System and method for single ion mass analysis using time information

Abstract

The present disclosure generally relates to mass spectrometers, including, but not limited to, mass spectrometers that can release ions at determinable times.In some embodiments, the time between the time that ions leave an ion source and the time that ions reach a detector can be determined with relatively high time resolution, which can be useful for certain applications, such as biopolymer sequencing.In addition, in some cases, a relatively large number of ions that leave an ion source, for example, at least 50% or more of the ions that are generated, can be determined by the detector.Other embodiments generally relate to systems and methods for using such mass spectrometers, techniques involving such mass spectrometers, and the like.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 179,046, entitled “System and Methods for Single-Ion Mass Spectrometry with Temporal Information,” by Stein et al., filed April 23, 2021, which is incorporated by reference in its entirety.

[0002] FIELD OF THE DISCLOSURE This disclosure relates generally to mass spectrometers, including but not limited to mass spectrometers that are capable of ejecting ions at determinable times. [Background technology]

[0003] Mass spectrometry may be suitable for protein sequencing because it is an analytical technique that can identify all 20 amino acids. Generally, mass spectrometers measure ions using a single ion detector and a mass filter that sweeps a narrow mass transmission window in time. For example, a quadrupole mass filter can only pass ions within a narrow m / z range, and the specific m / z range is controlled by a time-varying voltage applied to the four poles of the quadrupole. As the allowed m / z range is swept, the ion transmission rate is measured by the detector, and after at least one sweep, a mass spectrum is determined. Ions with m / z outside the transmission window do not pass through the filter. Therefore, a particular ion exiting the ion source is detected and identified only if it happens to pass through the filter when the window is centered on the ion's m / z. This cannot be guaranteed unless the order of the ion's m / z values ​​is known in advance. Therefore, such a system cannot be used for sequencing because it is not known when and in what order the ions will exit the ion source. Therefore, mass spectrometers could not be used for single protein sequencing. Summary of the Invention

[0004] The present disclosure relates generally to mass spectrometers, including, but not limited to, mass spectrometers capable of releasing ions at a determinable time. The subject matter of the present disclosure includes, in some cases, interrelated products, alternative solutions to a particular problem, and / or a plurality of different uses of one or more systems and / or articles.

[0005] One aspect generally relates to a mass spectrometer. In one set of embodiments, the mass spectrometer comprises an ion source including a capillary and an electrode proximate to the capillary, a magnetic mass filter downstream of the ion source, and an array of detectors downstream of the magnetic mass filter. In some cases, the capillary includes an opening having a cross-sectional dimension of less than 125 nm.

[0006] In another set of embodiments, a mass spectrometer comprises an ion source constructed and arranged to generate single ions or ion clusters, a magnetic mass filter positioned to accept the single ions or ion clusters from the ion source, a pump capable of generating a pressure of less than 100 mPa in an environment positioned between the ion source and the magnetic mass filter, and an array of detectors positioned to accept the single ions or ion clusters from the magnetic mass filter.

[0007] In yet another set of embodiments, a mass spectrometer comprises an ion source, a magnetic mass filter downstream of the ion source, and an array of detectors downstream of the magnetic mass filter.

[0008] Another aspect relates generally to a method for sequencing a biopolymer. According to one set of embodiments, the method includes ionizing a biopolymer contained within a fluid into ions or ion clusters, passing the ions or ion clusters through a magnetic mass filter, directing the ions or ion clusters to an array of detectors, and determining the ions or ion clusters with the array of detectors, thereby determining a sequence of the biopolymer.

[0009] In another set of embodiments, a method includes placing a fluid containing a biopolymer into a capillary tube defining an opening, applying an electric field to ionize the biopolymer proximate to the opening to generate ions or ion clusters, placing the ions or ion clusters directly into an environment having a pressure of 100 mPa or less, passing the ions or ion clusters through a magnetic mass filter, directing the ions or ion clusters to an array of detectors, and determining a sequence of the biopolymer by determining the ions or ion clusters using the array of detectors.

[0010] Yet another aspect relates generally to a method for determining a concentration. In one set of embodiments, the method includes ionizing molecules from a fluid as ions or ion clusters, passing the ions or ion clusters through a magnetic mass filter, directing the ions or ion clusters to an array of detectors, and determining the ions or ion clusters with the array of detectors, thereby determining the concentration of the molecules in the fluid.

[0011] Yet another aspect relates to a method that includes ionizing molecules from a fluid as ions or ion clusters, passing at least 50% of the ions or ion clusters through a magnetic mass filter, and directing the ions or ion clusters to a detector.

[0012] Another aspect relates to a method that includes ionizing molecules from a fluid as ions or ion clusters using an ion source, passing the ions or ion clusters through a mass filter, directing the ions or ion clusters to a detector, and determining a duration between the time the ions or ion clusters leave the ion source and the time the ions or ion clusters reach the detector.

[0013] Yet another embodiment includes ionizing molecules using an ion source to generate an array of ions or ion clusters, passing the array of ions or ion clusters through a mass filter, and directing the array of ions or ion clusters to an array of detectors. In some cases, at least 50% of the ions or ion clusters that reach the array of detectors arrive in the array. Additionally, in some cases, at least 90% of the ions or ion clusters that reach the array of detectors arrive in the array.

[0014] Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments of the present disclosure when considered in conjunction with the accompanying drawings.

[0015] Non-limiting embodiments of the present disclosure are described by way of example with reference to the accompanying drawings. These drawings are schematic and are not intended to be drawn to scale. In the drawings, each identical or nearly identical component shown is usually represented by a single numeral. For clarity, not every component is labeled in every drawing, and not every component of each embodiment of the present disclosure is shown if not necessary to enable a person skilled in the art to understand the present disclosure. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 illustrates a nanopore mass spectrometer according to one embodiment. [Diagram 2] 13A and 13B show mass spectra of positive amino acid ions delivered directly from a nanopore ion source into high vacuum in another embodiment. [Diagram 3] FIG. 2 is a schematic diagram of a mass spectrometer according to yet another embodiment. [Figure 4-1] FIG. 1A illustrates the operation of a nanopore mass spectrometer according to one embodiment. [Figure 4-2] 1B and 1C are diagrams illustrating the operation of a nanopore mass spectrometer according to one embodiment. [Figure 4-3]4D is a diagram illustrating the operation of a nanopore mass spectrometer according to an embodiment. [Figure 5-1] FIG. 2A shows a mass spectrum of a biomolecule according to some embodiments. [Figure 5-2] FIG. 2B shows a mass spectrum of a biomolecule according to some embodiments. [Figure 5-3] FIG. 2C shows a mass spectrum of a biomolecule according to some embodiments. [Figure 6-1] FIG. 1A is a diagram showing the generation or emission of ions from an ion source in yet another embodiment. [Figure 6-2] 1B and 1C are diagrams illustrating the generation or ejection of ions from an ion source in yet another embodiment. [Figure 6-3] 13D and 13E show the generation or ejection of ions from an ion source in yet another embodiment. [Figure 7] FIG. 13 illustrates a magnetic mass filter according to yet another embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present disclosure generally relates to mass spectrometers, including, but not limited to, mass spectrometers that can release ions at determinable times.In some embodiments, the time between the time that ions leave an ion source and the time that ions reach a detector can be determined with relatively high time resolution, which can be useful for certain applications, such as biopolymer sequencing.In addition, in some cases, a relatively large number of ions that leave an ion source, for example, at least 50% or more of the ions that are generated, can be determined by the detector.Other embodiments generally relate to systems and methods for using such mass spectrometers, techniques involving such mass spectrometers, and the like.

[0018] Conventional mass spectrometers typically measure ions using a single ion detector and a mass filter that sweeps a narrow mass transmission window in time. For example, when an allowed mass-to-charge ratio range is swept, only ions having mass-to-charge ratios within the allowed range can be measured. Thus, conventional mass spectrometers can only determine the mass-to-charge ratios of a small portion of the ejected ions. Furthermore, the capabilities of conventional mass spectrometers can be quite limited. For example, conventional mass spectrometers cannot be used to provide significant information regarding ion ordering, ion association, and / or ion sequence. Thus, certain aspects of the present disclosure relate to mass spectrometers that can be used to provide such information.

[0019] In one example, a mass spectrometer comprises a particular combination of components and / or configurations that give the spectrometer high detection and measurement capabilities. For example, a mass spectrometer comprises an ion source that can ionize molecules into single ions, a magnetic mass filter that can classify ions based on mass-to-charge ratio, and an array of detectors that can determine single ions. The combination of these features can advantageously enable the determination of the sequence, structure, and / or identity of a species of interest, such as a biopolymer. For example, mass spectrometers can be used for protein or nucleic acid sequencing. Compared to conventional mass spectrometers, the mass spectrometers described herein can advantageously have high time resolution (e.g., less than 1 microsecond) and high total ion transmission efficiency (e.g., about 0.8 or higher).

[0020] One non-limiting example of a mass spectrometer is shown in FIG. 1. In this example, ions (or ion clusters) are generated from an ion source, which in some cases can generate ions at a determinable time. The ion source can include a capillary tip that can allow for direct ion evaporation of the sample by an applied electric field. In some cases, the tip can have an opening with a cross section of less than 100 nm. Examples of such systems can be found in PCT application entitled "Nanotip Ion Sources and Methods," filed on even date herewith, and U.S. Patent Application No. 63 / 015,407, entitled "Nanotip Ion Sources and Methods," filed on April 24, 2020, each of which is incorporated herein by reference in its entirety. A fluid enters the ion source through a fluid inlet, and molecules in the fluid are converted to ions or ion clusters in the ion source. In addition, ions (or ion clusters) exiting the ion source can optionally pass through, for example, an ion lens or other suitable ion optics to focus the ions, and enter a relatively low pressure environment, for example a chamber having an (absolute) pressure of less than 100 mPa (a "vacuum" chamber). In some embodiments, single ions or ion clusters from the ion source can be ejected directly into the vacuum or low pressure environment. In some embodiments, the mass spectrometer includes a pump that is used to create such a vacuum or low pressure environment.

[0021] A mass filter can then be used to sort the ions, for example based on mass or mass-to-charge ratio. In some embodiments, a mass filter (e.g., a magnetic mass filter) can be downstream of the ion source. The mass filter can be positioned to accept single ions or ion clusters from the ion source. One non-limiting example is a magnetic sector mass filter. In some cases, ions or ion clusters with different mass-to-charge ratios can be deflected to different degrees by the magnetic field generated by the magnetic mass filter. Thus, ions with different masses or mass-to-charge ratios can have different trajectories when they exit the magnetic mass filter.

[0022] As the ions or ion filter exit the magnetic mass filter, they may be directed, for example, to a detector array positioned downstream of the mass filter (e.g., magnetic mass filter). The array of detectors may be arranged and configured to detect single ions or ion clusters displaced from the mass filter. Because ions may not necessarily have the same trajectory, an array of detectors positioned to receive ions with different trajectories may be used. Each detector may detect ions with a certain mass or mass-to-charge ratio, for example, versus time, and the position of the detector is related to the incident mass or mass-to-charge ratio of the ions arriving at the detector. Thus, because the time at which the ions or ion clusters exit the ion source is known and there are no substantial collisions (e.g., with air molecules) between the ion source and the detector array, the travel time between the ion source and the detector array may be determined with a relatively high time resolution, for example, within a few microseconds or less. In contrast, mass spectrometry systems that include air and / or time-varying voltages cannot achieve such time resolution or perform sequencing.

[0023] Thus, in some embodiments, the mass spectrometers described herein can have a relatively high time resolution, hi some embodiments, the mass spectrometer has a time resolution of 1 microsecond or less (500 nanoseconds or less, 250 nanoseconds or less, 100 nanoseconds or less, 50 nanoseconds or less, 10 nanoseconds or less, etc.).

[0024] In some cases, information about the travel time of the ions can be used for certain types of sequencing, where the monomers that form a polymer, such as, for example, amino acids in a protein or nucleotides in a nucleic acid, can be sequentially ionized and transferred to a detector, and such information is used to reconstruct or "sequence" the original polymer. Figure 3 shows another non-limiting embodiment of a mass spectrometer, for example, for sequencing a polymer, such as a protein in this example. As shown, the mass spectrometer includes an ion source (e.g., having a nanopore) that can emit single ions and ion clusters into a vacuum chamber, an electrode in close proximity to the ion source, a magnetic mass filter downstream of the ion source, and an array of detectors (e.g., single ion detectors) downstream of the magnetic mass filter.

[0025] Additionally, in some embodiments, a relatively large number of ions or ion clusters generated by the ion source can pass through a magnetic mass filter to a detector, for example, due to the relatively low pressure present in the mass spectrometer. In some cases, at least 50% or more of the generated ions can reach one of the detectors. This can also be useful, for example, to determine the concentration of certain ionizable molecules in a fluid.

[0026] Accordingly, certain aspects of the present disclosure relate to systems and methods related to mass spectrometers. In some embodiments, the mass spectrometer comprises an ion source configured and arranged to generate single ions or ion clusters. The ion clusters can include a single ion and some solvent molecules. As an example, the ion clusters can include ions with only one or two solvent molecules (e.g., water).

[0027] The ion source may be any of a variety of ion sources capable of, for example, ionizing a species of interest (e.g., a biopolymer) into single ions or ion clusters. In one set of embodiments, for example, an ion source is described herein that includes a capillary and an electrode in close proximity to the capillary. The capillary includes an opening having a cross-sectional dimension of less than 125 nm (e.g., less than 100 nm, less than 60 nm, etc.). In some embodiments, the electrode is used to apply an electric field to the fluid in the capillary such that molecules from the species of interest in the fluid can be ionized as single ions or ion clusters. Below, specific configurations and components of the ion source are disclosed in more detail.

[0028] For example, in some embodiments, the ion source can include a capillary and an electrode, which may be annular in some cases, between which a voltage is applied to generate ions. In some cases, the capillary can have a tip inner diameter of less than 125 nm or less than 100 nm. This allows ions to evaporate directly from the meniscus of the fluid in the capillary, avoiding the wasteful droplet evaporation process. In this manner, ion evaporation can dominate the ion current, and in some cases, this mode of emission can be achieved using solutions with relatively low salt concentrations. In some embodiments, a tip with an inner diameter of less than 125 nm or less than 100 nm can generate a high percentage of bare ions or ion clusters that contain only a small number of solvent molecules, e.g., one or two solvent molecules. In some cases, the small area of ​​the liquid-vacuum interface can prevent significant evaporation heat loss, and in some cases allows the use of volatile solvents such as water. In some embodiments, such methods can be used to analyze molecules or ions, e.g., biomolecules such as amino acids, nucleic acids, peptides, or proteins. In some cases, ion sources as described herein can improve the sensitivity of mass spectrometry experiments, enabling single molecule protein sequencing or single cell proteomic analysis. Other applications are possible, as described below.

[0029] For example, some embodiments generally relate to an ion source that includes a capillary and an electrode. Using the electrode, ionized molecules can be generated directly from the fluid in the capillary into a low pressure environment, for example, at a pressure of 100 mPa or other pressures described herein, or into a vacuum. In some embodiments, the opening of the capillary is sized such that when an electric field is applied, the fluid in the capillary forms a charged meniscus, and species in the fluid exit the charged meniscus, for example, primarily via ion evaporation. The use of a capillary with a submicron opening (e.g., less than 100 nm) can be advantageous for ionization of the fluid via ion evaporation, where species exiting the capillary directly ionize into single charged ions or clusters of charged ions. This ionization is in contrast to electrospray ionization, where species exiting the capillary exit through a liquid jet, which breaks into charged droplets, and the charged droplets further break into charged ions in the presence of a background gas. However, it should be understood that in some cases, some electrospray ionization may still be performed. Ion evaporation may be preferred in certain applications, for example, that require the efficient use or generation of single ions from a fluid, for example, certain embodiments relate to ion sources in mass spectrometry that directly generate and can then detect singly charged ions.

[0030] According to one set of embodiments, the ion source includes a capillary tube defining an opening having a cross-sectional dimension (e.g., the inner diameter of the capillary tube) of less than 100 nm. In some cases, the opening may be sized such that ion evaporation dominates over liquid jet formation when an electric field is applied. For example, in certain embodiments, at least 50% of the emitting species can exit via ion evaporation or in the form of ions or ion clusters. For example, nanoscale capillaries allow ions to evaporate directly from the fluid meniscus. In some embodiments, a fluid can enter a capillary tube with such an opening and be delivered directly to a low pressure or vacuum environment (e.g., having a pressure of 100 mPa or less) in the form of ions and ion clusters. The ions and ion clusters can be analyzed by a mass filter and ion detector of a mass spectrometer or applied to other applications as described herein.

[0031] It should be understood that other types of ion sources may be used, for example, the ion source may include a pulsed laser capable of ionizing molecules from a species of interest as single ions or ion clusters.

[0032] Certain embodiments include ionizing molecules contained within the fluid as ions or ion clusters. In some embodiments, the molecules may be ionized into single ions or ion clusters (i.e., single ions clustered with solvent molecules). In some embodiments, the molecules may be ionized as single ions with few, if any, ion clusters.

[0033] In one set of embodiments, the molecules contained within the fluid may be molecules from a polymer or biopolymer (e.g., proteins, polypeptides, nucleic acids, etc.). In another set of embodiments, the molecules may be small molecules contained within the fluid (e.g., monomers, biomonomers, salt ions, etc.) that can be ionized from the fluid.

[0034] Molecules can be ionized into single ions or ion clusters by any suitable ion source. In one set of embodiments, molecules can be ionized from a fluid using an ion source described herein. For example, the ion source can include a capillary with a relatively small opening. In some embodiments, a fluid containing a species of interest (e.g., a biopolymer) can be placed into the opening of the capillary. By applying an electric field to the fluid in proximity to the opening, molecules within the species of interest (e.g., a biopolymer) can be ionized to produce single ions or ion clusters. Specific embodiments related to such ion sources are described below.

[0035] Although the above embodiments have described an ion source that includes a capillary tube, it should be understood that any type of ion source can be used in the mass spectrometer, so long as the ion source is capable of producing single ions or ion clusters. For example, in one set of embodiments, molecules can be ionized into single ions or ion clusters by a pulsed laser.

[0036] In some embodiments, polymers such as biopolymers contained in a fluid can be ionized into ions or ion clusters. Examples of biopolymers include, but are not limited to, proteins, peptides, nucleic acids such as DNA or RNA, carbohydrates, polysaccharides, etc. These can be ionized into monomeric components such as amino acids, nucleotides, sugar units, or monosaccharides. In some embodiments, single ions or ion clusters can be released sequentially from the biopolymer upon ionization. For example, in some cases, a single ion can be released from the biopolymer at a time. The ability to ionize a biopolymer into single ions or ion clusters and release them sequentially can advantageously reveal spatial and / or temporal information regarding the order or sequence of ions in a molecule. For example, a biopolymer can be ionized into a sequence of ions or ion clusters (e.g., ionized monomers) that correspond to the sequence of base components (e.g., monomers) associated with the biopolymer prior to ionization.

[0037] 3 shows a non-limiting example of such an embodiment. As shown, an ion source (e.g., a nanopore) can be used to ionize a biopolymer (e.g., a protein) into single ions (e.g., amino acids) or ion clusters (e.g., amino acids with solvent molecules). The single ions can be sequentially ejected from the biopolymer as an array of ions or ion clusters. As shown, the sequence of ejected ions can correspond to the sequence of ions in the biopolymer prior to ionization.

[0038] In addition, ions or ion clusters can be generated by the ion source at any of a variety of rates. In some cases, it is advantageous to be able to generate ions or ion clusters at a relatively high rate. In some embodiments, ions or ion clusters are generated at a rate of 1 or more (ions or ion clusters) per microsecond, 5 or more (ions or ion clusters) per microsecond, 10 or more (ions or ion clusters) per microsecond, 25 or more (ions or ion clusters) per microsecond, 50 or more (ions or ion clusters) per microsecond, or 75 or more (ions or ion clusters) per microsecond. In some embodiments, ions or ion clusters are generated at a rate of 100 or less (ions or ion clusters) per microsecond, 75 or less (ions or ion clusters) per microsecond, 50 or less (ions or ion clusters) per microsecond, 25 or less (ions or ion clusters) per microsecond, 10 or less (ions or ion clusters) per microsecond, or 5 or less (ions or ion clusters) per microsecond. Combinations of the above ranges are possible (e.g., 1 or more (ions or ion clusters) per microsecond and 100 or less (ions or ion clusters) per microsecond). Other ranges are possible.

[0039] In some cases, the relatively fast ionization may be useful for sequencing polymers, such as biopolymers. In some embodiments, for example, the biopolymer is a protein, e.g., comprising a sequence of amino acids. In some embodiments, the protein may be ionized (i.e., generated) at any suitable rate in one or more ranges above, e.g., to generate amino acids (or portions thereof) that may be analyzed as described herein to determine the sequence of the protein. As another example, in some embodiments, the biopolymer is a nucleic acid, such as DNA, RNA, etc. In some embodiments, the nucleic acid may be ionized (i.e., generated) at any suitable rate in one or more ranges above, e.g., to generate nucleotides or other nucleic acid fragments that may be analyzed as described herein to determine the sequence of the nucleic acid. For example, the amino acids may be ionized at a rate of at least 1 base per microsecond (at least a base per microsecond, at least 100 bases per microsecond, etc.).

[0040] In some aspects, the ions or ion clusters are released directly into an environment having a relatively low pressure (e.g., a vacuum chamber) after exiting the ion source. The environment can have any of a variety of pressures or configurations, as described in more detail below. For example, in some cases, the environment can be an environment having a pressure of 100 mPa or less (e.g., 10 mPa or less, 1 mPa or less, 0.1 mPa or less, etc.). In some embodiments, the ions or ion clusters from the fluid enter the vacuum environment directly, for example, from the ion source. In addition, it should be understood that the vacuum environment does not have to be a perfect vacuum.

[0041] In some aspects, the ejected ions or ion clusters may pass through a mass filter, such as those described herein, contained within a relatively low pressure environment, for example. Additionally, in some embodiments, the ejected ions may optionally pass through ion optics, as described below, before passing through the mass filter.

[0042] In some embodiments, the mass filter is a magnetic mass filter. In some embodiments, the magnetic mass filter separates ions or ion clusters by mass-to-charge ratio by applying a magnetic field. The magnetic mass filter may be capable of directing (e.g., bending) incident ions or ion clusters in different directions (e.g., angles) according to their mass-to-charge ratio. For example, as shown in FIG. 3, incident ions and ion clusters having different mass-to-charge ratios may be directed or bent in different directions under the magnetic field generated by the magnetic mass filter. In addition, in some embodiments, a relatively large percentage of the emitted ions or ion clusters may pass through the magnetic mass filter. For example, at least 50% (e.g., at least 70%, at least 80%, at least 90%, at least 95%, or all) of the emitted ions or ion clusters may pass through the magnetic mass filter. Systems and methods for determining a relatively high percentage of ions generated by an ion source are described in more detail herein.

[0043] A variety of mass filters can be used, non-limiting examples of which include, but are not limited to, quadrupole mass filters, magnetic sector mass filters, and the like.

[0044] For example, as previously discussed, in some embodiments the mass filter may include a magnetic mass filter. In some embodiments the magnetic mass filter includes a magnet and a yoke associated with (e.g., housing) the magnet. The magnetic mass filter may include a magnet formed from any of a variety of magnetic materials including, but not limited to, rare earth elements, magnetic metal elements, magnetic composite materials (e.g., ferrite), and the like. In one set of embodiments the magnet is a permanent magnet including neodymium. In one set of embodiments the yoke includes iron.

[0045] In some embodiments, a magnetic field passes through an aperture in a magnetic mass filter, and incident ions and ion clusters pass through the magnetic mass filter, where the incident ions and ion clusters are deflected to different degrees by the magnetic field generated by the magnetic mass filter. For example, as shown in Figure 7, a magnetic mass filter has a central aperture within which a magnetic field exists in an axial direction. As the ions and ion clusters pass through the central aperture, they are deflected by the magnetic field to different degrees, for example according to their mass or mass-to-charge ratio.

[0046] The openings of the magnetic mass filter can have any of a variety of sizes and shapes. In some cases, the openings can have shapes such as cylindrical, square, rectangular, etc. The openings can have a first cross-sectional dimension (e.g., diameter, width, length) and a second cross-sectional dimension (e.g., height). In one set of embodiments, the openings have a first cross-sectional dimension (e.g., diameter) that is greater than the second cross-sectional dimension (e.g., height). In some embodiments, the openings can have a first dimension (e.g., diameter) of at least 4 cm (e.g., at least 5 cm, at least 6 cm, at least 8 cm, at least 10 cm, etc.). In some embodiments, the openings can have a second dimension (e.g., height) of at least 1 cm (e.g., at least 2 cm, at least 3 cm, at least 4 cm, at least 5 cm, etc.). In some embodiments, the openings can have a ratio of the first dimension to the second dimension of at least 2 (e.g., at least 3, at least 4, at least 5, at least 6, at least 8, at least 10, etc.).

[0047] The magnetic mass filter can generate a magnetic field having any of a variety of field strengths, hi some embodiments, the magnetic field strength can be at least about 0.1 T, at least 0.2 T, at least 0.3 T, at least about 0.5 T, at least about 0.7 T, at least about 1 T, at least about 5 T, etc.

[0048] In some embodiments, the ions and ion clusters may optionally pass through an ion bender after exiting the mass filter. The ion bender may be configured to deflect the ions and ion clusters exiting the mass filter towards a detector. For example, by way of non-limiting example, the ions or ion clusters pass from the ion bender to a detector. In some embodiments, the detector may be used to determine the ions or ion clusters.

[0049] In some embodiments, ions or ion clusters passing through a mass filter (e.g., a magnetic mass filter) can be directed to one or more detectors, such as an array of detectors (e.g., single ion detectors) as described herein. For example, as shown in FIG. 3, the magnetic mass filter bends the ions or ion clusters toward the array of detectors.

[0050] In some embodiments, ions or ion clusters having a particular mass or mass-to-charge ratio can be directed to a corresponding detector in an array of detectors, for example, based on the amount of deflection that occurs when the ions or ion clusters pass through a mass filter. Ions or ion clusters with a high charge can be deflected more than ions or ion clusters with a low charge. Thus, one or more detectors can be positioned to receive ions or ion clusters with different amounts of deflection, which can then be used to determine the mass or mass-to-charge ratio of the incident ions or ion clusters. Thus, for example, an array of detectors can be positioned to determine the mass or mass-to-weight ratio of an ion or ion cluster based on the location at which various ions or ion clusters strike various detectors in the array. Additionally, in some cases, the array of detectors can include detectors capable of determining the arrival time of each ion or ion cluster.

[0051] In some embodiments, one or more detectors may be positioned further downstream of the mass filter. The detectors may include any suitable detector capable of detecting ions or ion clusters. When there is more than one detector, each detector may be independently the same or different. Examples of specific detectors include, but are not limited to, Faraday cups, electron multipliers, dynodes, charge-coupled devices (CCDs), CMOS sensors, fluorescent screens, and the like.

[0052] As previously mentioned, in some embodiments, the mass spectrometer includes a detector or an array of detectors. In some embodiments, the array of detectors includes channel electron multipliers (e.g., Channeltron® detectors) and / or dynodes. Additional non-limiting examples of detectors include imaging detectors such as microchannel plate (MCP) arrays, CCDs, and / or CMOS sensors. Two or more of these and / or other detectors may be present in the array.

[0053] The array of detectors can include any number of detectors for determining ions and / or ion clusters. For example, in some embodiments, the array of detectors can include at least 2, at least 3, at least 5, at least 10, at least 20, at least 25, at least 30, at least 40, at least 50, etc. detectors. The detectors can be positioned to receive ions or ion clusters that are deflected by passing through a mass filter (e.g., a magnetic mass filter). For example, ions or ion clusters can be deflected at various angles, and the detectors form an array positioned to receive such ions or ion clusters that are expected to be deflected at different angles. Thus, any suitable number of detectors can be used to determine the mass and / or mass-to-charge ratio of individual ejected ions or ion clusters. In some embodiments, the number of detectors can be related to the number of base components (e.g., monomers of amino acids, nucleotides, etc.) present in the biopolymer.

[0054] In some embodiments, a system as described herein may enable a mass spectrometer with relatively high ion transmission efficiency, for example, where ions generated in an ion source are determined using one or more detectors, for example, in a detector array. Without being bound by any theory, it is believed that due to the absence of air molecules in the mass spectrometer (e.g., due to a relatively low pressure environment) and / or the use of a mass filter that does not lose ions or ion clusters (e.g., as would a mass filter that sweeps through a narrow mass transmission window), many or even most of the ions generated by the ion source can be efficiently directed (e.g., using ion optics, magnetic mass filters, etc.) to the detector, resulting in a surprisingly low loss rate. Thus, in some embodiments, a mass spectrometer as described herein can have a total ion transmission efficiency (e.g., the ratio of ions and ion clusters detected to ions and ion clusters exiting the fluid at the opening of the capillary) of greater than 0.01, and in some cases the transmission is at least 0.02, at least 0.03, at least 0.05, at least 0.1, at least 0.15, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.75, at least 0.8, etc. In some cases, the total ion transmission is 1 or less, 0.9 or less, 0.8 or less, 0.75 or less, 0.7 or less, 0.6 or less, 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, 0.15 or less, 0.1 or less, 0.05 or less, or 0.02 or less. Combinations of the above ranges are possible (e.g., at least 0.02 and 0.9 or less, or at least 0.1 and 0.8 or less). Other ranges are possible. Total ion transmission can be measured by determining the ratio of the current detected by the detector to the current emitted from the ion source.

[0055] Thus, in some aspects, a system and method is described herein for determining molecular concentration based on such relatively high ion transmission efficiency. The molecule may be any molecule that can be ionized, for example, using the ion source described herein. In one set of embodiments, the molecule is dissolved in a fluid. As previously described, the molecule can be ionized from the fluid as ions or ion clusters, and the ions or ion clusters can then be passed through a magnetic mass filter and directed to an array of detectors as described herein. Examples of such molecules include, but are not limited to, monomers or biomonomers (amino acids, nucleotides, etc.) as described herein. In addition, it should be understood that molecules detected in such a manner may be ionized to form two or more ions or ion clusters, such that, for example, the concentration of the ions or ion clusters can be used to determine the concentration of the starting molecule.

[0056] Additionally, in one set of embodiments, a fluid can contain one or more types of molecules. In some embodiments, by determining the identity and / or arrival time of one or more types of molecules, the relative amount (e.g., concentration) of one or more types of molecules can also be determined.

[0057] In some embodiments, ions or ion clusters that reach the detector can be detected with relatively high time resolution. As mentioned above, the time that ions or ion clusters are generated in the ion source can be determined with relatively high resolution, and such ions or ion clusters can be directed to an array of detectors, such as those described herein, without substantial obstacles such as air molecules, narrow mass transmission windows, etc., that may make it difficult to determine the timing and / or path of ions or ion clusters traveling from the ion source to the detector. Thus, in some cases, such ions and / or ion clusters can be determined with a time resolution of, for example, greater than 100 nanoseconds (e.g., greater than 75 nanoseconds, greater than 50 nanoseconds, greater than 25 nanoseconds, greater than 10 nanoseconds). In one set of embodiments, the time resolution can be determined between two (or more) ions or ion clusters that strike different detectors of the array.

[0058] Thus, in some embodiments, methods for determining duration are described herein. For example, the duration between the time an ion or ion cluster leaves the ion source and the time the ion or ion cluster reaches the detector can be determined. In some embodiments, the duration can be determined by monitoring the time the ion or ion cluster reaches the detector and the time the ion is ejected from the ion source. Additionally, in some embodiments, the detector has an ion detection rate that is equal to or greater than the rate at which the ions or ion clusters are generated (i.e., ejected) by the ion source. In some cases, the detector may be capable of detecting each of the ejected ions or ion clusters that reach the detector.

[0059] In some embodiments, the duration between the time that an ion or ion cluster leaves the ion source and the time that an ion or ion cluster reaches the detector is 10 microseconds or more, 25 microseconds or more, 50 microseconds or more, 75 microseconds or more, and 100 microseconds or more. In some embodiments, it is 100 microseconds or less, 75 microseconds or less, 50 microseconds or less, 25 microseconds or less, and 10 microseconds or less. Combinations of the above ranges are possible (e.g., 10 microseconds or more and 100 microseconds or less). Other ranges are also possible.

[0060] The duration between the time an ion or ion cluster leaves the ion source and the time the ion or ion cluster reaches the detector can be determined with a relatively high time resolution, for example, the duration can be determined with a time resolution of greater than 1 microsecond, greater than 500 nanoseconds, greater than 250 nanoseconds, greater than 100 nanoseconds, greater than 50 nanoseconds, greater than 10 nanoseconds, greater than 5 nanoseconds, etc.

[0061] Certain aspects relate to sequencing polymers, such as biopolymers, using an instrument that includes an ion source, e.g., a mass spectrometer as described herein. For example, in some embodiments, the polymer may be the species of interest. The species of interest may be a biopolymer, e.g., a protein or peptide (including amino acids), or a nucleic acid sequence (e.g., DNA, RNA, etc.). In some cases, other types of biopolymers, such as carbohydrates or polysaccharides, may be used as the species of interest. In addition, it should be understood that in some cases, other types of polymers, such as, e.g., artificial or synthetic polymers, may also be sequenced. Furthermore, the structure of species of interest that are not polymers may be determined as well.

[0062] In some cases, for example, the structure, sequence, and / or identity of a species of interest (e.g., a polymer) can be determined by determining the ionized fragments using a detector. For example, the sequence of a species of interest can be detected by monitoring the time that individual ionized fragments (e.g., ions or ion clusters) resulting from ionizing a polymer and generating ions or ion clusters as described above arrive at a detector. Without being bound by any theory, it is believed that a species of interest, such as a polymer, can be ionized in a substantially linear fashion, for example, due to the size of the capillary opening, and the generated ions or ion clusters can then be determined by a detector as described herein, for example, in the order in which the ions or ion clusters are generated from the species of interest. In some embodiments, the capillary includes a carbon nanotube or a boron nitride nanotube, and the cross-sectional dimension (e.g., inner diameter) of the nanotube is sufficiently small, for example, 1 nm to 2 nm, so that the polymer molecules can be ionized in an order that reflects the primary structure of the polymer. Of course, larger diameters or other materials are possible, for example, in other embodiments described herein. It should be noted that in some cases, for example, when the ions or ion clusters enter a low pressure environment, the detector may be able to determine such ordering with relatively high fidelity, for example, due to the relative lack of collisions with gas molecules as the ions or ion clusters pass to the detector. Thus, based on the order in which the ions or ion clusters are determined, the structure or sequence of the species of interest can be determined.

[0063] In some embodiments, a method for sequencing a species of interest is described herein. In one set of embodiments, the sequence of a species of interest (e.g., a biopolymer) is determined by determining ions or ion clusters using an array of detectors. As previously described, the molecules of the species of interest (e.g., a biopolymer) can be ionized using an ion source to generate a sequence of ions or ion clusters (e.g., ions that include solvent molecules). In some cases, the ion source can generate mainly single ions, with few, if any, ion clusters.

[0064] In some embodiments, the sequence of ions or ion clusters (e.g., single ions) emitted from the ion source can substantially retain the sequence of the biopolymer before ionization. For example, at least 50% (at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, etc.) of the ions or ion clusters are emitted in sequence. Once the sequence of ions or ion clusters passes through a mass filter, such as a magnetic mass filter, the ions or ion clusters can be directed to an array of detectors. Thus, in some embodiments, the array of detectors can be used to determine the mass-to-charge ratio of the ions or ion clusters and / or the arrival time of the ions or ion clusters (e.g., time at detection).

[0065] According to some embodiments, the arrival time of an ion or ion cluster can advantageously provide information about the sequence of the ion or ion cluster at the time of detection. In some cases, a significant portion of the ions or ion clusters that reach the detector array (e.g., single ion detector) arrive in sequence, e.g., in the same sequence of the ions or ion clusters that are emitted from the ion source. For example, at least 50% (at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, etc.) of the ions or ion clusters that reach the detector array arrive in sequence. Thus, in some embodiments, the detector array can be used to determine the identity of the ion or ion cluster and / or the sequence of the ion or ion cluster. In some cases, the sequence of a species of interest (e.g., a biopolymer) can be determined based on the sequence of the ion or ion cluster.

[0066] Some aspects relate to mass spectrometers that include an ion source as described herein. In some cases, the mass spectrometer can include components such as a vacuum chamber (e.g., capable of generating any of the low pressures described herein), ion optics (e.g., one or more lenses such as an Einzel lens), mass filters (e.g., quadrupole mass filters, magnetic sector mass filters, etc.), detectors, ion benders, ion traps, etc. These and other components are described in more detail herein.

[0067] For example, in one set of embodiments, a mass spectrometer or other device described herein can include an ion source having a capillary tube as disclosed herein. The device can also have an electrode proximate to the capillary tube. For example, one embodiment relates to an ion source including a capillary tube defining an opening and an electrode positioned proximate to the opening. The capillary tube can have an opening at an end or tip of the capillary tube. The opening can have any of a variety of cross-sectional dimensions and can be any shape, e.g., circular, oval, square, etc. In some embodiments, the openings can have cross-sectional dimensions of less than 150 nm, less than 130 nm, less than 125 nm, less than 120 nm, less than 110 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 75 nm, less than 70 nm, less than 65 nm, less than 60 nm, less than 55 nm, less than 50 nm, less than 45 nm, less than 40 nm, less than 35 nm, less than 30 nm, less than 25 nm, less than 20 nm, less than 15 nm, less than 10 nm, less than 5 nm, less than 2 nm, etc. Additionally, in some cases, the openings can have cross-sectional dimensions of at least 1 nm, at least 5 nm, at least 10 nm, at least 15 nm, at least 20 nm, at least 25 nm, at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, etc. Combinations of these are also possible. For example, the openings can have cross-sectional dimensions of 50 nm to 100 nm. Although the above embodiments describe capillaries having an opening at the end or tip of the capillary, it should be understood that not all embodiments described herein are limited thereto, and in some embodiments, in addition or instead, the capillary may have multiple openings along the side of the capillary. Additionally, in some cases, the device may have one or more openings or openings, for example, of a channel or other structure. Thus, the opening does not have to be the opening of the capillary.

[0068] In some embodiments, the capillary is tapered at the opening. For example, the capillary can have a certain taper, such as, for example, the tip of the capillary is conical. Any suitable angle can be present. For example, the angle can be less than 15 degrees, less than 10 degrees, less than 9 degrees, less than 8 degrees, less than 7 degrees, less than 6 degrees, less than 5 degrees, less than 4 degrees, less than 3 degrees, less than 2 degrees, or less than 1 degree (0 degrees indicates no taper, i.e., the capillary is cylindrical). Additionally, in some cases, the angle of taper can be at least 1 degree, at least 3 degrees, at least 5 degrees, etc. in some cases. Combinations of these ranges are also possible, for example, the taper can be from 1 degree to 5 degrees.

[0069] In certain embodiments where the capillary is tapered at the opening, a laser pulling technique can be used to create the tapered opening. It should be understood that techniques other than laser pulling techniques may be used to create capillaries with tapered openings. It should also be understood that while the capillaries described herein have tapered openings, in other examples the opening of the capillary may not be tapered.

[0070] In certain embodiments, the capillary of the ion source comprises quartz. Additional examples of materials that can be used to fabricate the capillary include, but are not limited to, glass (e.g., borosilicate glass), plastic, metal, ceramic, semiconductor, carbon nanotubes, boron nitride nanotubes, and the like.

[0071] In some embodiments, the capillary has a relatively high aspect ratio, for example, the ratio between the length of the capillary and the cross-sectional dimension (e.g., diameter) of the opening of the capillary. For example, the capillary can have an aspect ratio of more than 10,000. However, it should be understood that the aspect ratio is not limited thereto. For example, in some cases, the aspect ratio between the length of the capillary and the cross-sectional dimension of the opening can be more than 10, more than 100, more than 1,000, more than 10,000, more than 100,000, or more than 1,000,000.

[0072] The capillaries can have a circular or non-circular cross section (e.g., square). In addition, in some embodiments, the capillaries can have a relatively small cross section, e.g., diameter. For example, the cross-sectional dimension of the capillaries can be less than 200 nm, less than 150 nm, less than 100 nm, less than 75 nm, less than 60 nm, less than 50 nm.

[0073] Some embodiments of the ion source also include an electrode positioned proximate the capillary, e.g., the opening of the capillary. The electrode can be used to apply an electric field (e.g., as described below) to the fluid in the capillary, e.g., to the meniscus. In some cases, the fluid in the capillary may be in contact with a counter electrode, e.g., such that a voltage difference between the electrode proximate the opening of the capillary and the counter electrode in the capillary can generate an electric field on the fluid. In some embodiments, the electrode can be positioned to generate a maximum electric field proximate the opening of the capillary. For example, in some embodiments, the electrode can be positioned within 50 mm, 40 mm, 30 mm, 20 mm, 15 mm, 10 mm, 5 mm, 3 mm, 2 mm, 1 mm, etc., of the opening of the capillary.

[0074] In some embodiments, electrodes may be positioned around the capillary or may be positioned in front of the capillary, for example in front of or downstream from the opening of the capillary.

[0075] The electrodes can have any suitable shape. In some cases, the electrodes are circular or circularly symmetric, or positioned symmetrically relative to the capillary. However, other shapes or configurations are possible.

[0076] In some embodiments, the electrode defines an opening (e.g., an aperture). Thus, in some cases, the electrode may be annular. The electrode may be positioned such that ions or ion clusters escaping from the fluid in the capillary pass through the central opening of the electrode. The central opening of the electrode may be any shape, including but not limited to a circular shape, which may be positioned annularly around the opening of the capillary. In some cases, the opening may be non-circular. In some embodiments, the opening of the electrode is positioned coaxially with respect to the opening of the capillary. That is, in some embodiments, the opening may be aligned with the opening of the capillary such that, for example, an imaginary line passing through the center of the cross section of the capillary passes through the central opening of the electrode. This may facilitate the application of an electric field to the fluid in the capillary, for example, to cause ions or ion clusters to exit the fluid, as described herein.

[0077] For example, in some embodiments, the electrode has a central opening with a cross-sectional dimension (e.g., inner diameter) larger than the cross-sectional dimension of the capillary opening, e.g., at the end or tip of the capillary. For example, according to some embodiments, the electrode has a central opening with a cross-sectional dimension (e.g., inner diameter) that is at least 5 times larger than the cross-sectional dimension of the capillary opening. However, it should be understood that the ratio of the cross-sectional dimension of the electrode central opening to the capillary opening is not limited. For example, in some cases, the cross-sectional dimension of the electrode central opening may be at least 2 times, at least 3 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 50 times, at least 75 times, or at least 100 times larger than the cross-sectional dimension of the capillary opening. In some cases, the electrode opening may have a cross-sectional dimension of less than 10 cm, less than 5 cm, less than 3 cm, less than 1 cm, less than 5 mm, less than 3 mm, less than 1 mm, etc. Additionally, in some embodiments, the front surface of the electrode is positioned in front of the capillary opening.

[0078] In addition, the electrode itself may be any shape (e.g., circular or non-circular). The electrode may be the same or different shape as its opening (if present). The electrode may have any suitable cross-sectional dimension. For example, the electrode may have a cross-sectional dimension of less than 10 cm, less than 5 cm, less than 3 cm, less than 1 cm, less than 5 mm, less than 3 mm, less than 1 mm, etc.

[0079] In some embodiments, the electrodes include steel. Other examples include copper, graphite, silver, aluminum, gold, conductive ceramics, and the like.

[0080] Thus, certain embodiments relate to electrodes capable of generating an electric field. In some cases, as described above, the electrodes can be positioned to generate a maximum electric field proximate the opening of the capillary. In some embodiments, when a fluid is contained in the capillary and an electric field is applied by an electrode proximate the opening of the capillary, molecules in the fluid can be ionized and exit the opening of the capillary as ions or ion clusters, e.g., as described herein. In some cases, for example, the electrodes and the capillary (e.g., the interior of the capillary) can be connectable to a voltage source, e.g., as described herein.

[0081] Thus, in some embodiments, a voltage source can be used in conjunction with electrodes to generate an electric field that causes ions or ion clusters to exit the fluid in the capillary, for example as described herein. In some embodiments, a voltage is applied to generate an electric field at least sufficient to ionize molecules in the fluid at the opening of the capillary, e.g., to generate ions or ion clusters. For example, in some embodiments, a voltage between 80V and 400V can be used to generate the electric field. In some cases, the voltage can be at least 40V, at least 60V, at least 80V, at least 100V, at least 120V, at least 140V, at least 160V, at least 180V, at least 200V, at least 220V, at least 240V, at least 260V, at least 280V, at least 300V, at least 320V, at least 340V, at least 360V, at least 380V, at least 400V, at least 450V, at least 500V, at least 600V, etc. Additionally, in some cases, the voltage may be 600V or less, 500V or less, 450V or less, 400V or less, 380V or less, 360V or less, 340V or less, 320V or less, 300V or less, 280V or less, 260V or less, 240V or less, 220V or less, 200V or less, 180V or less, 160V or less, 140V or less, 120V or less, 100V or less, 80V or less, 60V or less, etc. In some cases, combinations of these voltages are possible. For example, voltages such as 80V to 360V can be applied. The voltage may be applied as a constant voltage or, in some cases, as a varying or periodic voltage.

[0082] As previously discussed, a voltage can be applied to generate a maximum electric field adjacent to the opening of the capillary or the fluid within the capillary (e.g., at the meniscus of the opening). For example, a voltage can be applied to generate a maximum electric field of at least 0.5 V / nm, at least 0.7 V / nm, at least 1 V / nm, at least 1.1 V / nm, at least 1.3 V / nm, at least 1.5 V / nm, at least 2 V / nm, at least 2.5 V / nm, at least 3 V / nm, at least 3.5 V / nm, at least 4 V / nm, etc. In certain embodiments, the maximum electric field can be 5 V / nm or less, 4.5 V / nm or less, 4 V / nm or less, 3.5 V / nm or less, 3 V / nm or less, 2.5 V / nm or less, 2 V / nm or less, 1.5 V / nm or less, 1 V / nm or less. In some embodiments, combinations of these ranges are possible, for example, the electric field may be between 1.5 V / nm and 3.0 V / nm, between 1.5 V / nm and 4.0 V / nm, etc.

[0083] Without being bound by any theory, in certain embodiments, it is believed that when an electric field is applied, the fluid in the capillary forms a charged meniscus, and species exit the charged meniscus, e.g., as ions or ion clusters. In some cases, the capillary opening may be sized such that at least 10% of the exiting species exit via ion evaporation, e.g., as ions or ion clusters. In some cases, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, etc., of the exiting species exit via ion evaporation.

[0084] As previously mentioned, according to certain embodiments, a cone-shaped charged fluid meniscus can be induced at the opening of the capillary under an electric field, hi some embodiments, the cone-shaped fluid meniscus acts as a point source allowing species to exit as ions or ion clusters.

[0085] The fluid meniscus can generate exiting species by mechanisms such as charged droplets via electrospray ionization, and / or ions and ion clusters via ion evaporation. However, in the case of electrospray ionization, the exiting species exiting the liquid meniscus exits as charged droplets of the fluid containing the exiting species. This typically requires the presence of background gas to further break the droplets into individual ions via a Coulomb fission process. Ion evaporation, on the other hand, represents a process in which molecules are directly ionized into ions (e.g., bare ions) or ion clusters (e.g., ions containing solvent molecules) instead of charged droplets. An ion cluster can contain a single ion and multiple, usually a relatively small number of solvent molecules. For example, an ion cluster can contain 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less solvent molecules.

[0086] Thus, for example, in some embodiments, the capillary opening is sized such that the formation of charged droplets can be avoided and such that at least 50% of the exiting species ionize directly as ions or ion clusters from the conical fluid meniscus at the capillary opening (e.g., the cross-sectional dimension of the opening is less than 100 nm).

[0087] As discussed above, in some embodiments, capillaries having relatively small openings (e.g., cross-sectional dimensions less than 100 nm) can be associated with the generation of relatively few solvent molecules in ion clusters, for example as discussed above. In some embodiments, the openings of the capillaries can be sized such that the plurality of solvent molecules contains no more than a certain number of solvent molecules, e.g., less than 100 nm, such that, on average, the ion clusters generated by the ion source contain no more than 7, 6, 5, 4, 3, or 2 solvent molecules. In some embodiments, a significant number of the ion clusters (e.g., 50% or more, 60% or more, 70% or more, 80% or more, 80% or more, 90% or more, 95% or more, 99% or more, or all) contain one or two solvent molecules.

[0088] Additionally, as previously discussed, certain embodiments relate to methods of ionizing a fluid using an ion source to generate, for example, single ions or ion clusters, some of which include placing the fluid in a capillary defining an opening having a cross-sectional dimension of less than 100 nm, or other configuration as described herein.

[0089] In some embodiments, the fluid includes a sample and a solvent. The sample can include any species of interest that can be ionized from the opening of the capillary. For example, according to certain embodiments, the species of interest includes biopolymers (e.g., nucleic acids such as DNA or RNA, peptides or proteins, etc.). Other examples include other types of polymers, e.g., nylon, polyethylene, etc., or other species of interest that are not necessarily polymers, e.g., biomolecules. Non-limiting examples of biomolecules can include monomers such as amino acids, nucleotides, etc. In some cases, the species of interest is unknown and it is desirable to at least partially determine the structure of the species, e.g., by ionizing the species and detecting ion fragments, such as in mass spectrometry or other related techniques.

[0090] In some embodiments, the solvent may be any liquid that can be used to dissolve the sample or species of interest. For example, according to some embodiments, the solvent includes water. However, the solvent is not limited to water. In some cases, the solvent may be an aqueous solution having any of a variety of salt concentrations. In some embodiments, the aqueous solution may have a salt concentration of 10 mM or more, 20 mM or more, 30 mM or more, 50 mM or more, 100 mM or more, 150 mM or more, 200 mM or more, 300 mM or more, 400 mM or more, 500 mM or more, 750 mM or more, 1 M or more, 2 M or more, 5 M or more, or 7.5 M or more. In some embodiments, the aqueous solution can have a salt concentration of 10 M or less, 7.5 M or less, 5 M or less, 2 M or less, 1 M or less, 750 mM or less, 500 mM or less, 400 mM or less, 200 mM or less, 150 mM or less, 100 mM or less, 50 mM or less, 30 mM or less, 20 mM or less, 10 mM or less, etc. Combinations of the above ranges are possible (e.g., 100 mM or more and 10 M or less, or 150 mM or more and 1 M or less).

[0091] Additional examples of solvents that can be used include, but are not limited to, formamide, alcohols (e.g., ethanol, isopropanol, etc.), organic solvents (e.g., toluene, acetonitrile, acetone, hexane, etc.), ionic liquids, inorganic solvents (e.g., ammonia, sulfuryl chloride fluoride, liquid acids, and bases, etc.) In some cases, combinations of any of these and / or other solvents are also possible.

[0092] Additionally, in some embodiments, the fluid includes a solvent (e.g., water) having a relatively high volatility, for example, to facilitate the generation of ions or ion clusters. For example, in some cases, water having a boiling point of 100° C. is considered volatile. In some embodiments, a liquid having a boiling point close to room temperature can be used to facilitate the generation of ions or ion clusters. In some embodiments, a solvent that can be used to facilitate the generation of ions or ion clusters can have a boiling point of 100° C. or less, 80° C. or less, 60° C. or less, 40° C. or less, 20° C. or less, etc. Additionally, the solvent can have a boiling point of 10° C. or more, 30° C. or more, 50° C. or more, 70° C. or more, 90° C. or more, etc. Combinations of these are also possible, for example, the solvent can have a boiling point between 50° C. and 100° C. Additional examples of solvents having a relatively high volatility include, but are not limited to, acetone, isopropanol, hexane, etc.

[0093] In some embodiments, the temperature of the capillary (in addition to the type of fluid it contains) can be varied to control the number of solvent molecules in the resulting ion clusters. In some embodiments, the temperature of the capillary is set so that the plurality of solvent molecules contains no more than a certain number of solvent molecules, for example, so that on average, the ion clusters produced by the ion source contain no more than 7, 6, 5, 4, 3, or 2 solvent molecules. In some embodiments, the temperature is at least 20° C., at least 30° C., at least 40° C., at least 50° C., at least 60° C., or at least 70° C. In some embodiments, the temperature is no more than 80° C., no more than 70° C., no more than 60° C., no more than 50° C., no more than 40° C., no more than 30° C. Combinations of the above ranges are possible (e.g., no less than 20° C. and no more than 80° C.). In some cases, the temperature of the capillary is controlled by a resistive heater, a Peltier junction, an infrared heater, or the like.

[0094] In some embodiments, for example as described herein, an appropriate range of electric field and an appropriate range of capillary opening size can be selected so that at least a portion of the molecules exit as ions or ion clusters.

[0095] In certain embodiments, the ion sources described herein can be used with liquid chromatography mass spectrometry systems. For example, a liquid chromatograph can be coupled to the ion source to separate peptides or other molecules before ionizing and feeding them to a mass spectrometer. In some cases, such as in proteomics experiments, a mass spectrometer can be used to perform single or tandem (MS / MS) analysis to identify the ionized peptides or molecules. Advantageously, the ion sources described herein (having capillaries with nano-sized openings and / or tips) can be used to directly feed ions into a low pressure environment, improving instrument sensitivity, ion transmission efficiency of such systems, and eliminating the need for multiple pumping stages.

[0096] In other embodiments, the ion source described herein can be used as both a nanopipette and an ion source. For example, a capillary tube described herein (e.g., a drawn quartz capillary tube) with a nano-sized tip can be used to puncture a cell or tissue and extract the biomolecular contents. The capillary tube can then be inserted directly into a vacuum chamber, and the extracted molecules can be ionized and fed into a mass spectrometer. Such techniques can be used to sample relatively small liquid volumes, such as the contents of a single cell. For example, such techniques can be used for proteomic analysis of a single cell.

[0097] According to certain aspects, in addition to the ion source, various ion optics can be positioned downstream of the ion source to, in some cases, transport the exiting molecules (e.g., ions and ion clusters) along a path downstream of the ion source, i.e., the downstream direction is the direction in which the ions or ion clusters travel. In some embodiments, the ion optics include one or more Einzel lenses (e.g., a first Einzel lens and a second Einzel lens). Those skilled in the art will be familiar with the various ion optics used in mass spectrometry.

[0098] Certain aspects include directly placing ionized molecules from a fluid into a low pressure or vacuum environment. Without being bound by any theory, it is noted that techniques such as electrospray ionization typically require the presence of a background gas to further break droplets into individual ions via a Coulomb fission process. In contrast, according to certain embodiments, ions or ion clusters generated as described herein can directly enter such an environment without the need for significant amounts of background gas. Thus, certain techniques such as mass spectrometry can be performed using a low pressure or vacuum environment without necessarily needing to add background gas.

[0099] Thus, in one set of embodiments, the capillary can be positioned such that the ions or ion clusters can exit the opening and enter a low pressure or vacuum environment. In some cases, the environment can be an environment having a pressure of 100 mPa or less. In certain embodiments, the environment can have a pressure of 1000 mPa or less, 10 mPa or less, 1 mPa or less, 0.1 mPa or less, etc. In some embodiments, the ions or ion clusters from the fluid enter the vacuum environment directly.

[0100] It should be understood that some of the embodiments presented herein focus on placing ionized molecules from a fluid directly into an environment having a pressure of 100 mPa or less. However, it should be understood that the pressure in the environment is not limited to 100 mPa. In some embodiments, the pressure may be 100 mPa or more and 1 Pa or less.

[0101] As previously mentioned, in some embodiments, the mass spectrometer comprises a pump. For example, as described herein, a pump can be used to create a low pressure or vacuum environment. Non-limiting examples of pumps include diffusion pumps, molecular drag pumps, turbomolecular pumps, etc.

[0102] In some embodiments, there may be a relatively high pressure difference between the vacuum chamber and the fluid at the opening of the capillary. For example, the pressure may be about 1 atm where the fluid enters the capillary and about 100 mPa or other low pressure as described herein in the vacuum chamber where the opening of the capillary is located. However, in some cases as described herein, the fluid meniscus at the opening of the capillary may be relatively stable despite the relatively high pressure difference, for example, due to the surface tension of the fluid at the meniscus. For example, the pressure difference across the fluid meniscus at the opening of the capillary may be at least 0.1 atm, at least 0.2 atm, at least 0.3 atm, at least 0.4 atm, at least 0.5 atm, at least 0.6 atm, at least 0.7 atm, at least 0.8 atm, at least 0.9 atm, at least 1 atm, etc. Furthermore, in some embodiments, the hydraulic resistance of a fluid in a capillary as described herein (e.g., a capillary having an opening less than 100 nm) may be higher than the hydraulic resistance of an ion source used in electrospray ionization.

[0103] According to certain embodiments, the capillary opening is sized such that the solvent having a relatively high volatility remains unfrozen at the capillary opening when exposed to a relatively low pressure. In some embodiments, the capillary opening is small enough so that the solvent having a relatively high volatility remains unfrozen when it enters the surrounding environment. In some embodiments, the capillary opening is small enough so that the fluid including the sample and the solvent remains unfrozen when the species of interest ionize, and at least a portion of the species of interest ionize to form ions (e.g., single ions) or ion clusters.

[0104] U.S. Provisional Patent Application No. 63 / 015,407, by Stein et al., entitled "Nanotip Ion Sources and Methods," filed April 24, 2020, is hereby incorporated by reference in its entirety. Additionally, International Patent Application No. PCT / US2021 / 028954, by Stein et al., entitled "Nanotip Ion Sources and Methods," filed April 23, 2021, is hereby incorporated by reference in its entirety. Additionally, U.S. Provisional Patent Application No. 63 / 179,046, by Stein et al., entitled "System and Methods for Single-Ion Mass Spectrometry with Temporal Information," filed April 23, 2021, is hereby incorporated by reference in its entirety.

[0105] The following examples are intended to illustrate certain embodiments of the present disclosure, but do not exemplify the full scope of the disclosure. EXAMPLES

[0106] [Example 1] The following examples disclose certain systems and methods that allow for the measurement of samples (e.g., amino acids) directly released into a vacuum (i.e., low-pressure environment) via ion evaporation from the surface of a fluid, e.g., an aqueous solution in this example. The systems and methods in these examples were applicable to fluids with relatively low conductivity, e.g., corresponding to about 10 mM NaCl. In some embodiments, the methods also generated mostly bare ions or ion clusters with only one or two water molecules. One feature was related to the nanoscale size (<100 nm) of the ion source. The small size of the capillary tip created significant field enhancement and restricted the flow rate of the fluid such that ions from the sample (e.g., amino acids) exited directly into the vacuum via ion evaporation rather than from a sequence of droplet formation and Coulomb breakup. The small tip opening (sometimes called a "nanopore") also prevented significant amounts of solvent from evaporating into the vacuum chamber while avoiding freezing of the fluid, allowing the analysis of samples such as amino acids in volatile solvents such as water.

[0107] This example shows the various parts of the mass spectrometer instrument that was used to perform the experiments, according to one embodiment. The experiments described in the following examples were all performed on a custom-built instrument called a "nanopore mass spectrometer," shown diagrammatically in Figures 1A and 1B.

[0108] One component of the instrument used in this example is the ion source. The ion source includes a capillary with a tip internal diameter of less than 100 nm and a ring electrode located in front of the capillary tip in the system. The capillary was formed from quartz in this example, but in other embodiments the capillary may be formed from borosilicate glass, plastic, metal, ceramic, semiconductor, or other materials. The diameter of the capillary in this example gradually tapers in size closer to the tip such that if the tip were approximated to have a conical shape, the cone would have an opening angle of about 1 degree to 5 degrees. The length of the capillary was much longer than the width of the tip, giving the capillary a very high aspect ratio, typically greater than 10,000. Of course, as previously mentioned, other capillary shapes and / or dimensions may be used in other embodiments.

[0109] In this example, an electrode was used to induce an electric field at the opening at the tip of the capillary. This electric field was high enough to allow ions to exit directly from the meniscus of the fluid created at the opening at the tip of the capillary, at least in part by ion evaporation. The electrode was made of steel in this device, but may be made of another conductive material. The electrode in this example featured an opening through which ions could migrate, e.g., exit the meniscus of the fluid by ion evaporation. In this device, the electrode had the shape of a washer, i.e., a circular plate with a circular hole in the center, but other shapes may be used. In this device, the diameter of the central hole was about 1 cm, but the dimensions are not critical. For example, the diameter of the hole may be at least 10 times larger than the diameter of the capillary tip, or other dimensions as described herein. The outer diameter of the electrode was about 5 cm, but the dimensions are also not critical. The outer diameter of the electrode may be larger than the inner diameter of the hole. In this experiment, the front side of the electrode defined a plane and the tip of the capillary was located at a distance of about 1 mm to 5 mm behind the plane, with the axis of the capillary aligned with the axis of the electrode.

[0110] A voltage, typically in the range of 80V to 400V, was applied between the fluid and the electrode to cause the ions to exit the fluid. An Ag / AgCl wire in a capillary was used as the counter electrode. Two Einzel lenses were used to focus the exiting beam of ions through an aperture in the electrode. The ions were analyzed by the instrument's quadrupole mass filter, although different types of mass filters, e.g. a magnetic sector, may also be used.

[0111] [Example 2] This example shows a mass spectrometer capable of determining the mass-to-charge ratio (m / z) and precise moment of detection of ions emitted at any time from a nanopore ion source, according to an embodiment. Knowing the relative timing of ion detection allows for ion association, ion ordering, and determination of ion sequence. The instrument in this example exploits the ability to provide time information by combining a magnetic mass filter with an array of single ion detectors. This example shows that such an instrument can sense multiple different amino acids arriving simultaneously, with a time resolution of less than one microsecond.

[0112] Nanopore ion sources can deliver single amino acid ions directly from a solution of formamide or water into the high vacuum section of a mass spectrometer. See U.S. Provisional Patent Application No. 63 / 015,407, entitled "Nanotip Ion Sources and Methods," filed April 24, 2020, by Stein et al., and PCT application entitled "Nanotip Ion Sources and Methods," filed April 23, 2021, by Stein et al., both of which are fully incorporated by reference herein. For example, FIG. 2 shows mass spectra of 14 different amino acids delivered from an aqueous solution by a capillary tip with a diameter of less than 100 nm. It is noteworthy that the amino acid ions are primarily naked, as opposed to clustered by solvent molecules, which makes the data relatively easy to interpret. It is also noteworthy that these high-quality mass spectra were obtained using very low extraction voltages (~200 V) from very small ion emission currents (~10 pA).

[0113] Mass spectra of positive amino acid ions delivered directly from the nanopore ion source into high vacuum are shown in Figure 2. The amino acids were dissolved in aqueous solutions. In each case, the pH was adjusted using acetic acid to be below the isoelectric point of the amino acid in solution.

[0114] In this example, an instrument capable of determining single ions was designed and constructed. It is shown diagrammatically in FIG. 3. The instrument combines a nanopore ion source with a magnetic mass filter and an array of single ion detectors. The magnetic mass filter was constructed using a neodymium magnet and an iron yoke. The mass filter created a cylindrical region approximately 5 cm in diameter and 1 cm in height in which an axially oriented magnetic field existed. The magnetic field was approximately 0.6 T. Ions passed horizontally (perpendicular to the cylindrical axis) through the cylindrical region and experienced a magnetic force that fanned out the ions according to their m / z. The array of single ion detectors accepted the fanned ions and determined the m / z of each ion by the location of the collision.

[0115] The ion detector array can include a Channeltron® detector (e.g., an electron multiplier) and a dynode. The detector array can also include an imaging detector, such as a microchannel plate (MCP) array, a CCD, or a CMOS sensor. In this example, the instrument can utilize the time domain to establish the order in which the ions were ejected, in order to determine the mass of the ions based on where they strike the detector array. Thus, the instrument can determine the amino acid sequence by interpreting the order of the detector array pulses.

[0116] [Example 3] This example describes techniques for sequencing single proteins. Approaches for single protein sequencing based on fluorosequencing, nanopore, and tunneling spectroscopy are under development and are promising. However, only mass spectrometry (MS) has shown the ability to identify amino acids with minimal degeneracy. Existing MS ion sources have low ion transmission efficiency, disrupting the spatial ordering of ions. Presented herein is an ion source that includes a glass capillary with an orifice with a diameter of less than 100 nm, and ejects amino acid ions directly from an aqueous solution into high vacuum. Single ions travel collision-free trajectories before hitting a single ion detector. In this example, we measured unsolvated ions of 16 different amino acids and glutathione and two of its post-translationally modified variants. This example describes an approach for sequencing single proteins based on MS and a nanocapillary ion source.

[0117] Mass spectrometry has been a mainstay of proteomics research for decades, deriving its utility from its ability to distinguish amino acids by their mass and the availability of fragmentation techniques that allow for probing protein structure in tandem MS / MS measurements. The development of soft ionization techniques, particularly electrospray ionization (ESI), has been crucial to transfer peptide ions intact into the gas phase. However, ESI has low ion transfer efficiency, limiting the sensitivity of mass spectrometry, requiring millions to billions of copies of a protein to reach the detection limit of typical instruments.

[0118] As shown in Figure 4A, ESI delivers analytes to the mass spectrometer from a column of charged droplets that originates from an electrically induced liquid cone jet at the end of a capillary. Each droplet, carrying a large number of charges and analyte molecules, must undergo a series of evaporation and Coulomb explosion cycles before the ions become available for analysis in the gas phase. Ultimately, only a small fraction of the analyte molecules emerge as gas-phase ions, the majority of which collide with the walls of the transfer capillary before entering the low-pressure region where the mass filter and detector are housed. ESI typically transfers only 1 in 10 ions to the mass filter. Nano-electrospray ionization (nano-ESI) techniques increase the transmission efficiency to 0.1%-1% by using smaller capillaries (tip diameter of about 1 micrometer). Hydrodynamic ion focusing is another technique that significantly improves the transmission efficiency using an optimized aperture between the vacuum stages. However, even with transfer efficiencies approaching unity, all of the above techniques face another challenge toward sequencing. The use of atmospheric background gas to encourage ion desolvation creates an environment in which the mean free path of amino acids is less than 50 nm, and collisions rapidly disrupt the spatial ordering required for sequencing single proteins.

[0119] In this example, an ion source that directly releases amino acid ions into high vacuum is described (Figure 4B). At the core of the ion source is a drawn quartz capillary with a tip diameter of less than 100 nm. The smallness of the tip can affect the release in a variety of ways. First, the fluid flow rate can be about three orders of magnitude smaller than nanoESI and smaller than the minimum flow rate required for the formation of a stable cone-jet. The absence of a cone-jet can prevent the formation of charged droplets. In some cases, the surface tension of the water meniscus extending across the nanoscale opening can support many air pressures and maintain a stable liquid-vacuum interface. In addition, in some cases, the electric field is concentrated at a sharp conductive tip such as an electrolyte-filled nanocapillary. As a result, under certain conditions, an electric field of 1 V / nm or less can be achieved at the liquid meniscus. At that high electric field strength, ions escape the liquid at high speeds by a process of ion evaporation.

[0120] A schematic of the mass spectrometer described in this example is shown in Figure 4C. Ions were ejected from the ion source by applying a voltage between an Ag / AgCl electrode in the nanocapillary and a ring-shaped extraction electrode located approximately 5 mm in front of the nanocapillary tip. Ions were measured by a continuous dynode single ion detector after passing through focusing ion optics, a quadrupole mass filter (Extrel), and an ion bender. Background pressure in the instrument was typically 10 -6 Torr, and the mean free path of amino acids (>10 m) was more than an order of magnitude larger than the size of the instrument.

[0121] Aqueous solutions of 16 different amino acids were prepared at a concentration of 100 mM, with the exception of tryptophan, which was prepared at 50 mM due to its low solubility. The pH of each solution was lowered below the corresponding isoelectric point by adding acetic acid to generate positive amino acid ions. After pre-filling the nanocapillary with the amino acid solution, the nanocapillary was inserted into a vacuum chamber. An extraction voltage V of +260 V to +360 V was applied between the nanocapillary and the extraction electrode. e began emitting ion currents of several picoamps (Figure 4B). Typically, the onset was sudden and was accompanied by measuring ions hitting the instrument's detectors fast enough to collect unambiguous mass spectra within minutes to hours.

[0122] The mass spectrum of an arginine solution obtained using a nanocapillary ion source with a tip inner diameter of 41 nm is shown in FIG. 4D. Five peaks are clearly visible. The peak at 174 m / z corresponds to a singly charged arginine ion (Arg + ). The higher m / z peaks are all separated by 18 m / z, and this shift is induced by an additional water molecule. Thus, the other peaks correspond to the solvated state of arginine (Arg + (H2O) n ) where the solvation number n is 1 to 4.

[0123] Figure 4A is a schematic of conventional electrospray ionization showing the background gas stimulating the evaporation of solvent from the droplets and the transfer capillary where significant ion loss occurs. Figure 4B is a schematic of a nanocapillary ion source showing the liquid-filled nanocapillary tip, the extraction electrode, and the Ve applied between them. The inset shows an SEM image of the tip of a drawn quartz nanocapillary with an inner diameter of 30 nm. Figure 4C is a schematic of the mass spectrometer used in this analysis. Ion optics including the extraction electrode and Einzel lens extract ions from the liquid meniscus of the ion source and focus the ions through a quadrupole mass filter and electrostatic ion bender. Transmitted ions strike a channel electron multiplier detector. Figure 4D shows a mass spectrum of 100 mM arginine in aqueous solution obtained using a 41 nm inner diameter nanocapillary ion source on a quadrupole mass spectrometer.

[0124] The effect of tip diameter on arginine mass spectra is shown in Figure 5A. The mass spectra shown were obtained using nanocapillaries with tip inner diameters of 300 nm, 125 nm, and 20 nm. The largest nanotip was able to capture a bare arginine ion, a cluster of eight incrementally hydrated arginine ions, and an arginine dimer ion (Arg Arg+H). + The intermediate size tip produced a broad spectrum of peaks, including a peak at 349 m / z corresponding to arginine ion, six incrementally hydrated arginine ion clusters, and a relatively attenuated arginine dimer ion peak. The smallest tip produced mostly bare arginine ions, but attenuated peaks corresponding to singly and doubly hydrated arginine ion clusters were also seen in the spectrum. As shown in the baseline of the three spectra in Figure 5A, smaller tips tended to produce relatively stronger signals and less noisy spectra than larger tips. Some variation in the distribution of solvation states was seen between nanocapillaries with similar tip sizes (e.g., 20 nm shown in Figure 4D compared to 41 nm shown in Figure 5A). However, only nanocapillaries with tip inner diameters less than about 65 nm produced spectra in which the majority of amino acid ions were measured in the unsolvated state.

[0125] The nanocapillary ion source can generate ions of many different amino acids and small peptides for analysis. A fluid delivery system was used to periodically exchange the solution in the nanocapillary without interrupting the measurements. Figure 2B shows the mass spectra of 16 different amino acid aqueous solutions. Three different nanocapillaries with tip inner diameters of 20 nm, 25 nm, and 60 nm were used for these measurements. The most prominent peaks in all spectra corresponded to singly charged and unsolvated amino acid ions. The spectra of glycine, alanine, proline, valine, cysteine, glutamine, and phenylalanine did not show any additional peaks that could correspond to solvated amino acid ions. The spectra of serine, threonine, asparagine, lysine, methionine, histidine, arginine, and tryptophan showed a secondary peak 18 m / z to the right of the unsolvated peak, corresponding to a singly hydrated amino acid ion. Leucine showed a third and possibly a fourth peak corresponding to a higher solvation state. The spectrum of tryptophan showed a peak below 200 m / z, consistent with a hydronium ion hydration state, which also appeared in the control measurement of aqueous solutions with no amino acids present. Tryptophan has a lower solubility than the other amino acids analyzed, producing a relatively weak signal. Four amino acids are missing from this example: aspartic acid and glutamic acid are not included because their low isoelectric points require operation in negative ion mode, isoleucine is not analyzed because it cannot be distinguished from leucine based on m / z, and the low solubility of tyrosine would reduce its release characteristics.

[0126] Mass spectra of glutathione and two chemically modified variants, s-nitrosoglutathione and s-acetylglutathione, are shown in Figure 5C. Glutathione is a tripeptide with biological importance, and the chemically modified forms represent common post-translational modifications. The peptides were analyzed in aqueous solutions at 100 mM concentration, with the pH set at 3.1-3.9 by adding acetic acid. A nanocapillary with a tip of 20 nm inner diameter was used to generate the ions. The glutathione spectrum showed a single peak at 307 m / z, corresponding to a singly charged, unsolvated glutathione ion. The spectra of s-acetylglutathione and s-nitrosoglutathione showed major peaks at 349 m / z and 336 m / z, respectively, corresponding to singly charged, unhydrated peptide ions. The spectra also showed two gradually smaller peaks at 18 m / z and 36 m / z, corresponding to singly and doubly hydrated peptide ions, respectively, to the right of the major peak.

[0127] Figure 5A shows mass spectra of a 100 mM arginine solution in HO using a nanocapillary ion source with three different tip inner diameters. Figure 5B shows a collection of 16 amino acid mass spectra ordered by mass from top left to bottom right. All experiments were performed using nanocapillaries with tip inner diameters between 20 and 60 nm. Figure 5C shows overlaid mass spectra of glutathione and two of its PTM variants, s-nitrosoglutathione and s-acetylglutathione.

[0128] The conventional electrospray mechanism shown in Figure 4A was ruled out as the primary source of ions to be measured for two reasons. First, nanoscale water droplets in high vacuum removed only a small portion of their mass before potential heat losses caused the evaporation process to freeze. The instrument described in this example lacked the background gases necessary to sustain the evaporation of solvent and the release of ions in the electrospray process. Second, the small size of the nanocapillary used in this example limited the fluid flow rate exiting the tip to 0.1 nL / min or less, which is at least three orders of magnitude smaller than the minimum flow rate required to maintain a stable cone-jet.

[0129] Instead, ions may have been ejected directly from the meniscus at the tip of the ion source by ion evaporation. Figure 4B illustrates a pure ion mode ejection. The theory of ion ejection from a Taylor cone predicts that the pure ion mode will dominate when the ratio of liquid conductivity to flow rate, K / Q, is large. In liquid metals and ionic liquids, this is achieved at very high conductivities, K. As shown in this example, the same effect is achieved at very small flow rates, Q. The size of the nanocapillary obstructs the liquid flow, limiting the flow rate to less than 0.1 nL / min. Figure 6B shows the predicted flow rate through the nanocapillary shown in this example as a function of tip radius. Insufficient flow to form a stable cone jet results in a closed meniscus with a large curvature. In such a meniscus, the local electric field can reach values ​​of 1 V / nm or more, which is high enough for ion evaporation to occur. Figure 6C shows the characteristic maximum electric field at the surface of the cone jet as a function of r0. This field is proportional to the r -1 / 2 and the electric field required for ion evaporation is independent of r0. As a result, ion evaporation is predicted to be the dominant ion emission mechanism when r0 is very small.

[0130] A single molecule protein sequencing mass spectrometer was envisioned in conjunction with a mechanism for gradually cleaving amino acids from peptides, as shown in Figure 6D. Proteins are denatured near the tip of the nanocapillary, presumably due to interactions between positively charged protein sites and negatively charged silanol groups on the capillary surface. The denatured proteins can be fragmented into individual amino acids or small peptides by photolysis inside the nanocapillary ion source, which can then be released by ion evaporation. The ions are focused by a set of ion optics and pass through a magnetic sector that separates the ions by their mass-to-charge ratio. The ions strike an array of electron multiplier detectors, and by considering the timing and location of each ion detection event, the original sequence can be reconstructed. Using 19 detectors, all of the amino acids except leucine / isoleucine can be distinguished. Electron multipliers traditionally used in mass spectrometry can detect ions at rates of about 100 MHz.

[0131] FIG. 6A compares conventional electrospray cone-jet mode ion emission with pure ion mode emission from a nanocapillary. FIG. 6B shows a theoretical prediction of the flow rate through a tapered capillary as a function of tip inner radius using a truncated cone model. FIG. 6C shows a theoretical prediction of the characteristic electric field in the transition region of the cone-jet at the tip of the capillary as a function of capillary tip inner radius compared to the predicted electric field required to achieve ion evaporation from water. FIG. 6D is a schematic of a hypothetical mass spectrometer capable of single molecule protein sequencing. FIG. 6E shows a theoretical calculation of the cumulative probability of an emitted amino acid colliding with an evaporated water molecule or background gas molecule as a function of distance from the meniscus. The dashed line shows the calculated maximum possible water vapor density as a function of distance from the meniscus, calculated using the Hertz-Knudsen model with an evaporation coefficient of 1.

[0132] The example device measured stable emission currents as low as 10 pA, corresponding to an ion emission rate of 60 MHz. As a result, the detector is fast enough to identify every single ion emitted by the nanocapillary ion source. This sequencing method relies on the maintenance of sequence through the fragmentation, emission, and mass separation stages.

[0133] As shown in this example, the nanocapillary ion source can directly eject singly charged, unsolvated biomolecular ions into high vacuum. Sixteen of the 20 proteinogenic amino acids, as well as glutathione, s-nitrosoglutathione, and s-acetylglutathione, were ejected primarily in an unsolvated state, as determined by mass spectrometry.

[0134] Although several embodiments of the present disclosure have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein. Each such variation and / or modification is deemed to be within the scope of the present disclosure. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application or applications in which the teachings of the present disclosure are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present disclosure described herein. Thus, the above embodiments are presented by way of example only, and it will be understood that within the scope of the appended claims and their equivalents, the present disclosure may be practiced otherwise than as specifically described and claimed. The present disclosure relates to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods is within the scope of the disclosure, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0135] In the event that the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and / or inconsistent disclosure with respect to each other, the document having the later effective date shall control.

[0136] All definitions defined and used herein should be understood to supersede any dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meaning of the defined terms.

[0137] The indefinite articles "a" and "an," as used in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0138] The term "and / or" as used herein and in the claims should be understood to mean "either or both" of the elements so connected, i.e., elements that are conjunctive in some cases and disjunctive in other cases. Multiple elements listed with "and / or" should be construed in the same manner, i.e., "one or more" of the elements so connected. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, when used in conjunction with an open-ended term such as "comprising," a reference to "A and / or B" can refer in one embodiment to only A (optionally including elements other than B), in another embodiment to only B (optionally including elements other than A), in yet another embodiment to both A and B (optionally including other elements), and so forth.

[0139] As used herein and in the claims, "or" should be understood to have the same meaning as "and / or" above. For example, when separating items in a list, "or" or "and / or" should be interpreted as being inclusive, i.e., including at least one of an element or list of elements, but also including more than one, and optionally including additional items not listed. Only terms clearly indicated to the contrary, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," refer to the inclusion of exactly one element of an element or list of elements. In general, the term "or" as used herein should only be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of."

[0140] As used herein and in the claims, the phrase "at least one" in reference to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to the specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") can refer in one embodiment to at least one A, optionally including more than one, and no B (optionally including elements other than B), in another embodiment to at least one B, optionally including more than one, and no A (optionally including elements other than A), in yet another embodiment to at least one A, optionally including more than one, and at least one B, optionally including more than one (optionally including other elements), etc.

[0141] When the word "about" is used herein in connection with a number, it should be understood that further embodiments of the present disclosure include that number unmodified by the presence of the word "about."

[0142] It should also be understood that, unless expressly indicated to the contrary, in any method claimed herein that includes two or more steps or actions, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are described.

[0143] In the claims and the above specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "including," "involving," "holding," "consisting of," and the like, are to be understood to be open-ended, i.e., meaning including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

1. An ion source including a capillary and an electrode proximate to the capillary, the ion source comprising an opening in the capillary having a cross-sectional dimension of less than 125 nm, and a magnetic mass filter downstream of the ion source, and an array of detectors downstream of the magnetic mass filter A mass spectrometer comprising the same.

2. The mass spectrometer according to claim 1, further comprising a vacuum chamber housing the ion source, and optionally, the vacuum chamber having a pressure of 10 mPa or less.

3. The mass spectrometer according to claim 1 or 2, wherein the magnetic mass filter includes a permanent magnet.

4. The mass spectrometer according to claim 1 or 2, wherein the mass spectrometer has a time resolution of 1 microsecond or less.

5. The mass spectrometer according to claim 1 or 2, further comprising ion optical components downstream of the ion source and upstream of the magnetic mass filter, and optionally, the ion optical components including at least one Einzel lens.

6. The mass spectrometer according to claim 1 or 2, further comprising an ion bender configured to deflect ions exiting the mass filter towards the detector.

7. The mass spectrometer according to claim 1 or 2, wherein the detector array includes an electron multiplier tube, a dynode, a microchannel plate array, a CCD, a CMOS, and / or a SQUID.

8. The mass spectrometer according to claim 1 or 2, wherein the capillary has a cross-sectional dimension of less than 100 nm or less than 60 nm, and the capillary has an aspect ratio of length to cross-sectional dimension of 100 or more, 1,000 or more, and / or 10,000 or more.

9. The mass spectrometer according to claim 1 or 2, wherein the capillary tapers at the opening, and optionally, the taper is at an angle of less than 10° or less than 5°.

10. The mass spectrometer according to claim 1 or 2, wherein the opening of the capillary has a cross-sectional dimension of less than 65 nm, less than 50 nm, less than 30 nm, or less than 2 nm.

11. The mass spectrometer according to claim 1 or 2, wherein the capillary comprises quartz, glass, borosilicate glass, plastic, metal, semiconductor, carbon nanotube, and / or boron nitride nanotube.

12. The mass spectrometer according to claim 1 or 2, wherein the electrode defines a central opening, and optionally, the central opening of the electrode has a cross-sectional dimension of less than 5 cm or less than 1 cm, the central opening of the electrode is larger than the opening of the capillary, and / or the central opening of the electrode is at least 5 times or at least 10 times larger than the opening of the capillary.

13. The mass spectrometer according to claim 1 or 2, wherein the electrode comprises steel, the electrode is annular, and / or the electrode has a cross-sectional dimension of less than 5 cm.

14. The electrode defines a central opening, the electrode is positioned within 10 mm, 5 mm, or 2 mm of the opening of the capillary, the electrode is positioned around the capillary, and / or the electrode is positioned in front of the opening of the capillary. The mass spectrometer according to claim 1 or 2.

15. The mass spectrometer according to claim 1 or 2, wherein the electrode defines a central opening, and a virtual line passing through the center of the cross-section of the capillary passes through the central opening of the electrode.

16. The mass spectrometer according to claim 1 or 2, wherein the electrode and the capillary have an interior connected to a voltage source.

17. The voltage source can generate a voltage of less than 400 V, less than 360 V, and / or at least 80 V between the electrode and the capillary, and / or the voltage source can generate an electric field of at most 4 V / nm, 3 V / nm, at least 1.5 V / nm between the electrode and the capillary. The mass spectrometer according to claim 16.

18. The magnetic mass filter has a magnetic filter strength of at least about 0.5 T, and / or the magnetic mass filter includes a magnet containing neodymium, a yoke containing iron, an opening having a first dimension of at least about 5 cm, and / or an opening having a second dimension of at least about 1 cm. The mass spectrometer according to claim 1 or 2.

19. The detector is a single ion detector. The mass spectrometer according to claim 1 or 2.

20. A method for sequencing a biopolymer, Ionizing a biopolymer contained in a fluid into ions or ion clusters, Passing the ions or ion clusters through a magnetic mass filter, Directing the ions or ion clusters towards an array of detectors, Determining the sequence of the biopolymer by determining the ions or ion clusters using the array of detectors Including, method.

21. The biopolymer is a protein. The method according to claim 20.

22. Ionizing the amino acids of the protein at a rate of at least one amino acid per microsecond. The method according to claim 21.

23. The biopolymer is a nucleic acid. The method according to any one of claims 20 to 22.

24. The method according to claim 23, comprising ionizing the bases of the nucleic acid at a rate of at least 1 base per microsecond, at least 10 bases per microsecond, and / or at least 100 bases per microsecond.

25. The method according to any one of claims 20 to 22 or 24, wherein the ion or ion cluster has a total ion transmission efficiency of about 0.8 or more.

26. The method according to any one of claims 20 to 22 or 24, wherein the ion or ion cluster is generated at a rate of 1 or more ions or ion clusters per microsecond to 100 ions or ion clusters per microsecond.

27. The method according to any one of claims 20 to 22 or 24, wherein the time interval between the emission of the molecule as an ion or ion cluster close to the opening and the detection of the ion or ion cluster in the detector array is 10 microseconds or more and 100 microseconds or less.

28. The method according to any one of claims 20 to 22 or 24, wherein the ion or ion cluster can be detected with a time resolution of more than 100 nanoseconds.

29. Ionizing the biopolymer contained in the fluid into ions or ion clusters includes ionizing the biopolymer into single ions, passing the ion or ion cluster through a magnetic mass filter includes passing a single ion through a magnetic mass filter, and / or directing the ion or ion cluster towards an array of detectors includes directing the single ion towards an array of detectors. The method according to any one of claims 20 to 22 or 24.

30. The method according to any one of claims 20 to 22 or 24, wherein the detector array includes an electron multiplier tube, a dynode, a microchannel plate array, a CCD, and / or a CMOS sensor. **Claim 31**: The method according to any one of claims 20 to 22 or 24, further comprising passing a fluid containing the biopolymer through a capillary defining an opening before ionizing the biopolymer. **Claim 32**: The ions or ion clusters include monomers of the biopolymer, and the monomers are sequentially ionized. The method according to any one of claims 20 to 22 or 24. **Claim 33**: A method for determining concentration, comprising: ionizing molecules from a fluid as ions or ion clusters; passing the ions or ion clusters through a magnetic mass filter; directing the ions or ion clusters towards an array of detectors; determining the concentration of the molecules in the fluid by determining the ions or ion clusters using the array of detectors. A method comprising the above steps.

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