Systems and methods for analyzing peptide photodissociation for single molecule protein sequencing - Patents.com

Abstract

The present disclosure relates generally to systems and methods for analyzing peptide photodissociation for single molecule protein sequencing. In one aspect, the systems and methods relate to allowing single protein molecules in aqueous solution to be fragmented such that their amino acid composition and sequence can be determined by mass spectrometry, which may be useful for single molecule protein sequencing techniques.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 341,992, entitled "Systems and Methods for Analysis of Peptide Photodissociation for Single-Molecule Protein Sequencing," filed May 13, 2022, and U.S. Provisional Patent Application No. 63 / 235,601, entitled "Systems and Methods for Analysis of Peptide Photodissociation for Single-Molecule Protein Sequencing," filed August 20, 2021, each of which is incorporated by reference in its entirety.

[0002] The present disclosure relates generally to systems and methods for analyzing peptide photodissociation for single molecule protein sequencing. [Background technology]

[0003] Peptide sequencing is an essential tool in proteomics, widely used to identify proteins and map the protein content of cells. The ability to sequence single copies of proteins would significantly improve the analysis of single cells, enabling the study of low-abundance proteins and proteoforms that may be present in fewer than 10 copies per cell. Single-molecule techniques are required because proteins cannot be biochemically amplified like nucleic acids. Furthermore, proteoforms due to post-translational modifications and alternative mRNA splicing cannot be inferred from DNA or RNA sequences. Single-molecule protein sequencing may also lead to new diagnostic applications and drug therapies. There is growing interest in developing techniques for sequencing single proteins based on fluorescent tagging, N-terminal probing, and nanopores.

[0004] Mass spectrometry (MS) is the current mainstream technique for peptide sequencing. However, this technique typically requires 10 7 copies or more of the protein are required. Among any other components, the most limiting sensitivity is typically the MS ion source. In electrospray ionization, charged analyte-containing droplets collide with background gas to release analyte ions into the gas phase, which are then transported through the instrument to the detector. The combined efficiency of these processes is very low. Therefore, improvements are needed. Summary of the Invention

[0005] The present disclosure relates generally to systems and methods for analyzing peptide photodissociation for single molecule protein sequencing. The subject matter of the present disclosure includes, in some cases, interrelated products, alternative solutions to a particular problem, and / or multiple different uses of one or more systems and / or articles.

[0006] In one aspect, the system and method relate to allowing single protein molecules in aqueous solution to be fragmented so that their amino acid composition and sequence can be measured by mass spectrometry. This can be useful for single molecule protein sequencing technology. Single molecule protein sequencing is the next frontier in biomolecular diagnostics, and its development can help revolutionize the fields of biology and disease diagnosis.

[0007] In some cases, these can be implemented using hardware that can be commercialized as an add-on component of a mass spectrometry system. In addition, certain embodiments relate generally to single molecule protein sequencing instruments that can be used in settings such as biomedical research and clinical.

[0008] Certain methods and systems can allow proteins to be fragmented in aqueous solution rather than in the gas phase. In some embodiments, peptide bonds linking amino acids to parent peptides can be selectively cleaved. In some cases, amino acids can be released intact for analysis by mass spectrometry. Such methods can be compatible with single molecule protein sequencing strategies, for example, as discussed in International Patent Application Publication No. PCT / US2021 / 028954, filed April 23, 2021, and U.S. Patent Application No. 63 / 179,046, filed April 23, 2021. Each of these documents is incorporated herein by reference.

[0009] In one set of embodiments, the method involves fragmenting proteins into single molecules using light in a mass spectrometer.

[0010] According to another set of embodiments, the method includes arranging the protein in a substantially linear configuration on the nanotip; fragmenting the protein into amino acids by applying laser light to the protein; releasing the amino acids from the nanotip; and detecting the amino acids released from the nanotip.

[0011] In another set of embodiments, the method includes exposing the protein to light of a wavelength greater than or equal to 150 nm and less than or equal to 213 nm to cleave fragments from the protein; and sequencing the fragments using mass spectrometry.

[0012] In yet another set of embodiments, the method includes exposing the protein to light of a wavelength greater than or equal to 150 nm and less than or equal to 222 nm to cleave fragments from the protein; and sequencing the fragments using mass spectrometry.

[0013] According to yet another set of embodiments, the method includes directing laser light at the protein to cleave amino acids from the protein; and sequencing the amino acids using mass spectrometry.

[0014] According to yet another set of embodiments, the method includes exposing the peptide to light having a wavelength of greater than or equal to 150 nm and less than or equal to 213 nm to cleave fragments from the peptide; passing at least 50% of the fragments through a magnetic mass filter; and directing the fragments to a detector.

[0015] One aspect of the disclosure relates generally to a method for sequencing a protein. According to one set of embodiments, the method for sequencing a protein includes fragmenting the protein by exposing the protein to light with a wavelength of 150 nm or more and 213 nm or less to generate fragments, passing the fragments through a magnetic mass filter, directing the fragments to an array of detectors, and determining the sequence of the protein by determining the fragments with the array of detectors.

[0016] According to another set of embodiments, a method for sequencing a protein includes passing a fluid containing the protein through a capillary defining an opening; directing light of a wavelength of 150 nm or more and 213 nm or less to the protein proximate the opening to generate fragments; passing the fragments directly into an environment having a pressure of 100 mPa or less; passing the fragments through a magnetic mass filter; directing the fragments to an array of detectors; and determining the sequence of the protein by determining the fragments with the array of detectors.

[0017] One aspect of the present disclosure generally relates to a mass spectrometer. According to one set of embodiments, the mass spectrometer includes a nanotip that allows proteins to be arranged in a linear configuration; and a laser arranged to direct light to dissociate the proteins into fragments.

[0018] According to another set of embodiments, a mass spectrometer includes an ion source including a capillary; a light source directed toward the ion source, the light source capable of producing light with a wavelength of greater than or equal to 150 nm and less than or equal to 213 nm; a magnetic mass filter downstream of the ion source; and an array of detectors downstream of the magnetic mass filter.

[0019] According to yet another set of embodiments, a mass spectrometer includes an ion source including a capillary; a laser positioned to direct light at the capillary; and a detector positioned downstream of the ion source.

[0020] According to yet another set of embodiments, a mass spectrometer includes an ion source including a capillary and an electrode proximate the capillary, the capillary including an opening having a cross-sectional dimension of less than 125 nm; a magnetic mass filter downstream of the ion source; an array of detectors downstream of the magnetic mass filter; and a light source directed toward the ion source, the light source capable of producing light with a wavelength between 150 nm and 213 nm, inclusive.

[0021] In another aspect, the present disclosure encompasses methods for making one or more of the embodiments described herein. In yet another aspect, the present disclosure encompasses methods for using one or more of the embodiments described herein.

[0022] 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.

[0023] Non-limiting embodiments of the present disclosure will be described by way of example with reference to the accompanying drawings, which are schematic and are not intended to be drawn to scale. In the drawings, each identical or nearly identical component shown is typically represented by a single numeral. For clarity, not every component is labeled in every drawing, nor are every component of each embodiment of the present disclosure shown unless illustration is necessary to enable a person skilled in the art to understand the present disclosure. [Brief description of the drawings]

[0024] [Figure 1]Figure 1A shows a schematic of single molecule protein sequencing by nanopore mass spectrometry, and Figure 1B shows that an elongated peptide chain is photofragmented near the tip of a nanocapillary ion source. [Diagram 2] FIG. 1 shows the structure of a dipeptide, the structures of common photofragmentation products, and the approximate frequency with which laser light of different wavelength regions induces specific transformations in vacuum. [Diagram 3] Figure 3A shows the calculated heating profile in the nanocapillary under steady-state irradiation with 10.6 μm, 193 nm, and 222 nm light. Figure 3B shows the dependence of the maximum temperature rise in the nanocapillary on the incident laser power density for 10.6 micrometer (triangles), 193 nm (squares), and 222 nm (circles) light. Symbols show the results of finite element method calculations and curves are linear fits of the data. [Figure 4-1] Figure 4A shows the cumulative probability of peptide bond dissociation obtained from Equation 2 when exposed to 193 nm laser light of different intensities as indicated. Figure 4B shows the probability of amino acid uncleavage calculated according to Equation 3 as a function of exposure time to 193 nm laser light with ρ=10,000 Wm-2. [Figure 4-2] FIG. 4C shows the probability of selective amino acid release (i.e., fragmenting the two peptide bonds joining an amino acid to a peptide without damaging the amino acid) as a function of exposure time to 193 nm laser light with ρ=10,000 W m-2. [Figure 5-1] FIG. 5A is a schematic diagram of conventional electrospray ionization highlighting the background gas that stimulates evaporation of solvent from the droplets and the transfer capillary where significant ion losses occur. [Figure 5-2]FIG. 5B is a schematic diagram of a nanopore ion source showing the liquid-filled nanocapillary tip, the extraction electrode, and the extraction voltage V applied between them. The inset shows an SEM image of the tip of a pulled quartz nanocapillary with a tip inner diameter of 30 nm. FIG. 5C is a schematic diagram of the mass spectrometer used in Example 2. Ion optics, including an extraction electrode and an Einzel lens, extract ions from the liquid meniscus of the ion source and focus them with a quadrupole mass filter and an electrostatic ion bender. Transmitted ions impinge on a single-ion sensitive channel electron multiplier detector. [Figure 5-3] FIG. 5D shows a mass spectrum of 100 mM arginine in aqueous solution obtained using a 41 nm inner diameter nanopore ion source on a quadrupole mass spectrometer described herein. [Figure 6-1] FIG. 6A shows mass spectra of a 100 mM arginine solution in HO using nanopore ion sources with three different tip inner diameters (20 nm, 125 nm, and 300 nm). [Figure 6-2] Figure 6B shows a list of mass spectra of 16 amino acids arranged in order of mass from top left to bottom right. All experiments were carried out using nanocapillaries with tip inner diameters of 20 to 60 nm. [Figure 6-3] FIG. 6C shows the overlaid mass spectra of glutathione and two of its PTM variants, s-nitrosoglutathione and s-acetylglutathione. [Figure 7-1]FIG. 7A shows the experimental setup for measuring the ion transmission efficiency of a nanopore ion source. A voltage VT is applied to the nanopore ion source by a source measurement unit, generating a tip current IT. The emitted ions are focused by ion optics and impinge on a Faraday cup where the collection current IC is measured by a current-to-voltage preamplifier. The experiment is carried out in a vacuum chamber at a pressure of about 10-7 torr. The ion transmission efficiency is the ratio of the collection current to the tip current IC / IT. FIG. 7B shows a plot of the ion transmission efficiency measured over several minutes from a nanopore ion source using 100 mM NaI in water. The inset shows IT and IC plotted over the same period. [Figure 7-2] FIG. 7C shows the experimental setup used to measure the relative fractions of ion current I ions and droplet current I droplets. A magnetic sector (diameter=6 cm, B field strength=0.54 T) is placed after the ion optics and deflects the emitted particles by their mass-to-charge ratio. I ions are collected by a wide Faraday plate and I droplets are collected by a Faraday cup. FIG. 7D shows the ion fractions of I ions, I droplets, and total measured current for a 2 min measurement performed using a 28 nm tip filled with a 100 mM aqueous solution of NaI. [Figure 8] Figure 1 shows the probability of an amino acid ion with a hydration shell colliding with a gas molecule based on the kinetic theory of gas molecules. The curve shows the cumulative probability that an ejected amino acid with a small hydration shell (radius = 7 Å) will collide with an evaporated water molecule or a background N2 molecule as a function of the distance r from the meniscus. The line shows the calculated maximum possible water vapor density as a function of the distance from the meniscus. The vapor pressure on the vacuum side of the meniscus was conservatively assumed to be 8.75 torr, half the equilibrium vapor pressure of water at room temperature. The inset shows a schematic of the evaporated water molecule distribution near the meniscus. [Figure 9-1] FIG. 9A illustrates water evaporating from a hemispherical meniscus, the tip radius r0, and the distance r. [Figure 9-2]Figure 9B is a diagram showing the plot of Cp(r) (solid line), and the separate contributions of water molecules (dashed line) and background gas (dash-dotted line) to the total probability of causing at least one collision. The dashed curve shows the contribution of the evaporated water molecules, the dash-dotted curve shows the contribution of the background gas, and the solid curve shows the total collision probability obtained by summing the contributions of water and the background gas. For the calculation, nb = 2.25×1015 m-3, nw0 = 6.44×1023 m-3, aw = 1.325 Å, ab = 1.82 Å, ai = 7 Å, and r0 = 30 nm were used. The plot is extended up to r = 0.5 m, which is the total distance from the meniscus to the detector. [Figure 10] It is a diagram showing a plot of fluid conductance measurements fitted to the theoretical prediction using the semi-infinite frustum model of the nanopore, with the semi-apex angle θ used as the fitting parameter. [Figure 11-1] Figure 11A is a diagram showing the simulation results of the deflection angle of monovalent ions against m / z. The region between the dashed lines represents the position and range of the Faraday plate detector, and ions with 70 < m / Z < 325 should collide with the Faraday plate. Figure 11B is a diagram showing the simulation results of the deflection angle of charged water droplets up to the Rayleigh limit against the droplet radius. The region below the dashed line represents the position and range of the Faraday cup aperture, and fully charged droplets larger than 15 nm should collide with the Faraday cup. [Figure 11-2] Figure 11C shows the selected trajectory simulations of five monovalent ions with masses from 60 amu to 460 amu. The arrows indicate the direction of increasing mass, and the circles represent the magnetic sector. Figure 11D shows the selected trajectory simulations of five droplets charged up to the Rayleigh limit with radii from 5 to 25 nm. The arrows indicate the direction of increasing radius. [Figure 12] It is a diagram showing a schematic of a multiplex mass spectrometer including a plurality of nanopore ion sources according to some embodiments.

Mode for Carrying Out the Invention

[0025] The present disclosure relates generally to systems and methods for analyzing peptide photodissociation for single molecule protein sequencing. In one aspect, the systems and methods relate to allowing single protein molecules in aqueous solution to be fragmented such that their amino acid composition and sequence can be determined by mass spectrometry, which may be useful for single molecule protein sequencing techniques.

[0026] Certain aspects of the present disclosure relate to mass spectrometers and related methods that allow for single molecule fragmentation and sequencing of species of interest (e.g., polymers, biopolymers, etc.). In some cases, the species of interest is a protein, and methods related to single molecule protein sequencing are disclosed herein. It should be understood that while some embodiments of the present disclosure relate to methods for analyzing and / or sequencing peptides and proteins, the disclosure is not limited thereto, and in other embodiments, the methods can be used to analyze any of a variety of molecules and / or ions, including, but not limited to, salt ions, macromolecules, and the like.

[0027] In some embodiments, disclosed herein is a mass spectrometer that includes a light source configured to fragment a species of interest (e.g., a protein) into individual components (e.g., amino acids). In some such embodiments, the mass spectrometer is a nanopore mass spectrometer that includes an ion source capable of ionizing the species of interest into a vacuum, as described in more detail below, and other associated components including, but not limited to, a vacuum, a magnetic mass filter, and one or more detectors. The combination of the light source, ion source, etc. can advantageously allow for the fragmentation of the species of interest (e.g., a biopolymer such as a protein) into its basic fragments (e.g., monomers such as amino acids) and for sequencing it. The ion source, magnetic mass filter, one or more detectors may have any of the properties, configurations, and / or arrangements, as described in more detail below.

[0028] In one set of embodiments, the light source (e.g., a laser) may be positioned adjacent to an ion source including a capillary, for example, such that the light source is directed toward the ion source or a portion thereof. Non-limiting examples of mass spectrometers including such light sources are shown in FIGS. 1A-1B. As shown, mass spectrometer 10 includes a light source 15 (e.g., a laser, such as a UV laser) directed toward an ion source 20 including a capillary 30 filled with a fluid 52 including a species of interest 50 (e.g., a protein or peptide). The fluid may include any suitable solvent described elsewhere herein, e.g., water, formamide, highly volatile solvents, aqueous solutions, etc. In some cases, the light source may be directed toward a capillary tip (e.g., a nanotip) of the ion source. In some embodiments, the light source may be directed toward a fluid including a species of interest (e.g., a protein or peptide) disposed within the capillary tip (e.g., a nanotip). For example, as shown in FIGS. 1A-1B, a light source 15 may be directed toward a fluid 52 containing a species of interest 50 within a capillary tip 34 (eg, a nanotip) of an ion source 20.

[0029] In some embodiments, the light source may be configured to be directed toward a fluid containing the species of interest to fragment the species of interest (e.g., a protein) in the fluid in the capillary into fragmented individual components or single molecules (e.g., individual amino acids). The fragmented individual components or single molecules may then be ionized into a vacuum from an opening at the capillary tip (e.g., a nanotip). In some embodiments, the vacuum chamber houses the nanotip. For example, as shown in FIG. 1B, the light source 15 may be configured to be directed toward a fluid 52 containing the species of interest 50 to fragment the species of interest 50 (e.g., a protein or peptide) in the fluid 52 in the capillary tip 34 (e.g., a nanotip) into fragmented individual components or single molecules 54 (e.g., amino acids). For example, light 62 (e.g., UV photons) emitted by the light source 15 may result in fragmentation 64 of portions of the species of interest 50 exposed to the light 62. The fragmented individual components or single molecules 54 may then be ionized through the opening 36 of the capillary tip 34 into the vacuum 80 containing the capillary tip 34. Methods for fragmenting species of interest are described in more detail below.

[0030] In some embodiments, methods are disclosed herein that relate to fragmenting a species of interest (e.g., a biopolymer such as a protein) into individual components (e.g., individual amino acids) and sequencing the same. It should be noted that such methods may be particularly beneficial for sequencing biopolymers (e.g., proteins) where single molecule accuracy is required.

[0031] In some embodiments, the method includes disposing a species of interest (e.g., a protein) in a substantially linear configuration at a nanotip of a capillary. In some cases, the nanotip of the capillary may be dimensioned such that the species of interest is driven to dispose itself in a linear configuration. As shown in FIGS. 1A-1B, the capillary tip 34 may be dimensioned such that the species of interest 50 (e.g., a protein or peptide) is disposed in a linear configuration at the tip of the capillary (e.g., by having a cross-sectional dimension (e.g., maximum cross-sectional dimension) of less than 200 nm, less than 150 nm, less than 120 nm, less than 100 nm, less than 80 nm, less than 65 nm, less than 60 nm, less than 50 nm, less than 30 nm, less than 25 nm, and / or a minimum of 20 nm (e.g., minimum 15 nm, minimum 10 nm, minimum 5 nm, minimum 4 nm, minimum 3 nm, minimum 2 nm, minimum 1 nm, etc.). In one set of embodiments, the nanotip may have a cross-sectional dimension of between 1 nm and 5 nm. Advantageously, such a linear arrangement can allow exposure of the individual bonds (e.g., peptide bonds) between the basic fragments (e.g., amino acids) that form the species of interest, which in turn can facilitate fragmentation of the species of interest into its basic fragments.

[0032] In some embodiments, the method includes fragmenting a species of interest (e.g., a protein) into basic fragments (e.g., amino acids) by applying laser light to the species of interest within a capillary tip (e.g., a nanotip). As shown in FIGS. 1A-1B, light 62 (e.g., laser light) generated by a light source 15 (e.g., a laser) adjacent to a capillary tip 34 (e.g., a nanotip) may be applied to a solution containing the species of interest 50. In some such embodiments, the species of interest is linearly positioned such that the laser light can sever bonds between individual components (e.g., amino acids) from the species of interest (e.g., a protein). For example, referring to FIG. 1B, the species of interest 50 may be linearly positioned within the capillary tip 34 (e.g., a nanotip) such that the light 62 (e.g., laser light) can sever bonds between individual components or molecules (e.g., amino acids) from the species of interest 50 (e.g., a protein or peptide).

[0033] In some embodiments, once a species of interest (e.g., a protein) has been fragmented into its base fragments (e.g., amino acids), the base fragments can be released (e.g., ionized) through an aperture of the nanotip. According to some embodiments, the nanotip can be dimensioned, for example, such that the base fragments are released sequentially while preserving the order of the base fragments within the species of interest. For example, as shown in FIGS. 1A-1B, the base fragments 54 (e.g., amino acids) can be released (e.g., one at a time) through the aperture 36 of the nanotip 34 in sequential order. In some embodiments, the released base fragments (e.g., amino acids) can pass through various components of a mass spectrometer (e.g., vacuum, ion optics, magnetic mass filter) and then be detected by one or more detectors. In some embodiments, the one or more detectors can be configured to determine the sequence of the species of interest (e.g., a protein) by determining the released fragments (e.g., amino acids). 1A-1B, as a non-limiting example, the released base fragments 54 (e.g., amino acids) may pass through various components of the mass spectrometer (e.g., vacuum 80, ion optics 100, mass filter 90 (e.g., magnetic mass filter)) and then be detected by one or more detectors 70. The one or more detectors 70 may be configured to determine the sequence of the species of interest (e.g., protein) by determining the released fragments 54 (e.g., amino acids). Details relating to the various components and methods of release, transmission, and detection of the base fragments are described in more detail below.

[0034] In some embodiments, the capillary may have a particularly advantageous configuration that allows for fragmentation of the species of interest into individual components within the capillary. A capillary having such a configuration may, for example, advantageously reduce mixing and diffusion of the fragmented individual components within the capillary (e.g., within the nanotip of the capillary) and / or may preserve the sequence of the individual components with respect to the original sequence before fragmentation of the species of interest, and / or may allow linear and sequential subsequent ionization of the fragmented individual components at the tip of the capillary. For example, in one set of embodiments, the capillary may include a body portion and a tip portion (e.g., a nanotip) fluidly connected to the body portion and adjacent the opening of the capillary. For example, as shown in FIG. 1B, the capillary 30 may include a body portion 32 and a tip portion 34 (e.g., a nanotip) adjacent the opening 36 of the capillary 30. The tip portion 34 may be fluidly connected to the body portion 32.

[0035] In some embodiments, the tip portion of the capillary may be substantially transparent to light having a certain wavelength or wavelength range emitted by the light source. As used herein, a tip portion that is "substantially transparent" to light of a certain wavelength means that more than 50% (e.g., more than 60%, more than 70%, more than 80%, more than 90%) and / or up to 95% (e.g., up to 99%, or up to 100%) of light having a certain wavelength or wavelength range can pass through to the tip portion of the capillary. For example, the tip portion of the capillary (e.g., nanotip) may be transparent to light having a wavelength of 213 nm or less (e.g., 213 nm or less, 200 nm or less, 193 nm or less, 185 nm or less, 180 nm or less, 175 nm or less, 150 nm or less) and / or at least 170 nm (e.g., at least 160 nm, at least 155 nm, or at least 150 nm). The wavelength values ​​referenced above may have a deviation of + / - 5 nm, + / - 10 nm, or + / - 15 nm. Combinations of the ranges referenced above are also possible (e.g., up to 220 nm + / - 5 nm and min 150 nm + / - 5 nm, up to 213 nm + / - 5 nm and min 150 nm + / - 5 nm, up to 193 nm + / - 5 nm and min 160 nm + / - 5 nm, or up to 185 nm + / - 5 nm and min 150 nm + / - 5 nm). Other ranges are also possible.

[0036] In some embodiments, the body portion of the capillary may be substantially opaque to light having a certain wavelength or wavelength range emitted by the light source. As used herein, a body portion that is "substantially opaque" to light of a certain wavelength means that less than 50% (e.g., less than 40%, less than 30%, less than 20%, less than 10%) and / or at least 5% (e.g., at least 1%, or at least 0%) of light having a certain wavelength or wavelength range can pass through the body portion of the capillary. For example, the body portion of the capillary may be opaque to light having a wavelength of 213 nm or less (e.g., at least 200 nm, at least 193 nm, at least 185 nm, at least 180 nm, at least 175 nm, at least 150 nm) and / or at least 170 nm (e.g., at least 165 nm, at least 160 nm, at least 155 nm, or at least 150 nm). The wavelength values ​​referred to above may have a deviation of + / -5 nm, + / -10 nm, or + / -15 nm. Combinations of the above referenced ranges are also possible (e.g., up to 220 nm + / - 5 nm and min 150 nm + / - 5 nm, up to 213 nm + / - 5 nm and min 150 nm + / - 5 nm, up to 193 nm + / - 5 nm and min 150 nm + / - 5 nm, or up to 185 nm and min 150 nm + / - 5 nm). Other ranges are also possible.

[0037] As a non-limiting example, with reference to FIGS. 1A-1B, the tip portion 34 (e.g., the nanotip) may be transparent to light having one or more ranges of wavelengths as described above for the tip portion of the capillary, and the body portion 32 may be substantially opaque to light having one or more ranges of wavelengths as described above for the body portion of the capillary.

[0038] In some embodiments, a majority of the target species contained within the tip portion of the capillary (e.g., the substantially transparent tip portion) is fragmented into individual components, while a small amount, if any, of the target species contained within the body portion of the capillary (e.g., the substantially impermeable body portion) is fragmented into individual components. For example, more than 50% (e.g., more than 60%, more than 70%, more than 80%, more than 90%) and / or up to 95% (e.g., up to 99%, or up to 100%) of the target species contained within the tip portion of the capillary may be fragmented into individual components, for example, by the incident light. Additionally, less than 50% (e.g., less than 40%, less than 30%, less than 20%, less than 10%) and / or at least 5% (e.g., at least 1%, or at least 0%) of the target species contained within the body portion of the capillary may be fragmented into individual components.

[0039] 1A-1B, most of the target species 50 (e.g., a protein or peptide) contained within the permeable tip portion 34 of the capillary 30 is fragmented into individual components 54 (e.g., amino acids) by light 62, while little, if any, of the target species 50 (e.g., a protein or peptide) contained within the impermeable body portion 32 of the capillary 30 is fragmented into individual components. In one set of embodiments, all (e.g., 100%) of the target species contained within the tip portion of the capillary is fragmented by light from the light source, while little or none (e.g., 0%) of the target species contained within the body portion of the capillary is fragmented by light from the light source.

[0040] In some embodiments, once the species of interest is fragmented into individual components at the tip portion of the capillary, the individual components diffuse towards the opening of the tip portion and can be ionized by an electrode near the capillary tip. The electrodes may have any of the properties and / or configurations described elsewhere herein. For example, as shown in FIG. 1B, the fragmented individual components 54 diffuse towards the opening 36 of the tip portion 34 and can be ionized by an electrode (not shown) near the capillary tip.

[0041] In some embodiments, the capillary advantageously has an impermeable body portion and a permeable tip portion, thereby allowing fragmentation of the species of interest only at the permeable tip portion adjacent the opening of the capillary. As described elsewhere herein, the tip portion can be dimensioned such that the species of interest contained within the tip portion are arranged in a substantially linear fashion. The capillaries described herein allow fragmentation of the species of interest while still arranged in a linear fashion at the tip portion, limiting mixing of the individual fragmented components prior to ionization at the opening of the tip portion, thus allowing ionization and detection of the individual fragmented components in the original sequence of the species of interest.

[0042] The tip portion of the capillary may have one or more of the size ranges described herein for the opening of the capillary. In some embodiments, the tip portion comprises a cross-sectional dimension (e.g., maximum cross-sectional dimension) of 150 nm or less, 130 nm or less, 125 nm or less, 120 nm or less, 110 nm or less, 100 nm or less, 90 nm or less, 80 nm or less, 75 nm or less, 70 nm or less, 65 nm or less, 60 nm or less, 55 nm or less, 50 nm or less, 45 nm or less, 40 nm or less, 35 nm or less, 30 nm or less, 25 nm or less, 20 nm or less, 15 nm or less, 10 nm or less, 5 nm or less, 3 nm or less, 2 nm or less, etc. In addition, the tip portion may have a cross-sectional dimension of at least 1 nm, at least 2 nm, at least 3 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 65 nm, at least 70 nm, at least 80 nm, at least 90 nm, etc. Combinations of these are also possible. For example, the opening may have a cross-sectional dimension of 50 nm to 100 nm, or 20 to 65 nm, 1 nm to 5 nm, 1 nm to 3 nm, etc. In some embodiments, the tip portion (e.g., nanotip) of the capillary may be nano-sized, e.g., formed from nanotubes. Non-limiting examples of nanotubes include carbon nanotubes and / or nitride nanotubes (e.g., boron nitride nanotubes). In some cases, the opening of the nanotip may have a cross-sectional dimension of 1 nm to 5 nm (e.g., 1 nm to 3 nm).

[0043] In some embodiments, the body portion of the capillary may be fabricated and / or coated with a material that is substantially opaque to a wavelength of light emitted by the light source. For example, the body portion of the capillary may be substantially opaque to light having one or more ranges of wavelengths described above for the body portion of the capillary. In some cases, the capillary wall of the body portion may be constructed using an opaque material (e.g., metal). Alternatively or in addition, the capillary wall may be formed of an originally transparent material and then coated with an opaque layer (e.g., a metal coating). The body portion of the capillary may be coated using any of a variety of suitable coating techniques.

[0044] In some embodiments, the capillary may include capillary walls that are variable in thickness along the length of the capillary. For example, the capillary walls may be thinnest near the opening of the nanotip and thicker further away from the nanotip. In one set of embodiments, the thickness of the capillary wall through which light passes may increase substantially linearly with increasing distance from the nanotip (e.g., toward the body of the capillary). This, in turn, may lead to a decrease in the amount of light entering the capillary with increasing distance from the nanotip. In some embodiments, a nanotip with a thinner capillary wall may be substantially transparent to a particular wavelength of light, while a capillary body with a thicker capillary wall may be substantially opaque to that wavelength of light.

[0045] In some cases, a particularly beneficial type of laser light and / or a particularly beneficial type of wavelength of light can be used in the mass spectrometer. In some such embodiments, the light source is a UV light source and the laser light is ultraviolet light. In some cases, the use of ultraviolet light can lead to more favorable conditions for protein sequencing compared to other wavelengths of light (e.g., X-rays). Such favorable conditions include, but are not limited to, more efficient fragmentation of the species of interest (e.g., proteins) into single molecules (single amino acids), the use of lower power lasers (which allows for more practical and safer operation), reduced heating of the fluid containing the species of interest (which reduces thermal degradation of the species), etc. In embodiments where the species of interest is a protein, the use of such laser light and / or laser light of such wavelengths can result in a relatively high probability of peptide backbone cleavage, thereby allowing the formation of individual amino acids. In some cases, other types of light sources (e.g., IR) with different wavelengths can also be used.

[0046] In some embodiments, the light source is configured to generate light of any of a variety of suitable wavelengths. In some embodiments, the light source may have a wavelength of at least 150 nm (e.g., at least 155 nm, at least 157 nm, at least 160 nm, at least 165 nm, at least 170 nm, at least 175 nm, at least 180 nm, at least 185 nm, at least 190 nm, at least 193 nm, at least 195 nm, at least 200 nm, at least 210 nm, at least 213 nm, at least 220 nm, etc.). In some embodiments, the light source may have a wavelength of 230 nm or less (e.g., 222 nm or less, 220 nm or less, 213 nm or less, 210 nm or less, 200 nm or less, 195 nm or less, 193 nm or less, 190 nm or less, 185 nm or less, 180 nm or less, 175 nm or less, 165 nm or less, 160 nm or less, 157 nm or less). The wavelength values ​​referenced above may have a deviation of + / -5 nm, + / -10 nm, or + / -15 nm. Any of the ranges referenced above may be possible (e.g., at least 150 nm + / - 5 nm and not more than 213 nm + / - 5 nm, at least 160 nm + / - 5 nm and not more than 213 nm + / - 5 nm, or at least 157 nm + / - 5 nm and not more than 193 nm + / - 5 nm, at least 180 nm + / - 5 nm and not more than 213 nm + / - 5 nm, at least 193 nm + / - 5 nm and not more than 213 nm + / - 5 nm). Other ranges are possible.

[0047] In some embodiments, by using the light sources described herein, relatively high fragmentation efficiencies can be achieved. The term fragmentation efficiency, as used herein, refers to the probability that a particular basic fragment (e.g., a particular amino acid) can be cleaved from a species of interest as a single molecule. In some such embodiments, for the majority of amino acids, the fragmentation efficiency is between 60% and 95%, or between 65% and 92%.

[0048] In addition, the present disclosure generally relates to the creation of ionized molecules, in certain embodiments, for example for detection in mass spectrometers, or for other uses such as lithography, sputtering machines, propulsion, etc. Some embodiments include an ion source that includes a capillary tip that can enable direct ion evaporation of a sample with an applied electric field. In some cases, the tip may have an opening with a cross-sectional dimension (e.g., diameter) of less than 125 nm or less than 100 nm, etc. In addition, certain aspects relate to the use of capillary tips that enable detection and, in some cases, sequencing of samples (e.g., amino acids). For example, some embodiments relate to enabling rapid evaporation of single ions and ion clusters directly from aqueous samples in mass spectrometers. Other aspects relate to methods for making or using such ionized molecules, or methods for making or using devices for creating such ionized molecules, etc.

[0049] For example, some embodiments relate to an ion source that generally includes a capillary and an electrode, which may be annular in some cases, between which ions are generated when a voltage is applied. In some cases, the capillary may have a tip inner diameter of less than 125 nm or less than 100 nm, etc. This may allow ions to be evaporated directly from the meniscus of the fluid in the capillary, avoiding the wasteful droplet evaporation process. In this manner, ion evaporation may dominate the ion current, and this mode of emission may be achieved in some cases with solutions of relatively low salt concentration. In some embodiments, a tip with an inner diameter of less than 125 nm or less than 100 nm (e.g., 65 nm or less or 60 nm or less, etc.) may be able to generate a high percentage of bare ions or ion clusters, for example, with a small number of solvent molecules, e.g., as few as one or two solvent molecules. The small area of ​​the liquid-vacuum interface may prevent significant evaporation heat loss in some cases and may allow the use of volatile solvents such as water in certain cases. In some embodiments, methods such as these 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 such as those described herein can improve the sensitivity of mass spectrometry experiments, enabling single molecule protein sequencing or single cell proteomics studies. Other applications, such as those described below, are also possible.

[0050] For example, some embodiments generally relate to an ion source including a capillary and an electrode. The electrode can be used to generate ionized molecules directly from a fluid in the capillary, for example, to a reduced pressure environment or vacuum, for example, at a pressure of 100 mPa, or other pressures described herein. In certain embodiments, the opening of the capillary is dimensioned 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 by ion evaporation. The use of a capillary with a submicron opening (e.g., less than 125 nm or less than 100 nm, etc.) can be advantageous for ionizing the fluid by ion evaporation. In this case, in contrast to electrospray ionization, in which species exiting the capillary exit the capillary through a liquid jet and break up into charged droplets, which are further broken up into charged ions in the presence of background gas, the species exiting the capillary are directly ionized into single charged ions or charged ion clusters, although it should be understood that in some cases, some degree of electrospray ionization may still occur. Ion evaporation may be preferred in certain applications, such as those requiring 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 subsequently detect singly charged ions.

[0051] According to one set of embodiments, the ion source includes a capillary that defines an opening having a cross-sectional dimension (e.g., the inner diameter of the capillary) of less than 100 nm. The opening can also be dimensioned in some cases such that ion evaporation predominates over liquid jet formation when an electric field is applied. For example, in certain embodiments, at least 50% of the exiting species may be present due to ion evaporation or may be present in the form of ions or ion clusters. For example, a nanoscale capillary can enable direct evaporation of ions from the fluid meniscus. In some embodiments, a fluid can be passed through a capillary having such an opening and delivered directly to a reduced 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 may be analyzed in a mass filter and ion detector of a mass spectrometer or applied to other applications such as those described herein.

[0052] In addition, certain aspects relate to methods for sequencing (e.g., by mass spectrometry) molecules or polymers, such as biopolymers, from a fluid in a capillary. In some embodiments, the fluid includes a polymer, such as a biopolymer, dissolved in a solvent (e.g., water) with a relatively high vapor pressure. Typically, evaporation of a highly volatile solvent (such as water) can lead to freezing of the fluid at the opening of the capillary, thus limiting the ability of the mass spectrometer to successfully evaporate ions from the fluid. However, the opening of the capillary can be dimensioned, for example, as discussed herein, such that the fluid meniscus at the opening can have a smaller area, reducing such effects. Thus, using a capillary with a small opening in the ion source of a mass spectrometer can enable the study of molecules in aqueous solutions, for example, polymers or biopolymers such as amino acids, nucleic acids, and peptides or proteins. In certain embodiments, molecules that are not polymers can also be studied.

[0053] To sequence molecules such as polymers (e.g., biopolymers), certain embodiments involve applying an electric field to ionize the molecules near the opening of the capillary to generate ionized fragments. In certain embodiments, the ionized fragments are delivered directly from the fluid to the reduced pressure environment. In some cases, the ionized fragments may include, for example, single ions or ion clusters with a small number of solvent molecules (e.g., water), as discussed herein. The opening can be sized such that the ionized fragments exit the opening in sequential order according to the sequence of the molecule. For example, certain embodiments allow for the determination of the sequence of the molecule by determining the ionized fragments generated by ionizing the molecule in a detector.

[0054] In addition, certain aspects relate to an apparatus including an ion source having a capillary as disclosed herein. The apparatus may also have an electrode in the vicinity of the capillary. In some embodiments, the use of the ion source in a mass spectrometer is disclosed, but it should be noted that the use of the ion source as disclosed herein does not apply only to mass spectrometers. The ion source may also be used, for example, in lithography machines, sputtering machines, space propulsion systems, etc., as discussed herein.

[0055] Certain aspects relate to an ion source having a capillary defining an opening and an electrode disposed proximate the opening. The capillary may have an opening at an end or tip of the capillary. The opening may have any of a variety of cross-sectional dimensions and may be any shape, such as circular, elliptical, square, etc. In some embodiments, the opening includes a cross-sectional dimension of 150 nm or less, 130 nm or less, 125 nm or less, 120 nm or less, 110 nm or less, 100 nm or less, 90 nm or less, 80 nm or less, 75 nm or less, 70 nm or less, 65 nm or less, 60 nm or less, 55 nm or less, 50 nm or less, 45 nm or less, 40 nm or less, 35 nm or less, 30 nm or less, 25 nm or less, 20 nm or less, 15 nm or less, 10 nm or less, 5 nm or less, or 2 nm or less. In addition, the opening may have a cross-sectional dimension 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, 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 65 nm, at least 70 nm, at least 80 nm, at least 90 nm, etc. Combinations of these are also possible. For example, the opening may have a cross-sectional dimension of 50 nm to 100 nm, 20 to 65 nm, 1 nm to 5 nm, or 1 nm to 3 nm, etc. While the above embodiments describe a capillary having an opening at the end or tip of the capillary, it should be understood that not all embodiments described herein are so limited, and in certain embodiments, the capillary may additionally or alternatively have multiple openings along the side of the capillary. Additionally, in some cases, the device may have one or more apertures or openings, for example, in a channel or other structure. Thus, the opening need not be an opening of the capillary.

[0056] In some embodiments, the capillary is tapered at the opening. For example, the capillary may have a certain taper, such as, for example, the tip of the capillary is conical. Any suitable angle may be present. For example, the angle may 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 tapering, i.e., the capillary is cylindrical). Additionally, in some cases, the angle of taper may be at least 1 degree, at least 3 degrees, at least 5 degrees, etc. in certain cases. Combinations of these ranges are also possible, for example, the taper may be between 1 degree and 5 degrees.

[0057] In certain embodiments where the capillary is tapered at the opening, a laser pulling technique can be used to produce the tapered opening. It should be understood that techniques other than the laser pulling technique can be used to produce capillaries with tapered openings. It should also be understood that while the capillaries discussed herein have tapered openings, in other examples, the opening of the capillary may not be tapered.

[0058] 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.

[0059] In some embodiments, the capillary has a relatively high aspect ratio, such as the ratio of the length of the capillary to the cross-sectional dimension (e.g., diameter) of the opening of the capillary. For example, the capillary may have an aspect ratio of greater than 10,000. However, it should be understood that the aspect ratio is not so limited. For example, in some cases, the aspect ratio of the length of the capillary to the cross-sectional dimension of the opening may be greater than 10, greater than 100, greater than 1,000, greater than 10,000, greater than 100,000, or greater than 1,000,000.

[0060] The capillary may have a circular or non-circular cross section (e.g., square). In addition, in some embodiments, the capillary may have a relatively small cross section, e.g., diameter. For example, the cross-sectional dimension of the capillary may be less than 200 nm, less than 150 nm, less than 75 nm, less than 60 nm, or less than 50 nm.

[0061] Certain embodiments of the ion source also include an electrode positioned near the capillary, e.g., the opening of the capillary. The electrode can be used to apply an electric field to the fluid in the capillary (e.g., as described below), e.g., to the meniscus. In some cases, the fluid in the capillary can be contacted with a counter electrode, e.g., such that a voltage difference between the electrode near the opening of the capillary and a counter electrode in the capillary can generate an electric field for the fluid. In some embodiments, the electrode can be positioned such that the electric field is greatest near the opening of the capillary. For example, in some embodiments, the electrode can be positioned within 50 mm, within 40 mm, within 30 mm, within 20 mm, within 15 mm, within 10 mm, within 5 mm, within 3 mm, within 2 mm, within 1 mm, etc., of the opening of the capillary.

[0062] In some embodiments, the electrodes may be positioned around the capillary or nanotip, or in front of the capillary or nanotip, e.g., in front of or downstream of the opening of the capillary. For example, and referring to Figures 1A-1B as a non-limiting example, electrodes (not shown) may be positioned around the capillary 30 or nanotip 34, in front of the capillary 30 or nanotip 34, in front of the opening 36 of the capillary 30, or downstream of the capillary 30.

[0063] The electrodes may 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.

[0064] 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 ejected 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. Also, in some cases, the opening may be non-circular. In some embodiments, the opening of the electrode is positioned coaxially with the opening of the capillary. That is, in certain 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 application of an electric field to the fluid in the capillary, e.g., to cause the exit of ions or ion clusters from the fluid, as discussed herein.

[0065] For example, in some embodiments, the electrode has a central opening, e.g., at the end or tip of the capillary, with a cross-sectional dimension (e.g., inner diameter) larger than the cross-sectional dimension of the capillary opening. For example, according to certain embodiments, the electrode has a central opening with a cross-sectional dimension (e.g., inner diameter) 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 certain 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. In addition, in some embodiments, the front face of the electrode is positioned in front of the capillary opening.

[0066] In addition, the electrode itself may be any shape (e.g., circular or non-circular). The electrode may have the same shape as its opening (if present), or may have a different shape. 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.

[0067] In some embodiments, the electrodes include steel. Other examples include copper, graphite, silver, aluminum, gold, or conductive ceramics.

[0068] Thus, certain embodiments relate to electrodes that can generate an electric field. In some embodiments, as described above, the electrodes may be positioned such that an electric field maximum is created near the opening of the capillary. In some embodiments, the fluid is contained in the capillary such that when an electric field is applied by the electrodes near the opening of the capillary, molecules in the fluid can be ionized and exit the opening of the capillary as ions or ion clusters, such as those discussed herein. In some cases, for example, the electrodes and the capillary (e.g., the interior of the capillary) may be connectable to a voltage source, such as those discussed herein.

[0069] Thus, in certain embodiments, a voltage source can be used in conjunction with electrodes to generate an electric field to cause ions or ion clusters to exit the fluid in the capillary, e.g., as discussed 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 certain embodiments, a voltage in the range of 80V to 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, 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 such voltages are possible. For example, a voltage between 80V and 360V may be applied. The voltage may be applied as a constant voltage, or in certain cases as a varying or cyclic voltage.

[0070] As mentioned, a voltage may be applied to create an electric field maximum near the opening of the capillary or in the fluid within the capillary (e.g., at the meniscus of the opening). For example, a voltage may be applied to create an electric field maximum 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 electric field maximum may 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 also possible. For example, the electric field may be 1.5 V / nm to 3.0 V / nm, 1.5 V / nm to 4.0 V / nm, etc.

[0071] Without wishing to be 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 opening of the capillary may be dimensioned such that at least 10% of the exiting species exit by 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 by ion evaporation.

[0072] As previously described, according to certain embodiments, a cone-shaped charged fluid meniscus can be generated at the opening of the capillary under an electric field. In some embodiments, the cone-shaped fluid meniscus acts as a point source that allows species to exit as ions or ion clusters.

[0073] The fluid meniscus can generate exiting species by mechanisms such as charged droplets by electrospray ionization and / or ions and ion clusters by ion evaporation. However, in the case of electrospray ionization, the exiting species exiting the liquid meniscus will exit as charged droplets of the fluid containing the exiting species, which would typically require the presence of background gas to further break up the droplets into individual ions by a Coulomb fission process. Ion evaporation, on the other hand, refers to a process in which molecules are ionized directly into ions (e.g., bare ions) or ion clusters (e.g., ions with solvent molecules) rather than charged droplets. Ion clusters may contain single ions and multiple, usually relatively few, solvent molecules. For example, ion clusters may 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.

[0074] Thus, for example, in some embodiments, the capillary opening is dimensioned (e.g., the cross-sectional dimensions of the opening are less than 125 nm or less than 100 nm, etc.) so as to avoid the formation of charged droplets and so that at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 95%, at least 90%, at least 93%, at least 99%, or all) of the exiting species are directly ionized as ions or ion clusters from the conical fluid meniscus at the capillary opening.

[0075] As mentioned, in some embodiments, capillaries with relatively small openings (e.g., cross-sectional dimensions of less than 125 nm or less than 100 nm, etc.) may be associated with the production of relatively few solvent molecules in ion clusters, e.g., as described above. In some embodiments, the capillary openings may be dimensioned such that the solvent molecules contain no more than a certain number of solvent molecules, e.g., ion clusters produced by the ion source contain, on average, 7, 6, 5, 4, 3, or 2 solvent molecules or less (e.g., less than 125 nm or less than 100 nm, etc.). In some embodiments, a significant number of ion clusters (e.g., 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or all) contain one or two solvent molecules.

[0076] In some embodiments, a voltage can be applied to the tip of the capillary to generate a current. In some embodiments, the tip of the capillary may have a relatively low current. In some embodiments, the current at the tip of the capillary may be at least 0.1 pA (e.g., at least 0.5 pA, at least 1 pA, at least 2 pA, at least 3 pA, at least 5 pA, at least 10 pA, at least 15 pA, at least 20 pA, at least 50 pA, at least 100 pA, at least 150 pA, at least 200 pA, 500 pA, at least 1 nA, etc.). In some embodiments, the tip of the capillary may be 2nA or less (e.g., 1nA or less, 500pA or less, 200pA or less, 150pA or less, 100pA or less, 10pA or less, 20pA or less, 18pA or less, 15pA or less, 10pA or less, 5pA or less, 3pA or less, 2pA or less, 1pA, 0.5pA or less, etc.). Any of the ranges referenced above are possible (e.g., at least 0.1pA and 2nA or less, or at least 3pA and 20pA or less). Other ranges are possible.

[0077] Additionally, as discussed, certain aspects relate to methods for ionizing a fluid using an ion source to generate, for example, single ions or ion clusters. Certain embodiments include passing the fluid through a capillary defining an opening having a cross-sectional dimension, such as less than 125 or less than 100 nm, or other configurations, such as those discussed herein.

[0078] In some embodiments, the fluid includes a sample and a solvent. The sample may 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 a biopolymer (e.g., a nucleic acid such as DNA or RNA, a peptide, or a protein, etc.). Other examples include other types of polymers, such as nylon, polyethylene, etc., or other species of interest that are not necessarily polymers, such as 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 desired to at least partially determine the structure of the species, for example, by ionizing the species and detecting ion fragments, such as by mass spectrometry or other related techniques.

[0079] In some embodiments, the solvent may be any liquid that can be used to dissolve or suspend the sample or species of interest. For example, according to certain embodiments, the solvent includes water. However, the solvent is not limited to water. In some cases, the solvent may be, for example, 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, 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 may 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 referenced ranges are possible (e.g., 100 mM or more and 10 M or less, or 150 mM or more and 1 M or less).

[0080] 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 certain cases, combinations of any of these and / or other solvents are possible.

[0081] In some embodiments, the fluid includes a solvent having a relatively low pH value. For example, in some embodiments, the solvent may have a pH of at least 3 (e.g., at least 3.1, at least 3.2, at least 3.4, at least 3.6, at least 3.8, etc.). Additionally, in some embodiments, the solvent may have a pH of 4 or less (e.g., 3.9 or less, 3.8 or less, 3.6 or less, 3.4 or less, 3.2 or less, 3.1 or less, etc.). Combinations of the ranges referenced above are possible (e.g., at least 3 and 4 or less). Other ranges are possible.

[0082] Additionally, in some embodiments, the fluid includes a solvent (e.g., water) with relatively high volatility, for example, to facilitate the generation of ions or ion clusters. For example, water with a boiling point of 100° C. may be considered volatile in some cases. In some embodiments, a liquid with a boiling point near room temperature may 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 may 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 may 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 may have a boiling point between 50° C. and 100° C. Additional examples of solvents with relatively high volatility include, but are not limited to, acetone, isopropanol, hexane, etc.

[0083] In some embodiments, the temperature of the capillary (in addition to the type of fluid contained therein) 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, e.g., so that the ion clusters produced by the ion source contain, on average, 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 ranges referenced above 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.

[0084] In some embodiments, for example as discussed herein, an appropriate range of electric fields and an appropriate range of capillary opening sizes can be selected to cause at least a portion of the molecules to exit as ions or ion clusters.

[0085] Certain embodiments include sending the ionized molecules directly from the fluid to a reduced pressure or vacuum environment. Without wishing to be bound by any theory, it is noted that techniques such as electrospray ionization typically require the presence of background gas to further break up the droplets into individual ions by Coulomb fission processes. In contrast, according to certain embodiments, ions or ion clusters generated as discussed herein can be sent directly to such an environment without the need for significant amounts of background gas. Thus, certain techniques such as mass spectrometry can be performed using a reduced pressure or vacuum environment without necessarily requiring the addition of background gas.

[0086] Thus, in one set of embodiments, the capillary may be positioned to allow ions or ion clusters exiting the opening to enter a reduced pressure or vacuum environment. In some cases, the environment may be an environment having a pressure of 100 mPa or less. In certain embodiments, the environment may have a pressure of 1000 mPa or less, 300 mPa or less, 100 mPa or less, 30 mPa or less, 10 mPa or less, 3 mPa or less, 1.5 mPa or less, 1 mPa or less, 0.3 mPa or less, 0.1 mPa or less, etc. In some embodiments, ions or ion clusters from the fluid are delivered directly to the vacuum environment.

[0087] It should be understood that some of the embodiments provided herein have focused on directly delivering ionized molecules from a fluid to 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. Also, in some embodiments, the pressure may be 100 mPa or more and 1 Pa or less.

[0088] In some embodiments, the mass spectrometer includes a pump. The pump can be used to create a reduced pressure or vacuum environment, for example as discussed herein. Non-limiting examples of pumps include a diffusion pump, a molecular drag pump, or a turbomolecular pump.

[0089] In some embodiments, there may be a relatively high pressure difference between the vacuum chamber and the fluid at the capillary opening. For example, the pressure may be about 1 atmosphere where the fluid enters the capillary, and in the vacuum chamber where the capillary opening is located, it may be about 100 mPa or other reduced pressure such as those described herein. However, in some cases such as those described herein, the fluid meniscus at the capillary opening 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 capillary opening may be at least 0.1 atmosphere, at least 0.2 atmosphere, at least 0.3 atmosphere, at least 0.4 atmosphere, at least 0.5 atmosphere, at least 0.6 atmosphere, at least 0.7 atmosphere or more, at least 0.8 atmosphere, at least 0.9 atmosphere, at least 1 atmosphere, etc. Furthermore, in some embodiments, the hydrodynamic resistance of the fluid in a capillary such as those described herein (e.g., a capillary with an opening less than 100 nm) may be higher than the hydrodynamic resistance in an ion source used in electrospray ionization.

[0090] According to certain embodiments, the opening of the capillary is dimensioned such that the solvent having a relatively high volatility remains unfrozen even when exposed to a relatively low pressure at the opening of the capillary. In some embodiments, the opening of the capillary is small enough that the solvent having a relatively high volatility remains unfrozen as it enters the surrounding environment. In some embodiments, the opening of the capillary is small enough that the fluid including the sample and the solvent remains unfrozen when the species of interest are ionized, and at least a portion of the species of interest ionize to form ions (e.g., single ions) or ion clusters.

[0091] Some aspects relate to mass spectrometers including ion sources as described herein. However, ion sources such as those described herein are not limited to mass spectrometers, but can also be used in other applications such as lithography, sputtering machines, propulsion (e.g., space propulsion), etc. As a non-limiting example, in lithography, a focused ion beam (FIB) machine can be used to inspect and / or modify lithography masks and / or etch features into materials by sputtering. Sputtering is a process of removing atoms from the surface of a solid by ions colliding with high kinetic energy. In some embodiments, the ion sources described herein are present in a focused ion beam (FIB) machine and can be used to pattern and deliver molecules into a substrate material.

[0092] In certain embodiments, the ion source as described herein can be used in a liquid chromatography mass spectrometry system. For example, a liquid chromatograph can be connected to the ion source to separate peptides or other molecules before ionizing and delivering 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 use of the ion source as described herein (having a capillary with a nano-sized opening and / or tip) to deliver ions directly to a low pressure environment can improve the instrument sensitivity, ion transmission efficiency in such systems, and can eliminate the need for multiple pumping stages.

[0093] In certain embodiments, an ion source as described herein can be used as both a nanopipette and an ion source. For example, a capillary as described herein (e.g., a pre-drawn quartz capillary) with a nano-sized tip can be used to pierce a cell or tissue and extract its biomolecular contents. The capillary can then be directly inserted into a vacuum chamber (e.g., having a relatively high vacuum, such as a chamber with reduced pressure, such as those described herein), and the extracted molecules can be ionized and delivered to a mass spectrometer. Such techniques can be used, for example, to sample relatively small liquid volumes, such as the contents of a single cell. For example, such techniques can be used for single cell proteomics studies.

[0094] As another example, in some embodiments, the ion sources described herein are used for propulsion. For example, ejecting ions backwards can generate a force that propels an object forward. In some embodiments, ion sources as described herein are used in propulsion systems. This can be used to deliver high thrust compared to the weight of the ion source, for example due to the small size of the ion source. Additionally, in some cases, the propulsion system can be compact and consume relatively less fuel compared to conventional propulsion systems.

[0095] Additionally, some embodiments relate to mass spectrometers having an ion source as described herein. In some cases, the mass spectrometer may include components such as a vacuum chamber (e.g., capable of generating any of the reduced pressures described herein) in addition to an ion source such as those described herein, ion optics (e.g., one or more lenses such as an Einzel lens), a mass filter (e.g., a quadrupole mass filter, a magnetic sector mass filter, etc.), a detector, an ion bender, or an ion trap. Examples of specific detectors include, but are not limited to, Faraday cups, electron multipliers, dynodes, charge-coupled devices (CCDs), CMOS sensors, and fluorescent screens. Additional non-limiting examples of mass spectrometers are described in the provisional application entitled "Systems and Methods for Single-Ion Mass Spectrometry with Temporal Information," filed April 23, 2021, which is incorporated by reference in its entirety.

[0096] In addition to the ion source, various ion optics can be positioned downstream of the ion source so that the exiting molecules (e.g., ions and ion clusters) can be transported, in certain cases, along a path downstream of the ion source. For example, the downstream direction is the direction in which the ions or ion clusters travel. Referring again to FIGS. 1A-1B as a non-limiting example, the ion optics 100 can be positioned downstream of the ion source 20 so that the exiting molecules 54 can be transported along a path downstream of the ion source 20. Those skilled in the art will be familiar with various ion optics used in mass spectrometry. In some embodiments, the ion optics includes one or more Einzel lenses (e.g., a first Einzel lens and a second Einzel lens). When the ion optics transmits the molecules (e.g., ions or ion clusters) to the mass filter, the mass filter can analyze the mass-to-charge ratio (m / z) of the molecules (e.g., ions and ion clusters). In some cases, the mass filter can be positioned downstream of the ion optics. 1A-1B, a mass filter 100 (e.g., a magnetic mass filter) may be positioned downstream of the ion optics 100. Examples of mass filters include, but are not limited to, quadrupole mass filters, magnetic sector mass filters, and the like.

[0097] In some embodiments, one or more detectors may be positioned further downstream of the mass filter. Referring again to FIGS. 1A-1B as a non-limiting example, one or more detectors 70 may be positioned downstream of the magnetic filter 90. The detector may be any suitable detector capable of detecting ions or ion clusters. In some embodiments, ions and ion clusters having a mass-to-charge ratio (m / z) within the acceptance window of the mass filter are sent to an ion bender. The ion bender may be configured to deflect the ions and ion clusters away from the mass filter and towards a detector. For example, as a non-limiting example, the ions or ion clusters are sent from the ion bender to a detector. In some embodiments, the detector may be used to determine the ions or ion clusters.

[0098] In some embodiments, mass spectrometers as described herein may include an average or overall ion transmission ratio (e.g., the ratio of detected ions and ion clusters to ions and ion clusters exiting the fluid at the opening of the capillary) of greater than 0.01, in some cases 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, at least 0.9, at least 0.93, at least 0.95, at least 0.99, etc. In some cases, the overall ion transmissibility may be 1 or less, 0.99 or less, 0.95 or less, 0.93 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 ranges referenced above are possible (e.g., at least 0.02 and 0.9 or less, or at least 0.1 and 0.8 or less, at least 0.9 and 1 or less, etc.). Other ranges are possible.

[0099] In one set of embodiments, a mass spectrometer including a tip having an aperture with a cross-sectional dimension described herein (e.g., less than 100 nm, 65 nm or less, 60 nm or less, 50 nm or less, 40 nm or less, 30 nm or less, etc.) can exhibit an average ion transmission efficiency of at least 85% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least 99%, etc.). In some embodiments, the average ion transmission efficiency referenced above may have a deviation of + / -3%, + / -2%, or + / -1%.

[0100] In some embodiments, a tip having an inner diameter of 65 nm or less (e.g., 60 nm or less, 40 nm or less, 20 nm or less, etc.) can be used to generate a high percentage (e.g., at least 0.7, at least 0.8, at least 0.9, at least 0.95, or at least 0.99, or equal to 1) of bare ions, e.g., ions that are free of solvent molecules. In some embodiments, only bare ions are generated by the tips described above. In some cases, releasing bare ions as opposed to ion clusters or charged droplets can be particularly advantageous, as direct release of bare ions can allow for improved determination of various amino acids (e.g., including variants of amino acids with post-translational modifications).

[0101] In some embodiments, when ions and / or ion clusters (if present) are ejected from the tip of the capillary into a vacuum, the ions and / or ion clusters experience few, if any, collisions with gas molecules (e.g., background gas molecules). For example, in some embodiments, the probability that the ions and / or ion clusters will experience collisions with gas molecules is less than about 2% (e.g., less than about 1.5%, less than about 1%, less than about 0.5%, or equal to 0%).

[0102] Certain aspects relate to sequencing polymers, such as biopolymers, using instruments that include an ion source, such as a mass spectrometer as described herein.

[0103] For example, in some embodiments, a polymer may be the species of interest. The species of interest may be a biopolymer, such as 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 also be used as the species of interest. In addition, it should be understood that in some cases, other types of polymers, such as artificial or synthetic polymers, may also be sequenced. Furthermore, the structure of a species of interest that is not a polymer may be determined as well.

[0104] In some cases, for example, the structure, sequence, and / or identity of a species of interest (e.g., a polymer) can be determined by measuring 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) generated by, for example, ionizing a polymer and generating ions or ion clusters as discussed above reach a detector. Without wishing to be bound by any theory, it is believed that a species of interest, such as a polymer, can be ionized in a substantially linear manner, for example, by the size of the opening of the capillary, and the generated ions or ion clusters can then be determined by a detector as discussed herein, for example, in the order in which the ions or ion clusters were generated from the species of interest. In some embodiments, the capillary comprises a carbon nanotube or a boron nitride nanotube, the cross-sectional dimension (e.g., inner diameter) of the nanotube being small enough, for example, 1 nm to 2 nm, to be able to ionize the polymer molecules in a sequential order that reflects the primary structure of the polymer. Of course, larger diameters or other materials are possible in other embodiments, for example, as discussed herein. It should be noted that in some cases, for example when the ions or ion clusters are directed into a reduced pressure environment, the detector may be able to determine such order with relatively high fidelity, for example because the ions or ion clusters experience relatively few collisions with gas molecules as they pass through 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.

[0105] In some embodiments, the mass spectrometers described herein may include more than one ion source. For example, the mass spectrometer may include an ion source described herein along with one or more additional ion sources. For example, in some aspects of the present disclosure, multiplexed mass spectrometers are disclosed herein that include multiple ion sources. The multiple ion sources may be multiple identical (or different) ion sources. Some or all of the multiple ion sources may include a capillary that includes a species of interest suspended in a fluid. The multiplexed mass spectrometers, in some embodiments, may enable simultaneous detection and sequencing of multiple species of interest contained within the capillaries of the multiple ion sources.

[0106] In some embodiments, a multiplex mass spectrometer may include multiple ion sources, a magnetic mass filter downstream of the multiple ion sources, and an array of detectors downstream of the magnetic mass filters. A non-limiting schematic diagram of a multiplex mass spectrometer is shown in FIG. 12. As shown, a multiplex mass spectrometer 110 includes multiple ion sources 120, a mass filter 190 (e.g., a magnetic mass filter) downstream of the ion sources 120, and an array of detectors 170 (e.g., an imaging detector) downstream of the mass filter 190. Some or all of the multiple ion sources shown in FIG. 12 may be the same as the ion sources shown in FIGS. 1A-1B. For example, some or all of the ion sources may include a capillary and an electrode in the vicinity of the capillary or the nanotip of the capillary. The capillary may have any of the characteristics described elsewhere herein, such as, for example, a tip portion (e.g., a nanotip) having an opening, a body portion, and / or including a species of interest suspended or dissolved in a fluid. In some cases, the multiple ion sources may be arranged in a linear array. In some cases, some or all of the multiple capillaries may include nanotips arranged in a linear array. For example, as shown in FIG. 12, mass spectrometer 110 includes multiple capillaries 130a, 130b, and 130c, each having a nanotip, arranged in a linear array. In some embodiments, some or all of the multiple ion sources may include a species of interest within the capillary. The species of interest within the ion sources may be the same or different.

[0107] A multiplex mass spectrometer may include any suitable number of ion sources. For example, a multiplex mass spectrometer may include at least two (e.g., at least three, at least five, at least ten, at least twenty-five, at least fifty) and / or up to one hundred (e.g., up to two hundred, up to five hundred, or up to one thousand) ion sources. Combinations of the ranges referenced above are possible (e.g., at least two and up to one thousand). Other ranges are possible.

[0108] In some embodiments, the multiplex mass spectrometer may further include a light source directed toward the multiple ion sources. The light source may have any of the characteristics and / or configurations described elsewhere herein. For example, the multiplex mass spectrometer shown in FIG. 12 may include a light source (not shown) identical to the light source (e.g., a UV laser) shown in FIGS. 1A-1B. As described elsewhere herein, the light source may be configured to fragment a species of interest in a capillary (e.g., a tip portion of the capillary) of the ion source into individual components. The fragmented individual components may then be ionized from the tip of the capillary into a vacuum. For example, as shown in FIG. 12, the light source may be configured to fragment a species of interest in a capillary (e.g., a tip portion of capillaries 130a, 130b, 130c) of the ion source 120 into individual components, and then ionize the individual components into a vacuum 180. The trajectories of the individual components ionized from the nanotip are indicated by 154.

[0109] In some embodiments, the multiplex mass spectrometer includes a magnetic mass filter capable of simultaneously separating the individual ionized components exiting each ion source based on their mass-to-charge ratio. For example, the magnetic mass filter may be configured to transmit the individual ionized and fragmented components from each of the ion sources to an array of detectors downstream of the magnetic mass filter. The array of detectors may in turn be configured to simultaneously detect the individual ionized and fragmented components from each of the multiple ion sources. For example, as shown in FIG. 12, a magnetic mass filter 190 (e.g., a magnetic sector) may be used to separate the individual ionized components exiting each ion source based on their mass-to-charge ratio.

[0110] According to some embodiments, a magnetic mass filter can be used to separate and focus the ionized individual components in both the lateral and transverse directions before the individual components impact an array of imaging detectors. Referring to FIG. 12 as a non-limiting example, a magnetic mass filter 190 can be used to separate and focus the ionized individual components 154 in both the lateral and transverse directions before the individual components impact an array of detectors 170 (e.g., imaging detectors). According to some embodiments, the array of detectors can be arranged in a two-dimensional array capable of detecting the ionized individual components in both the lateral and transverse directions. A multiplexed mass spectrometer having the configuration described herein can enable simultaneous and high-throughput sequencing of various types of species of interest.

[0111] The multiplex mass spectrometer may include any suitable additional components described elsewhere herein. For example, the multiplex mass spectrometer may further include ion optics (e.g., optical lenses, etc.) positioned between the ion source and the mass filter. A non-limiting example of one embodiment of ion optics is illustrated in Figures 1A-1B, for example as shown by ion optics 100.

[0112] U.S. Provisional Patent Application No. 63 / 235,601, filed August 20, 2021, by Stein et al., entitled "System and Methods for Analysis of Peptide Photodissociation for Single-Molecule Protein Sequencing," is incorporated herein by reference in its entirety. Additionally, U.S. Provisional Patent Application No. 63 / 341,992, filed May 13, 2022, by Stein et al., entitled "System and Methods for Analysis of Peptide Photodissociation for Single-Molecule Protein Sequencing," is incorporated herein by reference in its entirety. The publication entitled "System and Methods for Analysis of Peptide Photodissociation for Single-Molecule Protein Sequencing" by Stein et al. is incorporated herein by reference in its entirety.

[0113] U.S. Provisional Patent Application No. 63 / 015,407, filed April 24, 2020, by Stein et al., and entitled "Nanotip Ion Sources and Methods," is hereby incorporated by reference in its entirety.

[0114] Additionally, U.S. Provisional Patent Application No. 63 / 179,064, filed April 23, 2021 by Stein et al., entitled "Systems and Methods for Single-Ion Mass Spectrometry with Temporal Information," is incorporated herein by reference in its entirety. International Patent Application Publication No. PCT / US2022 / 025902, filed April 22, 2022, entitled "Systems and Methods for Single-Ion Mass Spectrometry with Temporal Information," is incorporated herein by reference in its entirety.

[0115] International Patent Application Publication No. PCT / US2021 / 028954, filed April 23, 2021, is hereby incorporated by reference in its entirety.

[0116] The following examples are intended to illustrate certain embodiments of the present disclosure, but do not exemplify the complete scope of the disclosure.

[0117] [Example 1] In this example, we analyze the feasibility of using light to fragment peptides into their constituent amino acids for single molecule protein sequencing, followed by identification of the constituent amino acids by mass spectrometry (MS). Laser power considerations make it highly advantageous to photofragment the peptides in solution before they leave the ion source, rather than photofragmenting in the gas phase. Ultraviolet (UV) wavelengths around 200 nm are weakly absorbed by water, and single photons can selectively cleave the peptide bonds that link amino acids together. These properties make UV photofragmentation more promising than methods based on infrared or X-ray light. In this example, we develop a simple model of the probability that an amino acid will be released intact by cleaving the peptide bonds on either side of the amino acid before the light can damage the amino acid itself. 193 nm light is capable of releasing many amino acids with probabilities ranging from 0.65 to 0.92, while aromatic amino acids, as well as histidine, methionine, arginine, and lysine, which are relatively susceptible to photodamage, are predicted to be released intact with probabilities ranging from 0.004 to 0.330. These findings suggest that UV photofragmentation can reveal a significant amount of single-protein sequence information to a mass spectrometer.

[0118] We describe a method for sequencing single proteins based on a nanocapillary ion source. The basic idea is illustrated in Figure 1A and Figure 1B. A voltage applied to the source drives positively charged peptides toward the nanopore tip, which is small enough to force the peptide chain into a linear configuration. The ion source ejects the constituent amino acids from the liquid into the vacuum in sequential order. The amino acid ions pass through a magnetic mass filter that separates them based on their mass-to-charge ratio before colliding with an array of single-ion detectors. The location of the collision reveals the identity of the amino acids, and the timing of detection provides information about their original sequence. In one intermediate step, individual amino acids are separated from the parent peptide chain before mass filtering. Light is widely used to photofragment peptides in proteomics. In this example, we analyze the feasibility of using laser light to photofragment peptides in our single-molecule protein sequencing approach.

[0119] FIG. 1A is a schematic of single molecule protein sequencing by nanopore mass spectrometry. The schematic shows the trajectories of heavy and light amino acid ions emitted from a nanocapillary ion source. The ions pass through ion optics and a magnetic mass filter and impinge on an array of single ion detectors. An ultraviolet laser is used to fragment the peptides. FIG. 1B shows that an extended peptide chain is photofragmented near the tip of the nanocapillary ion source.

[0120] To fragment peptides before they pass through the mass filter, a laser beam may be directed into the path of the ions on the vacuum side of the ion source. The problem with this approach is that peptides pass through the beam very quickly. For example, consider the arginine dipeptide, which has a mass of 330 amu and a charge of +2e. If an extraction voltage of 300 V is applied (close to the lower limit required for the ion source), the dipeptide ion will gain 600 eV of kinetic energy and travel the entire distance from the ion source to the detector of our instrument in less than 20 microseconds, spending even less time in the beam path. This sets a lower limit on the irradiation power required to achieve a high probability of fragmentation. For comparison, previous studies have irradiated peptides with a 50 W CO2 laser for 10 ms or longer to induce photofragmentation in vacuum. To deliver the same amount of energy in 20 microseconds as a 50 W laser dose for 10 ms would require 25,000 W of laser power. Therefore, to fragment peptides on the vacuum side of the ion source would require impractically powerful lasers.

[0121] Another possibility is to aim a laser at the peptides while they are still in solution inside a nanocapillary ion source. In solution, peptides move at least seven orders of magnitude slower than in vacuum. The most rapid transport process is thought to be the electroconvection mechanism.

[0122] The flow of ions along the charged surface of the Taylor cone induces a circular fluid flow that reaches a maximum velocity near the tip. The maximum velocity of the nanocapillary is 10 -4m / s. Another relevant transport mechanism is electrophoresis, but in this example, even at the highest electric field of the instrument acting on the electrophoretically most mobile peptide, the resulting migration speed is an order of magnitude slower than electroconvection. Brownian motion is also a relatively slow transport process over distances comparable to the radius of the laser beam. Even the most diffusive peptide would take almost 3 min to diffuse a distance of 0.5 mm, two orders of magnitude longer than the time electroconvection would take to travel the same distance. Thus, peptides in liquids need to pass through the laser beam at a speed slow enough that a 0.1 W laser would deliver the same energy to the peptide as a 50 W laser would deliver in 10 ms. Clearly, targeting peptides still in solution reduces the demands on the incident laser power sufficiently that liquid photofragmentation is feasible and safe.

[0123] The wavelength of light influences the type of molecular fragments that result from photofragmentation. Figure 2 shows the structure of a dipeptide, the structure of common photofragmentation products, and the approximate frequency with which laser light of different wavelength regions induces specific transformations in vacuum. A parent dipeptide contains two amino acids that share a chemical backbone. The peptide bond in the backbone requires only approximately 4 eV to break, making it one of the most unstable bonds in the molecule. If that bond could be selectively broken, the resulting amino acids should be easily identified by their mass. Alternatively, breaking different bonds in the backbone would result in some shift in mass from one amino acid to its neighbors, but each amino acid should be able to be identified by taking that mass shift into account. However, if the photons damage or eject the amino acid side chains that are distinguishing features, our protein sequencing scheme could be significantly complicated.

[0124] Figure 2 shows a comparison of peptide photofragmentation by IR, UV, and soft X-ray light. Line thickness indicates the relative abundance of key fragmentation products, estimated from studies in vacuum.

[0125] The absorption of light by water further limits the properties of light that can be used for sequencing. The linear absorption coefficient of water at 10.6 micrometers (μ) is 10 5 m -1 , an incident IR laser beam will be absorbed over a feature distance of only 10 μm.35 This suggests that only a small fraction of the laser power will be absorbed by peptides in solution, and the incident power required to induce the multiphoton dissociation process may be quite high. Absorption of UV light in water is orders of magnitude weaker, with μ=10m at 193 nm. -1 and at 222 nm, μ = 1 m -1 and characteristic absorption distances are 10 cm and 1 m, respectively. Presumably, heating effects set an upper limit on the incident power density at a given wavelength. If the absorbed light heats the water in the nanocapillary to its boiling point, it is likely to disrupt the ion evaporation process.

[0126] The temperature rise that should result from the absorption of light at different wavelengths in the IR and UV was calculated numerically. The capillary was modeled as a truncated cone with a tip radius of 20 nm, a length of 500 micrometers, and a cone aperture of 6°. It was assumed that a 1 mm wide laser beam centered on the capillary tip was irradiated onto the cone from the side (light arriving perpendicular to the cone axis). This results in the entire cone being exposed to a uniform incident power density ρ. For example, a 1 W laser beam focused to a diameter of 1 mm would have a power density of 1.3 × 10 6 W / m 2 The steady-state temperature distribution was calculated by solving the steady-state heat equation.

[0127]

number

[0128] Figure 3A shows the normalized steady-state temperature distribution along the axis of the cone. The temperature increases monotonically from base to tip for all wavelengths studied, but at UV wavelengths of 193 nm and 222 nm, the increase is relatively steeper than at the IR wavelength of 10.6 micrometers.

[0129] In Figure 3B, the maximum temperature rise at the tip is plotted as a function of ρ. The maximum temperature rise is strongly wavelength dependent. Light at 10.6 micrometers causes significant heating at the tip, and a power density of only about 4 × 10 is required to reach the boiling point of water in room temperature experiments. 4 W / m 2 , which corresponds to a 32 mW laser with a beam diameter of 1 mm. In contrast, UV wavelengths cause minimal heating, at 10 7 W / m 2 Even at the very high power densities of 193 nm and 222 nm light should heat the tip by less than 10 K and 1 K, respectively. These results indicate that heating of the water limits the use of IR light in this sequencing scheme.

[0130] Figure 3A shows the calculated heating profile in the nanocapillary under steady-state irradiation with 10.6 μm, 193 nm, and 222 nm light. Figure 3B shows the dependence of the maximum temperature increase in the nanocapillary on the incident laser power density for 10.6 μm (triangles), 193 nm (squares), and 222 nm (circles) light. Symbols show the results of finite element method calculations and curves are linear fits of the data.

[0131] To summarize this comparison of wavelengths, UV light offers relatively specific cleavage of the peptide backbone, low power requirements, and low absorption by water, all of which are advantageous for sequencing single proteins.

[0132] In this example, we will now evaluate whether UV light can reliably separate amino acids from one another without damaging them so much that they cannot be identified. The probability that the backbone between two amino acids will be cleaved should increase with UV exposure, but the chance that other bonds will be cleaved should increase as well, which could complicate sequencing. To evaluate these trade-offs, we developed a simple stochastic model of the photofragmentation process.

[0133] Table 1 shows the UV absorption and photolysis properties of peptides and amino acids.

[0134] [Table 1]

[0135] Peptide bonds and amino acids behave as independent UV absorbers in aqueous solution. The molar extinction coefficient ε i is due to the respective extinction coefficients at 193 nm. Thus, a particular peptide bond or amino acid has an average ratio Jσ i where J is the local photon flux and σ i is ε i is the absorption cross section of absorber i, calculated from: Following absorption of a photon, the added energy can result either in molecular fragmentation or in vibrational dissipation processes in which the chemical bonds remain intact. The fraction of absorbed photons that cause photodissociation, Φ i is also called the quantum efficiency. Combining the absorption and dissociation processes, the average photodissociation rate of a particular species, Jσ i Φ i is obtained.

[0136] The cumulative probability P of a particular peptide or amino acid undergoing photodissociation d,i increases with time t according to the following formula:

[0137]

number

[0138] 15 of the 20 different amino acids and peptide bonds, as well as the ε and σ of hydroxyproline (a common modified form of the amino acid proline) i , and Φ i The experimental values ​​for are summarized in Table 1. The experimental values ​​for the amino acids asparagine (asn), cysteine ​​(cys), glutamine (gln), glutamic acid (glu), and isoleucine (ile) are unknown.

[0139] In Figure 4A, the cumulative probability of peptide bond dissociation P for different values ​​of ρ is shown. d,pep are being compared. d,pep rises and asymptotically approaches 1 with a characteristic timescale that is inversely related to ρ. The characteristic timescale corresponds approximately to a power density of ρ=10,000 W / m, which corresponds to a 10 mW, 1 mm wide laser beam. 2 In this case, it is 0.7 seconds.

[0140] It may also be useful to estimate the number of amino acids that can survive UV light exposure without undergoing photodissociation. The cumulative survival probability P s,i is as follows:

[0141]

number

[0142] In Figure 4B, r = 10,000 W / m 2 In the case of P of 16 different amino acids s,i is plotted. Residual capacity of amino acids as measured by characteristic degradation time (Jσ i Φ i ) -1is strongly dependent on its type. The aromatic amino acids tyrosine (Tyr), phenylalanine (Phe), and tryptophan (Trp) decay relatively rapidly, undergoing photolysis on timescales of 0.07, 0.17, and 0.20 seconds, respectively. Histidine (His) also decays relatively rapidly, with a characteristic decay time of 0.20 seconds. In comparison, the amino acids valine (Val), threonine (Thr), leucine (Leu), serine (Ser), proline (Pro), hydroxyproline (Hyp), glycine (Gly), alanine (Ala), and aspartic acid (Asp) are longer-lived, with decay timescales ranging from 7 to 56 seconds. The decay times of methionine (Met), arginine (Arg), and lysine (Lys) are between the long-lived and short-lived groups.

[0143] In this example, we also investigate the probability that a given amino acid will be released from the protein without damaging it beyond recognition. The most obvious mechanism is to fragment the two peptide bonds connecting amino acid i to the peptide chain without inducing photodissociation of the amino acid itself. The cumulative probability of such selective cleavage P sel,i is obtained by combining Equation 2 and Equation 3.

[0144] P sel,i =P S,i (P d,pep ) 2 (4)

[0145] In Figure 4C, ρ = 10,000 W / m 2 The 16 amino acids in sel,i The time evolution of is plotted. In all cases, P sel,i increases with time, peaks, and then decays. For amino acids with longer characteristic dissociation times, the peaks are higher and occur after longer UV exposure. P sel,iP peaks within 0.25 s in the range of 0.004–0.028 for aromatic amino acids and His. The long-lived amino acids peak in the range of 0.65–0.92 after exposure times ranging from 2–3 s. The intermediate groups (Met, Arg, and Lys) peak in the range of 0.086–1.33 after exposure times ranging from 0.5–1.5 s. sel, to reach i.

[0146] FIG. 4A shows the cumulative probability of peptide bond dissociation obtained from Equation 2 for exposure to 193 nm laser light of different intensities as indicated. FIG. 4B shows the cumulative probability of peptide bond dissociation calculated according to Equation 3, where ρ=10,000 Wm -2 Figure 4C shows the probability of selective amino acid release (i.e., fragmenting the two peptide bonds joining an amino acid to a peptide without damaging the amino acid) as a function of exposure time to 193 nm laser light at ρ=10,000 Wm -2 The probability is shown as a function of exposure time to 193 nm laser light. The probability was calculated according to Equation 4. In Figures 4B and 4C, the different amino acids are color coded and ordered by probability.

[0147] One finding is that it should be possible to completely liberate intact amino acids from proteins with a fairly high efficiency by UV light. A free amino acid is defined as resulting from the cleavage of two flanking peptide bonds. Many amino acids can be liberated with probabilities ranging from 65 to 92%. If one also considers the possibility that an amino acid may be liberated by cleavage of a different (i.e. non-peptide) bond along the backbone, or if an amino acid may still be identifiable by its mass after other photodissociation processes, the percentage of identifiable fragments should increase. For example, cleavage of the bond of the aromatic group of an amino acid does not change its mass, but may significantly change its optical absorption spectrum (a change that would be recognized as photodissociation in optical measurements).

[0148] In conclusion, UV light offers a promising route to fragment peptides into their constituent amino acids for single molecule analysis. Because ions travel very fast from the ion source to the detector in a vacuum, it is preferable to photofragment peptides while they are in solution before extracting the ions. UV wavelengths around 200 nm are most promising for sequencing due to their low absorption in water, relatively high selectivity in fragmenting peptide backbone bonds, and the modest laser power required to trigger the single-photon bond cleavage process. Calculations of the rates of competing photochemical processes indicate that it should be possible to cleave peptide bonds flanking many amino acids before the side chains used for identification are damaged. The accuracy of amino acid calling in sequencing can exceed 90% for the most stable side chains, but is expected to decrease with increasing side chain instability. Future measurements of photofragmentation products and the relative selectivities of various wavelengths can be used to optimize UV ​​photofragmentation for analyzing the composition and sequence of single proteins.

[0149] [Example 2] Introduction Mass spectrometry (MS) is a workhorse technique in proteomics research due to its ability to distinguish amino acids and small peptides by mass. Its usefulness also depends critically on the availability of soft ionization techniques to transfer peptide ions intact into the gas phase. In particular, electrospray ionization (ESI) transfers analytes to the mass spectrometer via a plume of charged droplets emerging from a liquid cone jet at the end of a voltage-biased capillary, as shown in Figure 5A. The droplets pass through a background gas, which induces a series of evaporation and Coulomb explosion cycles, ultimately releasing the analyte ions into the gas phase. However, the background gas required to liberate ions from the droplets is also responsible for significant sample loss that limits the sensitivity of MS.

[0150] The background gas and the plume of charged droplets it creates disperse the ions widely, most of which collide with the transfer capillary that bridges the ambient pressure ion source and the first pumping stage of the mass spectrometer or other hardware components upstream of the detector. Early ESI sources had emitter tips several hundred micrometers in diameter and were approximately 10 4 Only one ion in every 100 ions reaches the mass analyzer. Nanoelectrospray ionization (nanoESI) has increased the ion transmission efficiency to about 1% in typical measurements (and in some cases even to 12%) by using emitters with micrometer-scale tips that reduce the flow rate to the range of a few nL / min. However, ESI involves a process that physically separates different ion species in the plume, making it fundamentally difficult to optimize the efficiency for multiple analytes simultaneously. Even with state-of-the-art MS instruments, thousands to millions of copies of a protein are still required for identification. This sensitivity falls short of the sensitivity required for single-cell proteomics and single-molecule analysis. To achieve single-molecule sensitivity, an ion source is needed that avoids the loss mechanism of spraying charged droplets into the background gas.

[0151] Herein, a nanopore ion source is presented that directly ejects amino acid ions and small peptide ions from its tip into high vacuum (Figure 5B). The ion source included a predrawn quartz capillary with a tip with an inner tip diameter smaller than 100 nm. It is believed that the small size of the tip could potentially affect ion ejection in several ways. First, the surface tension of water can maintain a stable liquid-vacuum interface supporting many atmospheres of pressure even when stretched across a nanoscale opening. Second, the fluid flow rate, which scales with the inverse of the cube of the tip diameter, may be too low to form a stable electrospray cone-jet, thereby preventing the complete ejection of charged droplets. Third, the electric field can be concentrated at a sharp conductive tip such as an electrolyte-filled nanocapillary, reaching approximately 1 V / nm at the meniscus, and extract ions at high speeds through the process of ion evaporation.

[0152] In this example, characterization of ion emission from aqueous solutions directly into high vacuum by a nanopore ion source is described here. -6 Mass spectra of amino acids and small peptides were obtained using a custom quadrupole mass spectrometer operating at pressures below 1000 psig (Figure 5C). Separately, current transmission rates of greater than 93% were measured between the electrolyte-filled ion source and a downstream Faraday cup. Furthermore, we showed that the contributions of charged droplets and ions to the tip current can be separated using a magnetic sector, thereby forcing the nanopore ion source to release only ions. This example illustrates the simplicity of the nanopore ion source described herein to efficiently transfer ions into high vacuum without the complexities of traditional ESI, such as ion funnels, multiple pumping stages, transfer capillaries, and droplet plumes.

[0153] result Emission of amino acid ions from a nanopore ion source The release of amino acids from aqueous solutions was characterized with a custom quadrupole mass spectrometer, shown in Figure 5C. In a typical experiment, an extraction voltage V ranging from +260 V to +360 V was applied between the tip and the extraction electrode. E Ion emission from the nanopore ion source was initiated by applying a voltage of V E may be significantly lower than the voltage typically required to initiate electrospray in a conventional ESI or nanoESI source. The tip current I used T was typically in the range of 3-20 pA. The onset of the current could be sudden and is usually associated with the measurement of ions striking the instrument's detector. Even at these low tip currents, readily interpretable mass spectra were collected within minutes.

[0154] FIG. 5D shows the mass spectrum of a 100 mM solution of arginine in water. The spectrum was acquired in positive ion mode using a nanopore ion source with a tip inner diameter of 41 nm. Five peaks are clearly visible. The peak at 175 m / z corresponds to the singly charged arginine ion (Arg+ ). All the higher m / z peaks are separated by 18 m / z, and this shift is caused by an additional water molecule. Therefore, the other peaks correspond to the solvated state of arginine (Arg + (H2O) n ) where the solvation number n is in the range of 1 to 4.

[0155] Figure 6A shows how tip diameter affected the mass spectrum of arginine. The spectra shown were obtained using nanocapillaries with tip inner diameters of 20 nm, 125 nm, and 300 nm. The largest tip captured a bare arginine ion, a cluster of eight stepwise hydrated arginine ions, and an arginine dimer ion (Arg Arg+H). + The medium-sized tip produced a broad spectrum of peaks that included a peak at 349 m / z corresponding to arginine ions, six stepwise hydrated arginine ion clusters, and a relatively reduced arginine dimer ion peak. The smallest tip produced primarily bare arginine ions, but attenuated peaks corresponding to mono- and dihydrated arginine ion clusters were also visible in the spectrum. As can be seen by comparing the baselines of the three spectra in Figure 6A, the smaller tips tended to produce relatively stronger signals and less noisy spectra than the larger tips. Some differences were observed in the distribution of solvation states between nanocapillaries with similar tip sizes (e.g., when comparing the spectrum in Figure 5D produced by a 41 nm tip with the spectrum in Figure 6A produced by a 20 nm tip). However, only nanocapillaries with tip inner diameters smaller than about 65 nm produced spectra in which the majority of amino acid ions were measured in the unsolvated state.

[0156] Figure 6B shows mass spectra obtained from 16 different amino acid aqueous solutions, all at 100 mM concentration, except tryptophan, which was 50 mM. Four different nanocapillaries with tip internal diameters of 20, 25, 57, and 58 nm were used for these measurements. The most prominent amino acid peaks in all spectra shown in Figure 6B corresponded to singly charged unsolvated ions. The spectra of glycine, alanine, proline, valine, cysteine, glutamine, and phenylalanine did not show 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 monohydrated amino acid ion. Leucine showed a third and possibly a fourth peak corresponding to a higher solvation state. The tryptophan spectrum showed a peak below 200 m / z, consistent with a hydronium ion hydration state, which was also present in control measurements in aqueous solution with no amino acid present. Tryptophan, which is less soluble than the other amino acids studied, produced a relatively weak signal. The four protein-forming amino acids are absent from Figure 6B. Measurements of aspartic acid and glutamic acid in positive ion mode were not attempted due to their low isoelectric points. Also, isoleucine was ignored as it is indistinguishable from leucine at m / z, and tyrosine had poor emission characteristics, which are likely related to its low aqueous solubility.

[0157] Measurement of post-translationally modified peptides Figure 6C shows the mass spectra of glutathione and two chemically modified variants, s-nitrosoglutathione and s-acetylglutathione. Glutathione is a tripeptide found in high concentrations in most cells, and the variants studied here result from common post-translational modifications. An ion source with a 20 nm inner diameter tip generated peptide ions from a 100 mM aqueous solution at pH 3.1-3.9 adjusted by the addition of acetic acid. The glutathione spectrum shows a single peak at 307 m / z, which corresponds to a singly charged unsolvated glutathione ion. The spectra of s-acetylglutathione and s-nitrosoglutathione show major peaks at 349 m / z and 336 m / z, respectively, corresponding to singly charged unsolvated peptide ions, and each spectrum also shows two progressively smaller peaks 18 and 36 m / z to the right of the major peak, corresponding to monosolvated and disolvated peptide ions, respectively.

[0158] Ion Transmission Efficiency We measured the efficiency of ion passage from the nanopore source to the remote detector in a high vacuum environment (Figure 7A). Ions emitted from the ion source were focused onto the 2 cm opening of a Faraday cup located approximately 50 cm away. The current I collected by the Faraday cup was C I T The ratio of I to I was the ion transfer efficiency. Figure 7B shows the I measured over a 17 min experiment using a 39 nm i.d. tip filled with a 100 mM aqueous solution of sodium iodide. C , I T The average ion transfer efficiency measured was 93.4% + / - 1.7%. T fluctuates from about 780 pA to 840 pA on a time scale of several minutes, while I T The slow rise and fall of I C This reflects the fact that the transmission efficiency is relatively stable.

[0159] Separation of ions and charged droplets The possibility of the nanopore source releasing charged droplets in addition to ions was investigated by adding a magnetic sector to the flight path as shown in Figure 7C. A 0.54 T magnetic sector with a diameter of 6 cm deflected charged species based on their mass-to-charge ratio. Droplets larger than 15 nm in diameter, even when charged to the Rayleigh limit, were deflected by less than 2.7° and entered the Faraday cup. The Faraday cup was used to measure the current I from the charged droplets. 液滴 On the other hand, ions with m / z in the range of about 100 to about 350 were deflected to separate Faraday plates, and the ion current I イオン Figure 7D shows the I イオン , I 液滴 , and the ion fraction of the total measured current

[0160]

number

[0161] Calculating the probability of ion scattering Calculations showed that most ions followed collision-free trajectories from the ion source to the detector. Figure 8 shows the probability of collisions of the hydration-shelled amino acid ions with gas molecules in this example based on the kinetic theory of gas molecules. The ions were assumed to pass through a distribution of evaporating water molecules and a homogeneous low-pressure background of N2. The physical situation is shown diagrammatically in Figure 8, where the number density of gas molecules and the cumulative collision probability are plotted as a function of the distance from the meniscus. The cumulative probability of an ion colliding with a gas molecule over the entire 50 cm trajectory from the source to the detector was only 2.1%. This suggests that the majority of ions did not experience any collisions. Most of the collisions occurred within 200 nm of the liquid meniscus due to the high density of evaporated water molecules there. A detailed explanation of these calculations can be found in the Supporting Information below.

[0162] Consideration The conventional droplet-mediated electrospray mechanism (Figure 5A) was ruled out as the primary source of the measured ions on two grounds. First, no droplets larger than 10 nm were measured among the charged species delivered by the source (Figure 7D). Second, the instrument lacked the background gas that normally sustains the evaporation of water from droplets in electrospray. In high vacuum, nanoscale aqueous droplets shed only a small fraction of their mass before the evaporation process freezes due to latent heat loss. Thus, sustained release of ions from droplets cannot occur with this instrument.

[0163] These findings can be explained by an alternative ion release mechanism: direct evaporation of ions from the liquid-vacuum interface of the nanopore, as shown in Figure 5B. Ion evaporation is a thermal process in which ions escape from a liquid with the assistance of a strong electric field at the surface. Typically, a sufficiently strong electric field occurs when the ratio of the electrical conductivity K to the flow rate Q of the charged liquid is sufficiently large. Thus, previous studies have observed ion evaporation from highly conductive liquids such as liquid metals, ionic liquids, and concentrated electrolyte solutions in formamide. Although the measured amino acid solutions have relatively low electrical conductivities (in the range of 0.01-0.5 S / m), the resulting K / Q was still large because the flow rates generated in the nanocapillary under 1 atm pressure were very low (<10 pL / min). The K / Q values ​​in this example are of the same order of magnitude as those reported for the ionic liquid EMI-BF4, but show ion evaporation without droplet ejection. Furthermore, the distribution of ion solvation states measured in Figure 5D and elsewhere was similar to that measured for sodium iodide in formamide, which was also attributed to ion evaporation.

[0164] In Figures 6B-6C, it was observed that mostly bare ions were measured, rather than hydrated ion clusters. The experiments described here were performed to determine whether ions were i) emitted in a bare state or ii) emitted in a hydrated state and subsequently shed their hydration shell on their way to the detector. Collisions with gas molecules were ruled out as a mechanism for desolvating ion clusters, since only about 2% of the emitted ions would experience a single collision (Figure 8). Furthermore, the tip size appeared to affect the hydration state (Figure 6A). This suggested that it was the local environment at the source that controlled the hydration state, rather than processes occurring in flight.

[0165] The high ion transmission efficiency (Figure 7B) was a direct result of the emission mechanism of the nanopore ion source. Ion evaporation allowed individual ions to travel directly into the high vacuum environment, without colliding with background gas molecules or undergoing Coulomb explosions that would propel charged species in random directions. The trajectory of each ion emitted from the source was determined primarily by the electric field created by the ion optics.

[0166] In summary, a nanopore ion source capable of ejecting amino acid and small peptide ions directly into high vacuum is presented here. The ability to eject bare ions, as opposed to solvated ion clusters or charged droplets, facilitated the identification of various amino acids and post-translational modifications. Ions apparently evaporated directly from the liquid meniscus at the tip, thereby eliminating the need for background gas to liberate ions from the droplets. By eliminating background gas collisions and the need to transfer ions from ambient pressure to high vacuum, the nanopore ion source was able to eliminate the major mode of ion loss that characterizes electrospray ionization.

[0167] method Nanocapillary preparation Nanocapillaries were pulled from 7.5 cm long quartz capillaries (QF100-70-7.5 from Sutter Instruments) with an inner diameter of 0.7 mm and an outer diameter of 1 mm. A laser puller (P-2000, Sutter Instruments) was used to pull nanocapillaries with tips smaller than 100 nm following the single line recipe: heat=650, speed=45, delay=175, pull=190. Nanocapillaries were coated with 5 nm of carbon and imaged with a scanning electron microscope (LEO1530VP, Zeiss) to measure tip size. Nanocapillaries were plasma cleaned in air for 2 min using a plasma preen (Plasmatic Systems Inc.) before filling with analyte solution.

[0168] Amino Acid Solution Amino acid solutions were prepared by dissolving the amino acid of interest (Sigma-Aldrich) in DI water (Millipore) at 100 mM concentration, except for tryptophan, which was prepared at a concentration of 50 mM. 0.1–0.5% v / v glacial acetic acid (Sigma-Aldrich) was added to the amino acid solutions to reduce the pH below the isoelectric point of the amino acid. Glutathione, s-acetylglutathione, and s-nitrosoglutathione solutions were prepared by dissolving the peptides in deionized water at 100 mM concentration. Glutathione and s-acetylglutathione were purchased in powder form (Sigma-Aldrich), and S-nitrosoglutathione was synthesized in the laboratory from glutathione according to the protocol of TW Art. The pH and conductivity of each solution were measured using a pH meter (Ultrabasic Benchtop, Denver Instruments) and a conductivity meter (Sension+ EC71 GLP, Hach), respectively.

[0169] Delivery of solution to the ion source Sample solutions were delivered to the nanocapillary tip and flushed through a tube-in-tube system. A thin inner PEEK tube (ID 150 micrometers, OD 360 micrometers) (IDEX Health and Science) carried the sample solution, and a thicker PEEK tube (ID 0.04, OD 1 / 16'') (IDEX Health and Science) carried the used solution around the outside of the inner tube and away from the tip. A syringe pump (NE-300, New Era Pump Systems) was used to deliver fresh solution from the inner tube to the ion source. A VacuTight upchurch fitting (IDEX Health and Science) was used to create a seal around the base of the nanocapillary and the end of the outer tube to prevent solution from leaking into the vacuum. The tube-in-tube system was housed in a 1 / 4-inch diameter steel tube that was inserted into the vacuum chamber of the mass spectrometer, from a KF-40 to a Quick-Connect adapter (Lesker Vacuum).

[0170] quadrupole mass spectrometer The instrument used for all amino acid and peptide measurements shown in this example is a custom-built quadrupole mass spectrometer. The instrument contains a custom Einzel lens, a quadrupole mass filter (MAX-500, Extrel), an ion bender (Extrel), and a channel electron multiplier detector (DeTech 413) with a conversion dynode that is sensitive to single ions. The base pressure of the instrument is approximately 10 -8 When the nanopore ion source is introduced into the mass spectrometer, the pressure is typically 10 -7 ~10 -6 torr.

[0171] Amino acid and glutathione measurements Nanocapillaries were pre-filled with amino acid or peptide solutions using microfil flexible needles (World Precision Instruments). The filled nanocapillaries were then attached to a tube-in-tube system and inserted into the mass spectrometer. The solution at the tip was continuously refreshed by pumping the solution through the inner tube at a rate of 0.4 mL / h from a syringe pump (NE-300, New Era Pump Systems). A voltage of +100 V was applied to the electrode in the capillary using a high-voltage source meter (2657A, Keithley Instruments), and a negative voltage was slowly applied to the extraction electrode using a high-voltage power supply (Burle) until ionization was observed. The onset of release typically occurred when the total extraction voltage was 200-350 V.

[0172] Ion transfer efficiency measurement Ion transfer efficiency measurements were performed in a custom vacuum chamber containing a set of ion optics and a Faraday cup (Figure 7A). Emission current was measured with a 2410 SourceMeter (Keithley Instruments) that applied high voltage to the tip via an Ag electrode. Leakage current from the BNC cable connecting the sourcemeter to the tip was measured and subtracted from the measured emission current. The current flowing through the Faraday cup was measured using an SR570 current preamplifier (Stanford Research Systems) connected to an NI PCIe-6251 DAQ card (National Instruments). Optics voltage was controlled using an 8-channel high voltage power supply (CAEN DT8033). A custom Labview program was used to control the voltage applied to the tip and record the emission and transfer currents.

[0173] Magnetic Sector Measurement A rudimentary magnetic sector mass spectrometer was constructed by adding a magnet and a Faraday plate to the vacuum chamber described above. The magnet consisted of a neodymium magnet with a yoke constructed of low carbon magnetic iron (ASTM A848). The yoke focused the magnetic field on a flat circular area 6 cm in diameter and 1 cm high, which was located just downstream of the ion optics. A magnetometer was used to measure the magnetic field strength within the flat circular area, B=0.54+ / -0.02 T. The Faraday plate used was a 4 cm diameter, 0.02 inch thick stainless steel disk, connected directly to the electrical feedthrough by steel wire. The Faraday plate was placed at a 45° angle to the Faraday cup and the same distance from the center of the magnetic sector. The current emitted from the tip was measured using a 2410 Sourcemeter (Keithley) and the ion and droplet currents were each measured using separate SR570 current preamplifiers (Stanford Research Systems).

[0174] Supplementary Information: Single Amino Acid Ion Measurement Conditions Figure 6B shows the mass spectrum obtained from the aqueous amino acid solution, and Table 2 shows the relevant experimental parameters for these data.

[0175] [Table 2]

[0176] The spectra were measured in positive ion mode, which required lowering the pH below the isoelectric point of the dissolved amino acids. This was done by adding acetic acid. The pH and conductivity K of each solution are reported in Table 2. Ions were emitted directly from the nanopore ion source into high vacuum. The nanocapillaries had tip inner diameters ranging from 20 nm to 58 nm, and one tip was frequently used for measurements of multiple amino acid solutions. Table 2 reports the tip inner and outer diameters of each nanocapillary used to obtain the data in Figure 6B, and measurements obtained from the same tip are indicated by tip number. Table 2 also lists the time-averaged pressure P of the vacuum chamber, extraction voltage V, which were continuously monitored during the experiment. e , and the emission current I e has also been reported.

[0177] The probability of an emitted ion colliding with a gas molecule In Figure 6B, amino acid ions were detected primarily in a nonsolvated state. In conventional electrospray ionization, collisions with gas molecules are the mechanism by which solvent molecules are separated from ions. However, the instrument was operated under high vacuum conditions where collisions with gas molecules are expected to be rare. Kinetic theory of gas molecules was used to calculate the probability that an ejected ion cluster would collide with at least one gas molecule. This addresses the question of whether the ions emerged from the solution in an unsolvated state or whether they emerged with a solvation shell that was knocked off by collisions with gas molecules on the way to the detector.

[0178] The gas distribution in the vacuum chamber is density n b The uniform background of and the distribution of water molecules evaporated from the meniscus at the tip of the nanocapillary w The background gas pressure in this example is typically about 7×10 -8The pressure was 100 MPa (see Table 2). The mean free path of water molecules in nitrogen gas at this pressure was over 1 km, suggesting that the evaporating water molecules moved along ballistic trajectories away from the meniscus. As shown in Figure 9A, the meniscus was modeled as a hemisphere and the evaporating water molecules were modeled as moving radially outward. The density of water molecules is n w ∝r -2 where r is the distance from the center of the hemisphere. Although the rate at which water evaporates from a liquid meniscus into a vacuum could not be well established experimentally, it was known that the flux of water molecules evaporating from a liquid surface cannot exceed the flux of incoming molecules at equilibrium. Thus, by subtracting the incoming molecules, the highest possible density that the (outgoing) evaporating water molecules could achieve on the vacuum side of the liquid interface would be half the equilibrium water vapor density. The maximum probability that an ejected ion could collide with a gas molecule was determined by assuming the density of water vapor on the vacuum side of the meniscus to be:

[0179]

number

[0180] The cumulative probability C of an ion cluster undergoing at least one collision before reaching r p (r) was also decided. p To calculate (r), we use the probability that an ion survives a distance r without collision, P s (r)=1-C p It is easier to consider (r). s (r) is the stochastic process P c The probability of collision with gas molecules in the interval r-dr->r represented by (r-dr->r) is P s Associated with (r-dr). P s (r)=P s (r-dr)(1-Pc (r-dr->r)) (5)

[0181] In the limit of rarefied gas and small displacements,

[0182]

number

[0183]

number

[0184] Integrating Equation 7 from r0 to r, the boundary condition P s Apply (r0)=1 and P s Solving (r), we get:

[0185]

number

[0186] FIG. 9B shows the C p (r) is plotted. Cross-sectional area σ w and σ b are respectively π(a i +a w ) 2 and π(a i +a b) 2 In the formula, a w = 1.325 Å is the kinetic radius of water, and a b = 1.82 Å is the kinetic radius of the background gas, and a i = 7 Å is the approximate radius of an amino acid with a complete solvation shell of water. b =2.25×10 15 m -3 and n w,0 =6.44×10 23 are the number densities of the background gas and water molecules on the vacuum side of the meniscus, respectively. The meniscus radius is r0 = 30 nm. C p (r) increases rapidly over the first 100 nm and then saturates at 1.8%. p (r) increased slowly at centimeter-scale distances due to the finite density of background gas molecules: Cp(r) reached 2.1% at a source-to-detector distance of 50 cm.

[0187] These results indicated that, in contrast to conventional electrospray, collisions between gas particles and ion clusters emitted from the nanopore ion source were rare. Because the instrument was operated under high vacuum conditions, most ions followed collision-free trajectories from the ion source to the detector. The lack of collisions meant that ions were emitted in the same state as they were detected, leading to the conclusion that the nanopore ion source may be capable of releasing mainly unsolvated amino acid ions.

[0188] Nanopore ion sources operate at flow rates below the minimum stable flow rate for cone-jet electrospray. The minimum flow rate Q below which a stable cone-jet electrospray cannot exist e There may be a high or low Reynolds number R e Q effective for a conical jet of polar liquid having either e The formula for is shown here. When a cone jet of an aqueous amino acid solution is formed, R e > 1, so Q eThe appropriate formula for would be:

[0189]

number

[0190] The expected flow rate through the nanopore ion source is Q e The flow rate through the nanocapillaries was measured by spraying a water droplet into silicone grease at a constant applied pressure and measuring the growth rate of the droplet. The nanocapillary was first filled with water and its rear end was connected to a nitrogen cylinder via a pressure regulator. The tip of the capillary was immersed in a dish of silicone grease under an optical microscope. The back pressure from the nitrogen cylinder was slowly increased until it was able to overcome the Laplace pressure of the water-grease interface at the nanocapillary tip and start expanding the droplet. Movies of the droplet growing under constant back pressure were recorded at an image rate of 10 Hz. In Figure 10, the fluid conductance of 14 nanocapillaries is plotted as a function of the inner diameter of their tips. The flow rate through the smallest tip, with an inner diameter of 120 nm, measured in this way would be 30 pL / min at 1 atm applied pressure. Thus, even a larger tip of 100 nm diameter would only tolerate flow rates three orders of magnitude below the minimum flow rate required to sustain a stable conical jet.

[0191] The theoretical flow velocity through the nanocapillary is also plotted in Figure 10. In this calculation, the nanocapillary geometry is considered to be a semi-infinite truncated cone with tip inner radius r0. The apex half angle θ was used as a fitting parameter. Poiseuille flow through the cone yields the fluid conductance.

[0192]

number

[0193] Simulation of ion and droplet trajectories through the magnetic sector Simulations were performed using custom Python code to determine the m / z range of particles expected to impact the Faraday cup and Faraday plate detectors used in the magnetic sector experiment. Particles were assigned m / z ratios and initial kinetic energies qV T where q is the charge of the particle and V T is the tip voltage. The trajectory of the particle passing through the magnetic sector was then numerically calculated by solving the Lorentz force law using a fourth-order Runge-Kutta scheme. The magnetic field was assumed to be 0.54 T in the z direction within the boundary of the magnetic sector (a circle with a diameter of 6 cm) and 0 elsewhere. Taking into account the geometry of the instrument and the arrangement of the detectors relative to the magnetic sector, it is possible to calculate the minimum / maximum deflection angles required for a particle to impact the two detectors. The droplet current I impacting the Faraday cup and measured 液滴 To contribute to the , the particle needs to be deflected by 0° to 3.15°, which is (I イオン) It is necessary to deflect the particles by 30.96° to 59.04°. Particles with 75 < m / z < 315, corresponding to monovalent sodium ions with 3 to 16 additional water molecules attached, were found to collide with the Faraday plate. Also, it was found that particles with m / z > 37000 would collide with the Faraday cup. This corresponds to charged water droplets charged up to the Rayleigh limit with a radius > 15 nm. It should be noted that droplets charged below the Rayleigh limit have less deflection.

[0194] Although some 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 such functions and / or obtaining one or more of such results and / or advantages described herein, and each of such variations and / or modifications is considered to be within the scope of the present disclosure. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and that actual parameters, dimensions, materials, and / or configurations will depend on the particular one or more applications in which the teachings of the present disclosure are used. Those skilled in the art will recognize or be able to confirm using no more than routine experimentation that there are many equivalents to the particular embodiments of the disclosure described herein. Accordingly, the above-described embodiments are presented by way of example only, and it is to be understood that the present disclosure may be practiced otherwise than as particularly described and claimed within the scope of the appended claims and their equivalents. The present disclosure relates to each individual feature, system, article, material, kit, and / or method described herein. Additionally, combinations of two or more such features, systems, articles, materials, kits, and / or methods are included within the scope of the present disclosure if such features, systems, articles, materials, kits, and / or methods are not mutually incompatible.

[0195] If 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 mutually conflicting and / or inconsistent disclosure, the document having the later effective date shall control.

[0196] All definitions and as used herein should be understood to take precedence over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0197] The indefinite articles "a" and "an," as used in the specification and claims, unless expressly indicated otherwise, should be understood to mean "at least one."

[0198] The phrase "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 interpreted 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 the specifically identified elements. Thus, as a non-limiting example, a reference to "A and / or B", when used in conjunction with open-ended language such as "comprising", may in one embodiment refer only to A (optionally including elements other than B), in another embodiment may refer only to B (optionally including elements other than A), in yet another embodiment may refer to both A and B (optionally including other elements), etc.

[0199] When used in the present specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., including at least one of a number or list of elements, but including more than one, and optionally including additional unlisted items. Only terms that clearly indicate otherwise, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," shall refer to including exactly one element of a number or list of elements. In general, the term "or" when used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") when preceded by an exclusive term, such as "either," "one of," "only one of," or "exactly one of."

[0200] 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 of the 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 elements specifically identified. Thus, as non-limiting examples, "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 to, in one embodiment, that at least one A is present, optionally including more than one, but no B (and optionally including elements other than B); in another embodiment, that at least one B is present, optionally including more than one, but no A (and optionally including elements other than A); in yet another embodiment, that at least one A is present, and optionally including more than one, and at least one B is present (and optionally including other elements); etc.

[0201] When the word "about" is used herein in connection with a numerical value, it is to be understood that yet another embodiment of the present disclosure includes that numerical value unmodified by the presence of the word "about."

[0202] It should also be understood that in any method claimed herein that includes more than one step or act, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are described, unless expressly indicated otherwise.

[0203] In the claims and the above specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," and "composed of" are to be understood to be open-ended, i.e., to mean 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 defined in Section 2111.03 of the United States Patent Office Manual of Patent Examining Procedures. [Explanation of symbols]

[0204] 10 Mass spectrometer 15 light source 20 Ion source 30 Capillary 32 Main body part 34 Capillary tip 34 Tip part 34 Nanochip 36 Capillary tip 36 Opening 50 Target Species 52 Fluid 54 Single molecule 54 Basic Fragments 54 Emitted Fragments 54 Individual Components 54 exit molecule 62 light 64 Fragmentation 70 Detector 80 vacuum 90 Mass Filter 90 Magnetic Filter 100 Ion Optical System 110 Multiplex Mass Spectrometer 120 Multiple Ion Sources 130a Capillary 130b Capillary 130c Capillary 154 154 Individual Components 170 Detector 190 Magnetic Mass Filter

Claims

1. placing a protein in a substantially linear configuration on a nanopipette; fragmenting the protein into amino acids by applying laser light to the protein; releasing the amino acids from the nanopipette; and detecting the amino acids released from the nanopipette A method comprising the steps of:

2. the laser light is ultraviolet light and / or the laser light has a wavelength of at least 150 nm and 222 nm or less, the method according to claim 1.

3. having an average ion transfer efficiency of at least 85% and / or the nanopipette includes an aperture having a cross-sectional dimension of less than 100 nm, the method according to claim 1 or 2.

4. the released amino acids are in the form of bare ions and / or ion clusters and / or at least 80% of the released amino acids are in the form of bare ions and / or the released amino acids are released continuously, the method according to claim 1 or 2.

5. detecting the amino acids in the order in which the amino acids are released from the nanopipette, the method according to claim 1 or 2.

6. further comprising determining the sequence of the protein by determining the released amino acids with the detector, the method according to claim 1 or 2.

7. the protein is contained in a solution, and optionally the solution contains water and / or formaldehyde, the method according to claim 1 or 2.

8. an ion source including a capillary; a light source directed at the ion source, capable of generating light having a wavelength of 150 nm or more and 213 nm or less; a magnetic mass filter downstream of the ion source; and an array of detectors downstream of the magnetic mass filter A mass spectrometer comprising:

9. the light source is capable of generating light having a wavelength of 193 nm ± 5 nm and / or the light source is a laser, the mass spectrometer according to claim 8.

10. the ion source includes an electrode near the capillary, the mass spectrometer according to claim 8 or 9.

11. the capillary includes a body portion and a tip portion fluidly connected to the body portion, the mass spectrometer according to claim 8 or 9.

12. More than 50% of the light having a wavelength of 150 nm or more and 213 nm or less passes through the tip portion of the capillary and / or Less than 50% of the light having a wavelength of 150 nm or more and 213 nm or less passes through the body portion of the capillary. The mass spectrometer according to claim 11. **Claim 13** The tip portion of the capillary has a cross-sectional dimension of less than 100 nm, less than 80 nm, less than 65 nm, less than 25 nm, or less than 5 nm. The mass spectrometer according to claim 11. **Claim 14** The mass spectrometer according to claim 8 or 9, further comprising one or more additional ion sources adjacent to the ion source. **Claim 15** (a) The ion source and the one or more additional ion sources are arranged in a linear array positioned upstream of the magnetic mass filter and / or (b) At least one of the one or more additional ion sources includes a capillary and / or (c) At least one of the one or more additional ion sources includes an electrode in the vicinity of the capillary and / or (d) The array of detectors downstream of the magnetic mass filter is arranged in a two-dimensional array. The mass spectrometer according to claim 14.

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