of around -3 V, as expected from a contaminated MS. Further, at an optimal offset 1280 (around -3.5 V), the IQ1 ion current for the low-energy graph 1260 is around -1.0x10-10A, while the IQ1 currents for the high-energy graphs 1270 and 1280 are approximately the same and less than -0.2x10-10A. Generally, the optimum IQ1 offset is lower for the contaminated IQ1. Finally, similar to the clean MS, in the contaminated MS the rate of contamination (as measured by the IQ1 ion current) decreases from the low-energy mode (or default mode such as in graph 1260) with increasing ion energy until it reaches a plateau at a high-energy value (here around 5eV) and remains the same in higher energies at least up to 10eV in this case.

[0183] Some embodiments utilize the above discussed relation between rate of contamination, the ion current in one or more the optics elements, and the energy level of the ions to configure the mass spectrometer. Fig. 12C shows a flowchart for a configuration method 1295 according to some embodiments.

[0184] In particular, at step 1296, ions are directed into the mass spectrometer.

[0185] At step 1297, one or more parameters may be measured. The parameter may, for example, be the ion current in one or more of the ion optics such as the IQ1. The controller may determine, based on the value of the parameter which operational mode provides the correct contamination rate. For example, steps 1296 and 1297 may be performed in a normal mode. If the ion current in the optic exceeds a threshold value, the controller may determine that the rate of contamination is too high for prolonging the operation for the desired uptime.

[0186] At step 1298 the controller may select level for the operation of the MS. For example, if the ion current is above the threshold, the controller may determine that a more suitable operational mode would be the robust mode. The robust mode may be defined by one or more settings of the MS such as the energy level or the IQ1 offset as detailed earlier.

[0187] In some embodiments, the mass spectrometer may be configured by the manufacturer in the manner described in configuration method 1295. More specifically, the manufacturer may provide a setting in the mass spectrometer allowing the user to switch to a robust mode or to the high-energy mode.

[0188] In some embodiments, the behavior of the ion transmission rate as a function of IQ1 offset may be used for determining values of the IQ1 offset for optimal operation. To that end, Fig. 13 A illustrates determining an optimum IQ1 offset in a low energy mode according to some embodiments. Figs. 13B and 13C, on the other hand, illustrate determining an optimum IQ1 offset range in a high energy mode.

[0189] Fig. 13A includes a graph 1300 showing the ion transition as the function of IQ1 offset according to an embodiment. As seen before, low energy graph 1300 reaches a maximum point 1312 at an optimum IQ1 offset value around -2 V (marked as optimum offset 1320). Moreover, graph 1300 drops to a performance degradation level around degradation point 1314 for an IQ 1 offset value around +2V (marked as degradation offset 1330). The difference between degradation offset 1330 and optimum offset 1320 is named as a contamination induced offset shift 1340, which causes the intensity to drop from its maximum value to a very low value around or equal to zero.

[0190] Moving to Figs. 13B and 13C, in a high energy mode an optimum IQ1 offset range may be determined as a range of IQ1 offset through which the intensity remains high and close to the maximum intensity. In particular, Fig.l3B includes an intensity graph 1350 showing the behavior of the intensity as a function of the IQ1 offset in a high energy (robust) mode according to some embodiments. In particular, as mentioned before and unlike a low energy intensity graph such as graph 1300, high energy graph 1350 decreases slowly when IQ1 offset decreases the maximum intensity offset. The disclosed methods determine a region of the slowly decreasing shoulder as an optimum IQ1 offset range, as further detailed below.

[0191] To that end, Fig. 13C includes a flowchart of a method 1370 for determining the optimum IQ1 offset range in the robust mode, as depicted in Fig.l3B. In various embodiments, one or more parts of the mass spectrometer, such as the controller, may perform the steps of method 1370.

[0192] More specifically, at step 1372, the controller may determine different parameters related to the maximum intensity. In particular, as also depicted in intensity graph 1350, the controller may find a maximum intensity point 1352 in graph 1350. Accordingly, the controllermay determine a maximum intensity offset 1352-1 (in this case around -0.5 V) and a maximum intensity value 1352-2 (in this case around 4.5x105cps).

[0193] Next, at step 1374, the controller may determine a starting offset for the optimum offset range, which in Fig. 13B is marked as a starting offset 1356-1 (around -11 V). To that end, the controller may 1stdetermine the desired intensity at starting offset 1356-1 as a fraction of maximum intensity 1352-2. The fraction (fl) may be selected as a positive number below 1, for example 0.3, 0.5, 0.9, etc. The controller may then find the desired intensity at starting offset 1356-1 as fl multiplied by the maximum intensity and then find the starting point 1356 before maximum intensity point 1352 at which graph 1350 reaches the desired intensity and accordingly find starting offset 1356-1 as the offset value for starting point 1356.

[0194] Next, at step 1376, the controller may determine an ending offset for the optimum offset range, which in Fig. 13B is marked as ending offset 1360-1. To that and, the controller may 1stfind a degradation point 1358 after maximum intensity point 1352, at which intensity graph 1350 degrades to a very low intensity or to a value of 0. The controller may then determining the degradation offset 1358-1 (around +2.5 V in this case). The controller may then find the difference between maximum intensity offset 1352-1 and degradation offset 1358-1 as degradation distance 1358-2 (in this case around +3 V). The controller may then determine a step back distance 1360 as a factor of degradation distance 1358-2 by multiplying a 2ndfactor f2 by degradation distance 1358-2. The 2ndfactor may be 0 or a positive number such as 0.1, 0.2, etc. Thus, the controller may determine ending offset 1360-1 by subtracting step back distance 1360 from maximum intensity offset 1352-1. In the case of graph 1350, f2 is around 1.0, therefore step back distance 1360 is equal to the relation distance 1358-2 and ending offset 1360-1 is around - 3.5 V.

[0195] Next step 1378, the controller determines the optimum offset range as the range of IQ1 offset between starting offset 1356-1 and ending offset 1360-1, in this case between -11 V and -3.5 V.

[0196] In various embodiments, the rate of contamination of at least one ion optic of the mass spectrometer can be measured, for example, on a daily basis. Such measurements can be made, for example, by measuring an electrical current induced in the ion optic due to impact of ions on the ion optic. Alternatively, the rate of contamination of the at least one ion optic can be measured by periodically measuring a change in the offset voltage applied to the ion optic so as to maintain operational level of the mass spectrometer at the desired level, for example, ion intensity level as ions pass through the ion optic. If the rate of contamination exceeds some predetermined threshold, the operational mode of the mass spectrometer may be switched from the default mode to the robust mode.

[0197] In various embodiments, one or more of disclosed modules may be implemented via one or more computer programs for performing the functionality of the corresponding modules, or via computer processors executing those programs. In some embodiments, one or more of the disclosed modules may be implemented via one or more hardware units executing firmware for performing the functionality of the corresponding modules. In various embodiments, one or more of the disclosed modules may include storage media for storing data used by the module, or software or firmware programs executed by the module. In various embodiments, one or more of the disclosed modules or disclosed storage media may be internal or external to the disclosed systems. In some embodiments, one or more of the disclosed modules or storage media may be implemented via a computing “cloud”, to which the disclosed system connects via a network connection and accordingly uses the external module or storage medium. In some embodiments, the disclosed storage media for storing information may include non-transitory computer-readable media, such as a CD-ROM, a computer storage, e.g., a hard disk, or a flash memory. Further, in various embodiments, one or more of the storage media may be non-transitory computer-readable media that store data or computer programs executed by various modules, or implement various techniques or flow charts disclosed herein.

[0198] By way of example, FIG. 14 schematically depicts an example of an implementation of a module 1400 according to some embodiments. Module 1400 includes a system memory 1402 that may include a permanent memory module (e.g., ROM 1402a) and a transient memory module (e.g., RAM 1402b), an internal bus 1404, a processor 1410 (e.g., a microprocessor), an I / O interface 1412, and a communication interface 1414 (such as a network adapter). I / O interface 1412 may be in communication with one or more external input devices 1406 (such as a mouse, a keyboard, or a touch screen) or output devices 1408 (such as a display, a printer, or a speaker).

[0199] Processor 1410 and the system memory 1402 may be utilized to store and execute instructions performing the function of module 1400. Moreover, internal bus 1404 may enable communication between the processor and other parts of module 1400 such as system memory 1402, I / O interface 1412, or communication interface 1414.

[0200] Some or all of the method steps may be executed by (or using) a hardware apparatus, like for example, a processor, a microprocessor, a programmable computer or an electronic circuit. In some embodiments, some one or more of the most important method steps may be executed by such an apparatus.

[0201] Depending on certain implementation requirements, embodiments of the disclosure may be implemented in hardware and / or in software. The implementation may be performed using a non-transitory storage medium such as a digital storage medium, for example a floppy disc, a DVD, a Blu-Ray, a CD, a ROM, a PROM, and EPROM, an EEPROM or a FLASH memory,having electronically readable control signals stored thereon, which cooperate (or are capable of cooperating) with a programmable computer system such that the respective method is performed. Therefore, the digital storage medium may be computer readable.

[0202] Those having ordinary skill will appreciate that various changes may be made to the above embodiments without departing from the scope of the disclosure.

[0203] Although some aspects have been described in the context of a system or an apparatus, in some embodiments these aspects may also represent a description of the corresponding method, where a block or device corresponds to a method step or a feature of a method step. Analogously, aspects described in the context of a method step may also represent in some embodiments a description of a corresponding block or item or feature of a corresponding apparatus.

[0204] The foregoing description of the embodiments has been presented for purposes of illustration only. It is not exhaustive and does not limit the embodiments to the precise form disclosed. While several exemplary embodiments and features are described, modifications, adaptations, and other implementations may be possible, without departing from the spirit and scope of the embodiments. Accordingly, unless explicitly stated otherwise, the descriptions relate to one or more embodiments and should not be construed to limit the embodiments as a whole. This is true regardless of whether or not the disclosure states that a feature is related to “a,” “the,” “one,” “one or more,” “some,” or “various” embodiments. As used herein, the singular forms “a,” “an,” and “the” may include the plural forms unless the context clearly dictates otherwise. Further, the term “coupled” does not exclude the presence of intermediate elements between the coupled items. Also, stating that a feature may exist indicates that the feature exists in one or more embodiments but not necessarily in all embodiments.

[0205] In this disclosure, the terms “include,” “comprise,” “contain,” and “have,” when used after a set or a system, mean an open inclusion and do not exclude addition of other, nonenumerated, members to the set or to the system. Further, unless stated otherwise or deducted otherwise from the context, the conjunction “or,” if used, is not exclusive, but is instead inclusive to mean and / or.

[0206] Moreover, if these terms are used, a set may include one or more members, and a subset of a set may include one or more than one, including all, members of the set.

[0207] Further, if used in this disclosure, and unless stated or deducted otherwise, a first variable is an increasing function of a second variable if the first variable does not decrease and instead generally increases when the second variable increases. On the other hand, a first variable is a decreasing function of a second variable if the first variable does not increase and instead generally decreases when the second variable increases. In some embodiment, a first variable may be an increasing or a decreasing function of a second variable if, respectively, the first variable is directly or inversely proportional to the second variable.

[0208] The disclosed compositions, systems, methods, and apparatus are not limited to any specific aspect or feature or combinations thereof, nor do the disclosed compositions, systems, methods, and apparatus require that any one or more specific advantages be present or problems be solved. Any theories of operation are to facilitate explanation, but the disclosed compositions, systems, methods, and apparatus are not limited to such theories of operation.

[0209] Modifications and variations are possible in light of the above teachings or may be acquired from practicing the embodiments. For example, the described steps need not be performed in the same sequence discussed or with the same degree of separation. Likewise various steps may be omitted, repeated, combined, or performed in parallel, as necessary, to achieve the same or similar objectives. Similarly, the systems described need not necessarilyinclude all parts described in the embodiments, and may also include other parts not described in the embodiments. Accordingly, the embodiments are not limited to the above-described details, but instead are defined by the appended claims in light of their full scope of equivalents.Moreover, subsets of elements or parts of different embodiments may be combined, if feasible, to create new embodiments not disclosed. Such embodiments may also include disclosed methods for which the elements or parts include method steps.

[0210] Further, the present disclosure is directed toward all novel and non-obvious features and aspects of the various disclosed embodiments, alone and in various combinations and subcombinations with one another.

[0211] While the present disclosure has been particularly described in conjunction with specific embodiments, many alternatives, modifications, and variations will be apparent in light of the foregoing description. It is therefore contemplated that the appended claims will embrace any such alternatives, modifications, and variations as falling within the true spirit and scope of the present disclosure.

Claims

1. CLAIMSWHAT IS CLAIMED IS:

1. A mass spectrometer, comprising:an ion source configured to generate sample ions in response to receiving a sample; anda mass filter positioned downstream of the ion source to receive the sample ions and configured to provide an ion transmission window to allow passage of precursor ions with m / z ratios within an m / z range of the ion transmission window;wherein:the mass spectrometer is configured to selectively operate in any of the following operational modes:a first operational mode characterized by a first m / z range of the ion transmission window and a first ion energy imparted to the precursor ions, and a second operational mode characterized by a second m / z range of the ion transmission window and a second ion energy imparted to the precursor ions,the second m / z range is greater than the first m / z range, andthe second ion energy is greater than the first ion energy.

2. The mass spectrometer of claim 1, wherein said second operational mode is further characterized by the mass filter operating in a Data Independent Acquisition (DIA) mode.

3. The mass spectrometer of any one of claims 1 and 102, wherein the first ion energy is in a range of about 0.5 eV to about 4 eV and the second ion energy is in a range of about 5 eV to about 15 eV.

4. The mass spectrometer of any one of claims 2 and 103, wherein said transmission window of the mass filter is scanned across m / z dimension in said DIA mode.

5. The mass spectrometer of any one of claims 2 and 103, wherein said transmission window of the mass filter is discretely varied across said m / z dimension in said DIA mode.

6. The mass spectrometer of any of the preceding claims, further comprising a controller, wherein the controller is configured to control operation of the mass spectrometer in the first and second operational modes.

7. The mass spectrometer of claim 6, further comprising:an ion guide positioned upstream of the mass filter;an ion lens separating the ion guide from the mass filter; anda DC voltage source operating under controller of the controller and configured to apply a DC voltage to the ion lens.

8. The mass spectrometer of claim 7, wherein the controller is configured to set the DC voltage so as to maintain a DC offset voltage between said ion lens and said ion guide within a target range predetermined for the second operational mode.

9. The mass spectrometer of claim 8, wherein:the target range is a first target range;the controller is further configured to set the DC voltage so as to maintain the DC offset voltage between said ion lens and said ion guide within a second target range predetermined for the first operational mode.

10. The mass spectrometer of any of the preceding claims, wherein the 1stm / z range is less than or equal to ITh and the 2ndm / z range is greater than 2 Th.

11. The mass spectrometer of any one of the preceding claims, wherein said mass filter is an RF / DC quadrupole mass filter.

12. A method of configuring an operational mode of a mass spectrometer, the method comprising:directing a plurality of ions into the mass spectrometer;measuring a parameter indicative of a level of contamination of at least one ion optic of the mass spectrometer; andin response to a value of the measured parameter, selecting an energy level for imparting to the ions.

13. The method of claim 12, further comprising:selecting a robust mode for the mass spectrometer when the value of the measured parameter exceeds a threshold, wherein the robust mode is related to the selected energy level;14. The method of claim 12, further comprising operating the mass spectrometer in [the] a high-energy mode related to the selected energy level.

15. The method of claim 12, wherein the parameter is an electrical current measured at the at least one ion optic.

16. The method of claim 12, wherein the selected energy level corresponds to a high-energy mode.

17. The method of claim 16, wherein the method further comprises determining an acceptable range for a voltage applied to the at least one ion optic in the high-energy mode.

18. The method of claim 17, wherein determining the acceptable range comprises:acquiring a plurality of intensities for the ions passing the mass filter to derive an intensity function as a function of the voltage applied to the component; determining a maximum intensity for the intensity function acquired at an optimal focusing voltage;determining a lower limit for the acceptable range that is lower than or equal to the optimal focusing voltage and at which the intensity function is equal to a predetermined fraction of the maximum intensity;determining a higher limit for the acceptable range that is higher than or equal to the lower limit and is further lower than or equal to the optimal focusing voltage.

19. The method of claim 18, determining the higher limit comprises:determining a degradation voltage larger than the optimal focusing voltage at which the intensity function reaches a degraded intensity that is lower than the maximum intensity;determining a 1st distance between the optimal focusing voltage and the degradation voltage;determining a degradation voltage larger than the optimal focusing voltage at which the intensity function reaches a degraded intensity that is lower than the maximum intensity;determining a 1st distance between the optimal focusing voltage and the degradation voltage;determining a 2nd distance that is equal to a factor multiplied by the 1st distance, wherein the factor is nonnegative; anddetermining the higher limit to be equal to the optimal focusing voltage minus the 2nd distance.

20. The method of claim 16, wherein:measuring the parameter is performed in a low-energy mode selected for operating the mass spectrometer; andthe low-energy mode is different from the high-energy mode.21.2 T A mass spectrometer comprising:an ion source; anda mass filter,wherein:the mass spectrometer is configured to operate two operational modes: a high-ion-energy mode and a low-ion-energy mode;in the high-ion-energy mode:the ion source is configured to direct a plurality of high-energy ions into the mass filter, wherein the plurality of high-energy ions are characterized by having, in the mass filter, kinetic energies that are greater than a high energy level;in the low-ion-energy mode:the ion source is configured to direct a plurality of low-energy ions into the mass filter, wherein the plurality of low-energy ions are characterized by having, in the mass filter, kinetic energies that are smaller than a low energy level; andthe low energy level is not larger than the high energy level.22.23-. A method of mass spectrometry comprising:selecting an operational mode of a mass spectrometer from two operational modes: a high-ion-energy mode and a low-ion-energy mode,wherein:the high-ion-energy mode comprises:directing a plurality of high-energy ions into a mass filter, wherein the plurality of high-energy ions are characterized by having, in the mass fdter, kinetic energies that are greater than a high energy level;the low-ion-energy mode comprises:directing a plurality of low-energy ions into the mass filter, wherein the plurality of low-energy ions are characterized by having, in the mass filter, kinetic energies that are smaller than a low energy level; andthe low energy level is not larger than the high energy level; and performing mass spectrometry in the selected operational mode.23.24? The method of claim 2223-. wherein the operational mode is a first operational mode selected to be the high-ion-energy mode, the method further comprising:selecting the low-ion-energy mode as a second operational mode; and performing mass spectrometry in the second operational mode.24.2? The method of claim 23 24. wherein performing mass spectrometry in the high-ion-energy mode is followed by performing mass spectrometry in the low-ion-energy mode. 25.26? The method of claim 23 24. wherein performing mass spectrometry in the low -ionenergy mode is followed by performing mass spectrometry in the high-ion-energy mode. 26.

27. The method of claim 2223-, wherein:in the high-ion-energy mode:the mass filter is configured to operate with a wide bandpass window for m / z selection so as to allow passage of a subset of the plurality of high- energy ions having m / z ratios within the wide bandpass window through the mass filter; andthe wide bandpass window is greater than an upper bandpass threshold; and in the low-ion-energy mode:the mass filter is configured to operate with a narrow bandpass window for m / z selection so as to allow passage of a subset of the plurality of low- energy ions having m / z ratios within the narrow bandpass window through the mass filter; andthe narrow bandpass window is smaller than a lower bandpass threshold. 27.2& T The method of claim 2622. wherein the upper bandpass threshold is substantially equal to the lower bandpass threshold.28.

29. The method of claim 2222. wherein the high energy level is substantially equal to the low energy level.29.

29. The method of claim 2222, wherein the high energy level is larger than n times the low energy level, wherein n is equal to 2, 3, or 4.302-9 The method of claim 2222, wherein the low energy level is determined to provide unit resolution.31.3 T The method of claim 3024. wherein the unit resolution is configured to detect mass spectrometry peaks with widths around 0.7 Th.

32. T The method of claim 2222, wherein the low energy level is determined based on a minimal attainable peak width, and one or more physical parameters of the mass filter.

33. T The method of claim 3222, wherein:the mass filter includes an RF + DC quadrupole; andthe low energy level is determined based on an equationVz= 2.5 x 10-10f2L2A(M / Z),in whichis the low energy level, f is an RF frequency of the quadrupole, L is a Mlength of the quadrupole, and A(— ) is a desired peak width.M34.35- The method of claim 33 34. wherein the desired peak width y) is calculated based on a desired resolution A( ) / ( ).

35. 4T The method of claim 2223, wherein the low-ion-energy mode is configured for isolation with a narrow bandpass and the high-ion-energy mode is configured for isolation with a wide bandpass.36.

37. The method of claim 35 34, wherein the narrow bandpass is <1 Th.37.30-. The method of claim 3637, wherein the wide bandpass is >2Th.38.34T The method of claim 2627, wherein:the high energy level is around 10 eV and the upper bandpass threshold is around 5 Th., andthe low energy level is around 1 eV and the lower bandpass threshold is around 2 Th..39.40T The method of claim 2223. wherein the mass filter comprises a plurality of rods arranged in a multipole configuration so as to provide an ion passageway for transit of ions through the mass filter.

40. 4T The method of claim 3940, wherein the multipole configuration comprises a quadrupole configuration.41.42 The method of claim 3940, wherein:the ion passageway has an axial length L;in the high-ion-energy mode the mass filter is operated at a radiofrequency RFH and the high-energy ions have an average axial velocity VH, based on which a high- energy oscillation number (NH) is defined as NH;in the low-ion-energy mode the mass filter is operated at a radiofrequency RFL and the low-energy ions have an average axial velocity VL, based on which a low- energy oscillation number (NL) is defined as NL; andthe NL is at least 1.5 times the NH.42.43 The method of claim 2223. wherein the high-ion-energy mode is utilized for a proteomics workflow.

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