CN118120042A

CN118120042A - System and method for peptide photolysis analysis of single molecule protein sequencing

Info

Publication number: CN118120042A
Application number: CN202280070218.7A
Authority: CN
Inventors: D·M·斯坦; J·S·维托里茨; N·德拉赫曼
Original assignee: Brown University
Current assignee: Brown University
Priority date: 2021-08-20
Filing date: 2022-08-18
Publication date: 2024-05-31

Abstract

The present disclosure relates generally to systems and methods for peptide photolysis analysis of single molecule protein sequencing. In one aspect, the systems and methods involve allowing one to fragment individual protein molecules in aqueous solution so that their amino acid composition and sequence can be measured by mass spectrometry. This may be useful for single molecule protein sequencing techniques.

Description

System and method for peptide photolysis analysis of single molecule protein sequencing

RELATED APPLICATIONS

The present application claims the benefit of U.S. provisional patent application Ser. No. 63/341,992, entitled "System and method for peptide photolysis analysis for Single molecule protein sequencing", filed on day 13 of 5.2022, and U.S. provisional patent application Ser. No. 63/235,601, entitled "System and method for peptide photolysis analysis for Single molecule protein sequencing", filed on day 8.20 of 2021, each of which is incorporated herein by reference in its entirety.

Technical Field

The present disclosure relates generally to systems and methods for peptide photolysis analysis of single molecule protein sequencing.

Background

Peptide sequencing is an important tool in proteomics, widely used to identify proteins and map the protein content of cells. The ability to sequence individual copies of a protein will significantly improve the analysis of single cells and will enable investigation of low abundance proteins and protein forms that may be present in less than 10 copies per cell. Single molecule technology is required because proteins cannot be biochemically amplified like nucleic acids. Furthermore, post-translational modifications and protein forms from alternative mRNA splicing cannot be deduced from DNA or RNA sequences. Single molecule protein sequencing may also lead to new diagnostic applications and drug therapies. There is increasing interest in developing single protein sequencing technologies based on fluorescent labels, N-terminal probes and nanopores.

Mass Spectrometry (MS) is the dominant force in current peptide sequencing. However, this technique typically requires 10 ⁷ or more copies of the protein to reach the limit of detection. MS ion sources are generally more sensitivity limiting than any other component. In electrospray ionization, charged droplets containing the analyte collide with a background gas to release analyte ions into the gas phase, which can then be transferred by the instrument to a detector; the overall efficiency of these processes is very low. Thus, improvements are needed.

Disclosure of Invention

The present disclosure relates generally to systems and methods for peptide photolysis analysis of single molecule protein sequencing. In some cases, the subject matter of the present disclosure relates to a variety of different uses of interrelated products, alternative solutions to particular problems, and/or one or more systems and/or articles.

In one aspect, the systems and methods involve allowing one to fragment individual protein molecules in aqueous solution so that their amino acid composition and sequence can be measured by mass spectrometry. This may be useful for single molecule protein sequencing techniques. Single molecule protein sequencing is the next leading field of biomolecular diagnostics, the development of which may be helpful in completely changing the fields of biology and disease diagnosis.

In some cases, these may be implemented using hardware that may be commercialized as an additional component of a mass spectrometry system. Furthermore, certain embodiments relate generally to single molecule protein sequencing instruments that may be used in environments such as biomedical research and clinical.

Certain methods and systems may allow protein fragmentation in aqueous solution rather than in the gas phase. In some embodiments, the peptide bond connecting the amino acid to the parent peptide may be selectively cleaved. In some cases, the amino acids may be released intact for analysis by mass spectrometry. These methods may be compatible with single molecule protein sequencing strategies, for example as discussed in International patent application publication No. PCT/US2021/028954 filed on month 4, 2021, 23 and U.S. patent application Ser. No. 63/179,046 filed on month 4, 2021, each of which is incorporated herein by reference.

In one set of embodiments, the method includes fragmenting a protein into individual molecules using light in a mass spectrometer.

According to another set of embodiments, the method includes arranging the proteins into a substantially linear configuration in the nanotip; fragmenting the protein into amino acids by irradiating laser light onto the protein; emitting an amino acid from the nanotip; and detecting the amino acid emitted from the nanotip.

In another set of embodiments, the method comprises applying light having a wavelength greater than or equal to 150nm and less than or equal to 213nm to the protein to cleave fragments from the protein; and sequencing the fragments using mass spectrometry.

According to yet another set of embodiments, the method comprises applying light having a wavelength greater than or equal to 150nm and less than or equal to 222nm to the protein to cleave fragments from the protein; and sequencing the fragments using mass spectrometry.

According to yet another set of embodiments, the method comprises applying a laser to the protein to cleave amino acids from the protein; and sequencing the amino acids using mass spectrometry.

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

One aspect of the present disclosure generally relates to a method of sequencing a protein. According to one set of embodiments, a method of sequencing a protein includes fragmenting a protein by applying light to the protein having a wavelength greater than or equal to 150nm and less than or equal to 213nm to produce fragments; passing the fragments through a magnetic mass filter; directing the fragments to a detector array; and determining the sequence of the protein by determining the fragments with a detector array.

According to another set of embodiments, a method of sequencing a protein includes delivering a fluid comprising a protein into a capillary defining an opening; applying light having a wavelength of 150nm or more and 213nm or less to the protein in the vicinity of the opening to produce fragments; directly delivering the fragments into an environment at a pressure of no more than 100 mPa; passing the fragments through a magnetic mass filter; directing the segments to a detector array; and determining the sequence of the protein by determining the fragments with a detector array.

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

According to another set of embodiments, a mass spectrometer includes an ion source comprising a capillary; a light source directed to the ion source, wherein the light source is capable of producing light having a wavelength greater than or equal to 150nm and less than or equal to 213 nm; a magnetic mass filter downstream of the ion source; and a detector array downstream of the magnetic filter.

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

According to yet another set of embodiments, a mass spectrometer comprises an ion source comprising a capillary and an electrode proximate the capillary, wherein the capillary comprises an opening having a cross-sectional dimension of less than 125 nm; a magnetic mass filter downstream of the ion source; a detector array downstream of the magnetic filter; and a light source directed to the ion source, wherein the light source is capable of producing light having a wavelength greater than or equal to 150nm and less than or equal to 213 nm.

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

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

Non-limiting embodiments of the present disclosure are 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 that is illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure nor is every component of every embodiment of the disclosure shown where illustration is not necessary for those of ordinary skill in the art to understand the disclosure.

Drawings

FIG. 1A shows a schematic representation of single molecule protein sequencing by nanopore mass spectrometry.

Fig. 1B shows the fragmentation of elongated peptide chain near the tip of a nanocapillary ion source.

Fig. 2 shows the structure of the dipeptide, the structure of the common photo-fragmentation product, and the approximate frequencies at which lasers of different wavelength ranges induce specific transformations in vacuo.

FIG. 3A shows calculated heating profiles in a nanocapillary under stable illumination with light at 10.6 μm, 193nm and 222 nm.

Fig. 3B shows the dependence of the maximum temperature increase in the nanocapillary on the incident laser power density for 10.6 μm (triangle), 193nm (square) and 222nm (dot) light. The symbols show the results of the finite element calculations, and the curve is a linear fit of the data.

FIG. 4A shows the cumulative probability of dissociation of peptide bonds obtained from equation 2 when exposed to 193nm laser radiation of varying intensities, as shown.

Fig. 4B shows the probability of amino acid non-decomposition as a function of exposure time for 193nm laser light for ρ=10,000 wm ^-2, calculated according to equation 3.

Fig. 4C shows the probability of selective amino acid release (i.e., fragmenting two peptide bonds linking an amino acid and a peptide without damaging the amino acid) as a function of exposure time of 193nm laser light for ρ=10,000 wm ^-2.

Fig. 5A is a schematic diagram of conventional electrospray ionization highlighting the background gas that stimulates solvent evaporation from the droplet and the transfer capillary in which significant ion loss occurs.

Fig. 5B is a schematic diagram of a nanopore ion source showing a liquid filled nanocapillary tip, an extraction electrode, and an extraction voltage V _e applied therebetween. The inset shows an SEM image of a drawn quartz nanocapillary tip with a tip inside diameter of 30 nm.

Fig. 5C is a schematic diagram of a mass spectrometer used in example 2. The ion optics include extraction electrodes and a single lens that extract ions from a liquid meniscus at the ion source and focus them through a quadrupole mass filter and an electrostatic ion bender. The emitted ions strike a channel electron multiplier detector that is sensitive to the individual ions.

FIG. 5D shows a mass spectrum of a 100mM arginine aqueous solution obtained with a 41nm inside diameter nanopore ion source in a quadrupole mass spectrometer as described herein.

FIG. 6A shows a mass spectrum of a 100mM arginine aqueous solution using nanopore ion sources with 3 different tip inner diameters (20 nm, 125nm, and 300 nm).

Fig. 6B shows a mass spectrum of 16 amino acids, ordered from top left to bottom right. All experiments were performed using a nano-capillary with a tip inside diameter of 20-60 nm.

FIG. 6C shows overlapping mass spectra of glutathione and its two PTM variants (s-nitrosoglutathione and s-acetyl glutathione).

Fig. 7A shows an experimental setup for measuring ion transport efficiency of a nanopore ion source. A voltage V _T is applied to the nanopore ion source by the source measurement unit to produce a tip current I _T. The emitted ions are focused by ion optics and strike a faraday cup, where the collected current I _C is measured by a current-voltage preamplifier. This experiment was performed in a vacuum chamber at a pressure of about 10 ^-7 torr. Ion transport efficiency is the ratio of the collected current to the tip current, I _C/I_T.

FIG. 7B shows a graph of ion transport efficiency measured from a nanopore ion source using a 100mM NaI aqueous solution over a few minutes. The inset shows I _T and I _C plotted over the same time period.

Fig. 7C shows experimental setup for measuring the relative fractions of ion current I _Ion and droplet current I _Drop. A sector magnetic field (diameter=6cm, b field strength=0.54T) is placed on one side of the ion-optical element to deflect the emitted particles at their mass-to-charge ratio. I _Ion is collected by a wide faraday plate and I _Drop is collected by a faraday cup.

FIG. 7D shows the ion fraction of the total measured current and I _Ion、I_Drop of a2 minute long measurement using a 28nm tip filled with 100mM NaI aqueous solution.

Fig. 8 shows the probability of amino acid ions with a hydrated shell colliding with gas molecules based on aerodynamic theory. The curve is with a small hydrated shellCumulative probability of collisions of the amino acids with evaporating water molecules or background N ₂ molecules as a function of distance r from the meniscus. The line shows the calculated maximum possible water vapor density as a function of distance from the meniscus. The vapor pressure on the vacuum side of the meniscus is conservatively assumed to be 8.75torr, half the vapor pressure of the water balance at room temperature. The inset shows a schematic of the distribution of evaporative water molecules near the meniscus.

Fig. 9A is a graph showing water evaporated from a hemispherical meniscus, tip radius r ₀, and distance r.

Fig. 9B shows a graph of the respective contributions of C _p (r) (solid line) and water molecules (dashed line) and background gas (dashed line) to the total probability of experiencing at least one collision. The dotted line shows the contribution of evaporated water molecules, the dash-dot line shows the contribution of background gas, and the solid line shows the total collision probability obtained by adding the contributions of water and background gas. The calculation used is that of n _b＝2.25×10¹⁵m^-3,n_w0＝6.44×10²³m^-3, And r ₀ = 30nm. The graph extends to r=0.5m, the total distance of the meniscus to the detector.

Fig. 10 shows a fitted curve of fluid conductance measured using a semi-infinite truncated model of a nano-capillary with a semi-apex angle θ used as a fitting parameter, versus theoretical predictions.

FIG. 11A shows the results of a simulation of deflection angle versus m/z for singly charged ions. The area between the dashed lines represents the position and extent of the faraday detector, so ions of 70< m/Z <325 should strike the faraday plate.

Fig. 11B shows simulation results of deflection angle and droplet radius of a water droplet charged to the rayleigh limit. The area under the dashed line represents the position and extent of the faraday cup aperture so that a fully charged droplet of greater than 15nm should strike the faraday cup.

Fig. 11C shows the selection of simulated trajectories of five singly charged ions with masses between 60amu and 460amu, with arrows pointing in the direction of mass increase, and circles representing the sector magnetic field.

Fig. 11D shows the selection of a simulated trajectory of 5 drops charged to the rayleigh limit with a radius between 5nm and 25nm, with the arrow pointing in the direction of increasing radius.

Fig. 12 shows a schematic diagram of a multi-mass spectrometer including a plurality of nanopore ion sources, according to some embodiments.

Detailed Description

Certain aspects of the present disclosure relate to mass spectrometers and related methods that allow single molecule fragmentation and sequencing of a substance of interest (e.g., a polymer, a biopolymer, etc.). In some cases, the substance of interest is a protein and methods related to single molecule protein sequencing are disclosed herein. While some embodiments of the present disclosure relate to methods for analyzing and/or sequencing peptides and proteins, it should be understood that the present disclosure is not so limited, and in other embodiments, the methods may be used to analyze any of a variety of molecules and/or ions including, but not limited to, salt ions, macromolecules, and the like.

In some embodiments, disclosed herein are mass spectrometers comprising a light source configured to fragment a substance of interest (e.g., a protein) into monomeric components (e.g., amino acids). In some such embodiments, the mass spectrometer is a nano Kong Zhipu instrument that includes an ion source capable of ionizing a substance of interest into a vacuum, as well as other related components, including, but not limited to, a vacuum, a magnetic filter, and one or more detectors, as described in more detail below. The combination of light sources, ion sources, etc. may advantageously allow for fragmentation of a substance of interest (e.g., a biopolymer such as a protein) into basic fragments (e.g., monomers such as amino acids) and sequencing thereof. The ion source, magnetic filter, one or more detectors may have any of the properties, configurations, and/or arrangements described in more detail below.

In one set of embodiments, a light source (e.g., a laser) may be positioned adjacent to an ion source comprising a capillary (e.g., such that the light source is directed at the ion source or a portion thereof). A non-limiting example of a mass spectrometer including such a light source is shown in fig. 1A-1B. As shown, the mass spectrometer 10 includes a light source 15 (e.g., a laser, such as a UV laser) directed at an ion source 20, the ion source 20 including a capillary 30 filled with a fluid 52 containing a substance 50 of interest (e.g., a protein or peptide). The fluid may include any suitable solvent described elsewhere herein, such as water, formamide, high volatility solvents, aqueous solutions, and the like. In some cases, the light source may be directed at a capillary tip (e.g., nanotip) of the ion source. In some embodiments, the light source may be directed at a fluid containing a substance of interest (e.g., a protein or peptide) disposed within the capillary tip (e.g., nanotip). 1A-1B, the light source 15 may be directed to a fluid 52 containing a substance 50 of interest within the capillary tip 34 (e.g., nanotip) of the ion source 20.

According to some embodiments, the light source may be configured to fragment a substance of interest (e.g., a protein) in a fluid within the capillary into fragmented individual components or individual molecules (e.g., individual amino acids) by being directed to the fluid containing the substance of interest. According to some embodiments, the fragmented individual components or individual molecules may then be ionized into a vacuum from the opening of the capillary tip (e.g., nanotip). In some embodiments, the vacuum chamber houses a nanotip. For example, as shown in fig. 1B, the light source 15, by being directed to a fluid 52 containing a substance of interest 50, may be configured to fragment the substance of interest 50 (e.g., a protein or peptide) into fragmented individual components or individual molecules 54 (e.g., amino acids) in the fluid 52 within the capillary tip 34 (e.g., nanotip). For example, light 62 (e.g., UV photons) emitted by the light source 15 may result in partial fragmentation 64 of the substance of interest 50 exposed to the light 62. The fragmented individual components or individual molecules 54 may then be ionized from the opening 36 of the capillary tip 36 into a vacuum 80 that holds the capillary tip 34. Methods for fragmenting a substance of interest are described in more detail below.

In some embodiments, disclosed herein are methods related to fragmenting a substance of interest (e.g., a biopolymer, such as a protein) into individual components (e.g., individual amino acids) and sequencing thereof. It should be noted that this approach may be particularly beneficial for sequencing of biopolymers (e.g., proteins) where single molecule precision is desired.

In some embodiments, the method includes the step of arranging the substance of interest (e.g., a protein) into a substantially linear configuration in the nanotip of the capillary. In some cases, the capillary nanotip may be sized such that it drives the substance of interest to align itself into a linear configuration. As shown in fig. 1A-1B, capillary tip 34 may have a size (e.g., by having a cross-sectional dimension (e.g., a maximum cross-sectional dimension) of less than 200nm, less than 150nm, less than 120nm, less than 100nm, less than 80nm, less than 65nm, less than 60nm, less than 50nm, less than 30nm, less than 25nm, and/or as low as 20nm (e.g., as low as 15nm, as low as 10nm, as low as 5nm, as low as 4nm, as low as 3nm, as low as 2nm, as low as 1nm, etc.) such that substance 50 of interest (e.g., protein or peptide) is linearly aligned at the tip of the capillary. In one set of embodiments, the nanotip may have a cross-sectional dimension between 1nm and 5 nm. Advantageously, such a linear arrangement may allow for exposure of individual bonds (e.g., peptide bonds) between the basic fragments (e.g., amino acids) forming the substance of interest, which may in turn facilitate fragmentation of the substance of interest into its basic fragments.

In some embodiments, the method includes fragmenting a substance of interest (e.g., a protein) into basic fragments (e.g., amino acids) by applying a laser to the substance of interest within a capillary tip (e.g., nanotip). As shown in fig. 1A-1B, light 62 (e.g., a laser) generated by a light source 15 (e.g., a laser) adjacent to capillary tip 34 (e.g., a nanotip) may be applied to solution 52 containing substance 50 of interest. In some such embodiments, the substances of interest are arranged linearly such that the laser is capable of cleaving bonds between individual components (e.g., amino acids) from the substances of interest (e.g., proteins). For example, referring to fig. 1B, the substance of interest 50 may be arranged linearly within the capillary tip 34 (e.g., nanotip) such that light 62 (e.g., laser) is capable of cleaving bonds between individual components or molecules (e.g., amino acids) from the substance of interest 50 (e.g., protein or peptide).

In some embodiments, when a substance of interest (e.g., a protein) breaks into its basic fragments (e.g., amino acids), the basic fragments may be emitted (e.g., ionized) from the opening of the nanotip. According to some embodiments, the nanotip may be sized such that the basic fragments are emitted in a continuous manner, e.g., preserving the order of the basic fragments within the substance of interest. For example, as shown in fig. 1A-1B, the base fragments 54 (e.g., amino acids) may be emitted from the openings 36 at the nanotips 34 (e.g., one after the other) in a sequential order. In some embodiments, the emitted basic fragments (e.g., amino acids) can be transferred to various components of a mass spectrometer (e.g., vacuum, ion optics, magnetic filters) and subsequently detected by one or more detectors. In some embodiments, one or more detectors may be configured to determine the sequence of a substance of interest (e.g., a protein) by determining the emitted fragments (e.g., amino acids). Referring again to fig. 1A-1B, as a non-limiting example, the emitted base fragments 54 (e.g., amino acids) may be transferred to various components of a mass spectrometer (e.g., vacuum 80, ion optics 100, filters 90 (e.g., magnetic filters)) and then detected by one or more detectors 70. The one or more detectors 70 may be configured to determine the sequence of a substance (e.g., protein) of interest by determining the emitted fragments 54 (e.g., amino acids). Details regarding the various components and methods of transmission, transmission and detection of basic fragments are described in more detail below.

In some embodiments, the capillary may have a particularly advantageous configuration that allows the substance of interest within the capillary to fragment into individual components. Capillaries having such a configuration can, for example, advantageously reduce mixing and diffusion of fragmented individual components within the capillary (e.g., within the nanotip of the capillary), and/or preserve the sequence of individual components relative to their original sequence prior to fragmentation of the substance of interest, and/or allow subsequent ionization of fragmented individual components at the capillary tip in a linear and continuous manner. For example, in one set of embodiments, a capillary tube may include a body portion and a tip portion (e.g., nanotip) adjacent an opening of the capillary tube in fluid connection with the body portion. As shown in fig. 1B, for example, capillary 30 may include a body portion 32 and a tip portion 34 (e.g., nanotip) adjacent an opening 36 of capillary 30. Tip portion 34 may be fluidly connected to body portion 32.

In some embodiments, the tip portion of the capillary tube may be substantially transparent to light emitted by the light source having a particular wavelength or range of wavelengths. As used herein, the tip portion being "substantially transparent" to the wavelength of light means that greater than 50% (e.g., greater than 60%, greater than 70%, greater than 80%, greater than 90%) and/or up to 95% (e.g., up to 99%, or up to 100%) of light having a particular wavelength or range of wavelengths can enter the tip portion of the capillary tube. For example, the tip portion (e.g., nanotip) of the capillary may be transparent to light having a wavelength of less than or equal to 213nm (e.g., less than or equal to 213nm, less than or equal to 200nm, less than or equal to 193nm, less than or equal to 185nm, less than or equal to 180nm, less than or equal to 175nm, less than or equal to 150 nm) and/or a minimum of 170nm (e.g., a minimum of 160nm, a minimum of 155nm, or a minimum of 150 nm). The wavelength references may have a deviation of +/-5nm, +/-10nm, or +/-15 nm. Combinations of the above-referenced ranges are also possible (e.g., less than or equal to 220nm +/-5nm and as low as 150nm +/-5nm, less than or equal to 213nm +/-5nm and as low as 150nm +/-5nm, less than or equal to 193nm +/-5nm and as low as 160nm +/-5nm, or less than or equal to 185nm +/-5nm and as low as 150nm +/-5 nm). Other ranges are also possible.

In some embodiments, the body portion of the capillary tube may be substantially opaque to light emitted by the light source having a particular wavelength or range of wavelengths. As used herein, the body portion being "substantially opaque" to the wavelength of light means that less than 50% (e.g., less than 40%, less than 30%, less than 20%, less than 10%) and/or as little as 5% (e.g., as little as 1% or as little as 0%) of light having a particular wavelength or range of wavelengths can enter the body portion of the capillary tube. For example, the body portion of the capillary tube may be opaque to light having a wavelength of less than or equal to 213nm (e.g., less than or equal to 200nm, less than or equal to 193nm, less than or equal to 185nm, less than or equal to 180nm, less than or equal to 175nm, less than or equal to 150 nm) and/or a minimum of up to 170nm (e.g., a minimum of up to 165nm, a minimum of up to 160nm, a minimum of up to 155nm, or a minimum of up to 150 nm). The wavelength references may have a deviation of +/-5nm, +/-10nm, or +/-15 nm. Combinations of the above ranges are also possible (e.g., less than or equal to 220nm +/-5nm and as low as 150nm +/-5nm, less than or equal to 213nm +/-5nm and as low as 150nm +/-5nm, less than or equal to 193nm +/-5nm and as low as 150nm +/-5nm, or less than or equal to 185nm and as low as 150nm +/-5 nm). Other ranges are also possible.

1A-1B, while the tip portion 34 (e.g., nanotip) may be transparent to light having wavelengths within one or more of the ranges described above with respect to the capillary tip portion, the body portion 32 may be substantially opaque to light having wavelengths within one or more of the ranges described above with respect to the body portion of the capillary.

In some embodiments, when a majority of the substance of interest contained within the tip portion (e.g., a substantially transparent tip portion) of the capillary is fragmented into individual components, a small amount (if any) of the substance of interest contained within the body portion (e.g., a substantially opaque body portion) of the capillary is fragmented into individual components. For example, greater than 50% (e.g., greater than 60%, greater than 70%, greater than 80%, greater than 90%) and/or up to 95% (e.g., up to 99% or up to 100%) of the substance of interest contained within the tip portion of the capillary tube may be fragmented into individual components, e.g., due to incident light. In addition, less than 50% (e.g., less than 40%, less than 30%, less than 20%, less than 10%) and/or as little as 5% (e.g., as little as 1% or as little as 0%) of the substance of interest contained within the body portion of the capillary tube may be fragmented into individual components.

1A-1B, when a majority of a substance of interest 50 (e.g., a protein or peptide) contained within the transparent tip portion 34 of the capillary 30 is fragmented into individual components 54 (e.g., amino acids) by light 62, a small amount (if any) of the substance of interest 50 (e.g., a protein or peptide) contained within the opaque body portion 32 of the capillary 30 is fragmented into individual components. In one set of embodiments, when all (e.g., 100%) of the substance of interest contained within the tip portion of the capillary is fragmented by the light sheet from the light source, little (e.g., 0%) of the substance of interest contained within the body portion of the capillary is fragmented by the light sheet from the light source.

In some embodiments, when a substance of interest is fragmented into individual components at the tip portion of the capillary, the individual components may diffuse toward the opening of the tip portion and be ionized by an electrode adjacent the tip of the capillary. The electrodes may have any of the characteristics and/or configurations described elsewhere herein. For example, as shown in fig. 1B, the fragmented individual component 54 may diffuse toward the opening 36 of the tip portion 34 and be ionized by an electrode (not shown) near the tip of the capillary.

According to some embodiments, the capillary tube may advantageously allow fragmentation of the substance of interest only at the transparent tip portion adjacent to the capillary opening by having an opaque body portion and a transparent tip portion. As described elsewhere herein, the tip portion may be sized such that the substances of interest contained within the tip portion are arranged in a substantially linear manner. The capillaries described herein can allow for fragmentation of a substance of interest while being arranged in a linear fashion in the tip portion, limiting mixing of fragmented individual components at the opening of the tip portion prior to being ionized, and thus allowing the fragmented individual components to be ionized and detected in their original sequence in the substance of interest.

The tip portion of the capillary tube may have dimensions within one or more of the ranges described herein with respect to the opening of the capillary tube. In some embodiments, the tip portion includes a cross-sectional dimension (e.g., maximum cross-sectional dimension) of less than or equal to 150nm, less than or equal to 130nm, less than or equal to 125nm, less than or equal to 120nm, less than or equal to 110nm, less than or equal to 100nm, less than or equal to 90nm, less than or equal to 80nm, less than or equal to 75nm, less than or equal to 70nm, less than or equal to 65nm, less than or equal to 60nm, less than or equal to 55nm, less than or equal to 50nm, less than or equal to 45nm, less than or equal to 40nm, less than or equal to 35nm, less than or equal to 30nm, less than or equal to 25nm, less than or equal to 20nm, less than or equal to 15nm, less than or equal to 10nm, less than or equal to 5nm, less than or equal to 3nm, less than or equal to 2nm, etc. In addition, in some cases, the tip portion may have a cross-sectional dimension of at least 1nm, at least 2nm, at least 3nm, at least 5nm, at least 10nm, at least 15nm, at least 20nm, at least 25nm, at least 30nm, at least 40nm, at least 50nm, at least 60nm, at least 65nm, at least 70nm, at least 80nm, at least 90nm, etc. Combinations of these are also possible; for example, the opening may have a cross-sectional dimension of between 50nm and 100nm, or between 20nm and 65nm, between 1nm and 5nm, between 1nm and 3nm, 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 between 1nm and 5nm (e.g., or between 1nm and 3 nm).

In some embodiments, the body portion of the capillary tube may be made of and/or coated with a material that is substantially opaque to the wavelength of light emitted by the light source. For example, the body portion of the capillary tube may be substantially opaque to light having wavelengths within one or more of the ranges described above with respect to the body portion of the capillary tube. In some cases, the capillary walls of the body portion may be constructed using an opaque material (e.g., metal). Alternatively or additionally, the capillary walls may be formed of an initially transparent material that is subsequently coated with an opaque layer (e.g., a metal coating). The body portion of the capillary tube may be coated using any of a variety of suitable coating techniques.

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

In some cases, particularly beneficial types of lasers and/or light wavelengths may be employed in a mass spectrometer. In some such embodiments, the light source is a UV light source and the laser is ultraviolet light. In some cases, the use of ultraviolet light may provide more favorable conditions for protein sequencing than other wavelengths of light (e.g., X-rays). Such advantageous conditions include, but are not limited to, more efficient fragmentation of a substance of interest (e.g., a protein) into individual molecules (e.g., individual amino acids), use of a lower power laser (thereby allowing for more practical and safer operation), reduced heating of a fluid containing the substance of interest (thereby reducing thermal degradation of the substance), and the like. In embodiments where the substance of interest is a protein, the use of such lasers and/or laser wavelengths may result in a relatively high probability of cleavage at the peptide backbone, allowing the formation of individual amino acids. In some cases, other types of light sources (e.g., IR) having different wavelengths may also be employed.

In some embodiments, the light source is configured to produce light of any of a number of suitable wavelengths. In some embodiments, the light source may have a wavelength of at least 150nm (e.g., at least 155nm, at least 157nm, at least 160nm, at least 165nm, at least 170nm, at least 175nm, at least 180nm, at least 185nm, at least 190nm, at least 193nm, at least 195nm, at least 200nm, at least 210nm, at least 213nm, at least 220nm, etc.). In some embodiments, the light source may have a wavelength of no greater than 230nm (e.g., no greater than or equal to 222nm, no greater than 220nm, no greater than 213nm, no greater than 210nm, no greater than 200nm, no greater than 195nm, no greater than 193nm, no greater than 190nm, no greater than 185nm, no greater than 180nm, no greater than 175nm, no greater than 165nm, no greater than 160nm, no greater than 157 nm). The wavelength references may have a deviation of +/-5nm, +/-10nm or +/-15 nm. Any of the above ranges are possible (e.g., at least 150nm +/-5nm and no greater than 213nm +/-5nm, at least 160nm +/-5nm and no greater than 213nm +/-5nm, or at least 157nm +/-5nm and no greater than 193nm +/-5nm, at least 180nm +/-5nm and no greater than 213nm +/-5nm, at least 193nm +/-5nm and no greater than 213nm +/-5 nm). Other ranges are also possible.

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

Furthermore, in certain embodiments, the present disclosure relates generally to the generation of ionized molecules, e.g., for detection in a mass spectrometer, or for other uses, such as photolithography, sputtering machines, propulsion, and the like. Some embodiments include an ion source that includes a capillary tip that can allow 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) less than 125nm or 100nm, etc. Furthermore, certain aspects relate to the use of such capillary tips: allowing detection of samples (e.g., amino acids) and, in some cases, sequencing. For example, some embodiments relate to allowing high-speed evaporation of individual ions and ion clusters directly from an aqueous sample in a mass spectrometer. Other aspects relate to methods for making or using such ionized molecules, methods for making or using devices to produce such ionized molecules, and the like.

For example, some embodiments generally relate to ion sources including capillaries and electrodes, which in some cases may be annular, between which a voltage is applied to generate ions. In some cases, the capillary tube may have a tip inner diameter of less than 125nm or 100nm, etc. This may allow ions to evaporate directly from the meniscus of the fluid in the capillary, bypassing the wasteful droplet evaporation process. In this case, ion evaporation may account for a majority of the ion current, and in some cases this emission pattern may be achieved using a relatively low salinity solution. In some embodiments, tips with an inner diameter less than 125nm or 100nm (e.g., less than or equal to 65nm or 60nm, etc.) may be capable of producing a high proportion of bare ions or ion clusters, e.g., containing a small number of solvent molecules, e.g., only 1 or 2 solvent molecules. In some cases, the small area of the liquid vacuum interface may prevent significant heat loss from evaporation, which allows the use of volatile solvents, such as water, in some cases. Methods such as these may be used in some embodiments to analyze molecules or ions, e.g., biomolecules such as amino acids, nucleic acids, peptides or proteins, and the like. In some cases, ion sources such as those described herein can increase the sensitivity of mass spectrometry experiments, allowing single molecule protein sequencing or single cell proteomics research. Other applications are possible as described below.

For example, some embodiments relate generally to ion sources that include capillaries and electrodes. The electrodes may be used to generate ionized molecules directly from the fluid within the capillary, e.g., into a reduced pressure environment or vacuum, e.g., at a pressure of 100mPa or other pressures described herein. In certain embodiments, the opening of the capillary is sized such that when an electric field is applied, the fluid within the capillary forms a charged meniscus and species within the fluid leave the charged meniscus, for example, primarily by ion evaporation. The use of capillaries with submicron openings (e.g., less than 125nm or 100nm, etc.) may be advantageous for fluid ionization by ion evaporation, where the material exiting the capillaries ionizes directly into single charged ions or clusters of charged ions, as opposed to electrospray ionization, where the material exiting the capillaries is expelled through a liquid jet that breaks up into charged droplets that in the presence of a background gas break up further into charged ions, although it should be appreciated that some electrospray ionization may still occur in some cases. In certain applications, such as applications requiring efficient use or the generation of individual ions from a fluid, ion evaporation may be preferred. For example, certain embodiments relate to ion sources in mass spectrometry where individual charged ions can be directly generated and subsequently detected.

According to one set of embodiments, the ion source includes a capillary tube defining an opening having a cross-sectional dimension (e.g., an inner diameter of the capillary tube) of less than 100 nm. In some cases, the openings may also be sized such that ion evaporation dominates over liquid jet formation when an electric field is applied. For example, in certain embodiments, at least 50% of the exiting species may exit by ion evaporation or in the form of ions or clusters of ions. For example, nanoscale capillaries may allow ions to evaporate directly from a fluid meniscus. In some embodiments, the fluid may be passed into a capillary having such openings and delivered directly to a reduced pressure or vacuum environment (e.g., having a pressure of no more than 100 mPa) in the form of ions and ion clusters. Ions and ion clusters may be analyzed by filters and ion detectors in mass spectrometers or applied to other applications such as those described herein.

In addition, certain aspects relate to methods of sequencing molecules or polymers (e.g., biopolymers) from a fluid within a capillary (e.g., by mass spectrometry). In some embodiments, the fluid comprises a polymer, such as a biopolymer, dissolved in a solvent (e.g., water) having a relatively high vapor pressure. In general, evaporation of a highly volatile solvent (e.g., water) can cause the fluid at the capillary opening to freeze as the solvent evaporates, thereby limiting the ability of the mass spectrometer to successfully evaporate ions from the fluid. However, the opening of the capillary tube may be sized such that the fluid meniscus at the opening may have a smaller area that may reduce these effects, as discussed herein. Thus, the use of capillaries with small openings in the ion source of a mass spectrometer may allow the investigation of molecules in aqueous solutions, for example polymers or biopolymers such as amino acids, nucleic acids and peptides or proteins. Non-polymeric molecules may also be studied in certain embodiments.

To sequence molecules such as polymers (e.g., biopolymers), certain embodiments involve applying an electric field to ionize the molecules near the capillary openings to produce ionized fragments. In certain embodiments, ionized debris from the fluid is transferred directly into the reduced pressure environment. In some cases, the ionized fragments may contain a single ion or cluster of ions, as discussed herein, e.g., with a small number of solvent molecules (e.g., water). The opening may be sized such that ionized fragments exit the opening in sequence according to the sequence of molecules. For example, certain embodiments allow the sequence of molecules to be determined by determining ionized fragments within a detector that are generated by ionizing the molecules.

In addition, certain aspects relate to devices comprising ion sources having capillaries as disclosed herein. The device may also have an electrode adjacent the capillary. It should be noted that while some embodiments disclose the use of ion sources in mass spectrometers, the use of ion sources disclosed herein is not only applicable to mass spectrometers. The ion source may also be used in, for example, a lithography machine, a sputter machine, a space-propelling system, etc., as discussed herein.

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

In some embodiments, the capillary tube tapers at the opening. For example, the capillary tube may have a constant taper, e.g., such that the tip of the capillary tube is tapered. 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 (where 0 degrees means no taper, i.e., the capillary is cylindrical.

In certain embodiments where the capillary tube tapers at the opening, a laser drawing technique may be used to fabricate the tapered opening. It should be understood that techniques other than laser drawing techniques may be used to produce capillaries with tapered openings. It should also be appreciated that while the capillaries discussed herein have tapered openings, in other examples, the openings of the capillaries may be non-tapered.

In certain embodiments, the capillary of the ion source comprises quartz. Other examples of materials that may 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.

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 capillary opening. For example, the capillary tube may have an aspect ratio greater than 10,000. However, it should be understood that the aspect ratio is not limited thereto. For example, in some examples, the aspect ratio of the capillary length to the cross-sectional dimension of the opening can 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.

The capillary tube may have a circular or non-circular cross-section (e.g., square). Additionally, in some embodiments, the capillary tube may have a relatively small cross-section, such as a diameter. For example, the cross-sectional dimensions of the capillary tube may be less than 200nm, less than 150nm, less than 75nm, less than 60nm, less than 50nm.

Certain embodiments of the ion source further comprise an electrode positioned adjacent to the capillary (e.g., an opening of the capillary). The electrodes may be used to apply an electric field (e.g., as described below) to the fluid within the capillary (e.g., to the meniscus). In some cases, the fluid within the capillary may be in contact with the counter electrode, e.g., such that a voltage difference between the electrode near the capillary opening and the counter electrode within the capillary is capable of generating an electric field acting on the fluid. In some embodiments, the electrode may be located near the opening of the capillary tube where the electric field is induced to be maximum. For example, in some embodiments, the electrodes may be located within 50mm, within 40mm, within 30mm, within 20mm, within 15mm, within 10mm, within 5mm, within 3mm, within 2mm, within 1mm, etc. of the capillary opening.

In some embodiments, the electrode may be located around the capillary or nanotip, or may be located in front of the capillary or nanotip, for example in front of the capillary opening, or in the downstream direction of the capillary. 1A-1B, electrodes (not shown) may be located around capillary 30 or nanotip 34, in front of opening 36 of capillary 30, or in a direction downstream of capillary 30, as non-limiting examples.

The electrodes may have any suitable shape. In some cases, the electrodes are circular or circularly symmetric, or symmetrically positioned with respect to the capillary. However, other shapes or arrangements are also possible.

In some embodiments, the electrodes define openings (e.g., holes). Thus, in some cases the electrode may be annular. The electrode may be positioned such that ions or clusters of ions escaping from the fluid in the capillary pass through the central opening of the electrode. The central opening of the electrode may be any shape including, but not limited to, a circular shape that may be positioned annularly about the opening of the capillary. In some cases, the opening may also be non-circular. In some embodiments, the opening of the electrode is positioned coaxially with the opening of the capillary. That is, in some embodiments, the opening may be aligned with the opening of the capillary, e.g., such that an imaginary line passing through the center of the capillary cross-section passes through the central opening of the electrode. This may be advantageous to apply an electric field to the fluid in the capillary, for example, to cause ions or ion clusters to leave the fluid, as discussed herein.

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

Furthermore, the electrodes themselves may be of any shape (e.g., circular or non-circular). The electrodes may have the same or a different shape than their openings (if present). The electrodes may have any suitable cross-sectional dimensions. For example, the electrodes may have cross-sectional dimensions of less than 10cm, less than 5cm, less than 3cm, less than 1cm, less than 5mm, less than 3mm, less than 1mm, etc.

In some embodiments, the electrode comprises steel. Other examples include copper, graphite, silver, aluminum, gold, conductive ceramics, and the like.

Accordingly, certain embodiments relate to electrodes capable of generating an electric field. In some cases, as noted, the electrodes may be positioned to produce an electric field maximum near the opening of the capillary. In some embodiments, the fluid is contained in a capillary tube such that when an electric field is applied by an electrode near the opening of the capillary tube, molecules within the fluid can ionize and exit from the opening of the capillary tube, for example as ions or clusters of ions, such as discussed herein. In some cases, for example, the electrodes and capillary (e.g., the interior of the capillary) may be connected to a voltage source, for example, as discussed herein.

Thus, in certain embodiments, a voltage source in combination with an electrode may be used to generate an electric field to move ions or ion clusters away from the fluid in the capillary, for example as discussed herein. In some embodiments, a voltage is applied to generate an electric field at least sufficient to ionize molecules within the fluid at the opening of the capillary, e.g., to generate ions or clusters of ions. For example, in certain embodiments, voltages in the range of 80V to 400V may be used to generate the electric field. In some cases, the voltage may 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. Further, in some cases, the voltage may be no more than 600V, no more than 500V, no more than 450V, no more than 400V, no more than 380V, no more than 360V, no more than 340V, no more than 320V, no more than 300V, no more than 280V, no more than 260V, no more than 240V, no more than 220V, no more than 200V, no more than 180V, no more than 160V, no more than 140V, no more than 120V, no more than 100V, no more than 80V, no more than 60V, and so forth. In some cases, a combination of these voltages is possible. For example, a voltage between 80V and 360V, etc. may be applied. The voltage may be applied as a constant voltage or, in some cases, as a varying or periodic voltage.

As mentioned, a voltage may be applied to generate an electric field maximum near the opening of the capillary or the fluid within the capillary (e.g., at the meniscus at the opening). For example, a voltage may be applied to produce an electric field maximum of at least 0.5V/nm, at least 0.7V/nm, at least 1V/nm, at least 1.1V/nm, at least 1.3V/nm, at least 1.5V/nm, at least 2V/nm, at least 2.5V/nm, at least 3V/nm, at least 3.5V/nm, at least 4V/nm, etc. In certain embodiments, the electric field maximum may be no more than 5V/nm, no more than 4.5V/nm, no more than 4V/nm, no more than 3.5V/nm, no more than 3V/nm, no more than 2.5V/nm, no more than 2V/nm, no more than 1.5V/nm, no more than 1V/nm. In some embodiments, combinations of these ranges are also possible; for example, the electric field may be between 1.5V/nm and 3.0V/nm, between 1.5V/nm and 4.0V/nm, etc.

Without wishing to be bound by any theory, it is believed that in certain embodiments, when an electric field is applied, the fluid within the capillary forms a charged meniscus and the species exits the charged meniscus, for example as ions or clusters of ions. In some cases, the opening of the capillary may be sized such that at least 10% of the exiting species exits by ion evaporation, for example, as ions or clusters of ions. 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 exits by ion evaporation.

As previously described, according to certain embodiments, a cone-shaped charged fluid meniscus may be generated at the opening of the capillary under an electric field. In some embodiments, the tapered fluid meniscus acts as a point source to allow the species to exit as ions or clusters of ions.

The fluid meniscus may generate the exiting species by mechanisms such as generation of charged droplets by electrospray ionization and/or generation of ions and ion clusters by ion evaporation. However, in the case of electrospray ionization, the exiting species exiting from the liquid meniscus will exit as charged droplets containing the exiting species, which will require the presence of a background gas to further break down the droplets into individual ions, typically by a coulomb fission process. Ion evaporation, on the other hand, describes the process of direct ionization of molecules into ions (e.g., bare ions) or clusters of ions (e.g., ions with solvent molecules) rather than charged droplets. Ion clusters can contain a single ion and multiple solvent molecules, typically in relatively small numbers. For example, an ion cluster may contain no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 solvent molecules.

Thus, for example, in some embodiments, the opening of the capillary can be sized (e.g., the cross-sectional dimension of the opening is less than 125nm or 100nm, etc.) such that formation of charged droplets can be avoided and such 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 ionize directly from the conical fluid meniscus at the capillary opening into ions or clusters of ions.

As described above, in some embodiments, capillaries with relatively small openings (e.g., cross-sectional dimensions less than 125nm or 100nm, etc.) can be associated with, for example, generating a relatively small number of solvent molecules in an ion cluster, as described above. In some embodiments, the openings of the capillaries can be sized (e.g., less than 125nm or 100nm, etc.) such that the plurality of solvent molecules comprises less than or equal to a number of solvent molecules, e.g., such that, on average, the ion clusters generated by the ion source comprise less than or equal to 7, 6, 5, 4, 3, or 2 solvent molecules. In some embodiments, a plurality (e.g., greater than or equal to 50%, greater than or equal to 60%, greater than or equal to 70%, greater than or equal to 80%, greater than or equal to 85%, greater than or equal to 90%, greater than or equal to 95%, greater than or equal to 99%, or all) of the ion clusters contain one or two solvent molecules.

In some embodiments, a voltage may 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 capillary tip may be at least 0.1pA (e.g., at least 0.5pA, at least 1pA, at least 2pA, at least 3pA, at least 5pA, at least 10pA, at least 15pA, at least 20pA, at least 50pA, at least 100pA, at least 150pA, at least 200pA, 500pA, at least 1nA, etc.). In some embodiments, the tip of the capillary may be no more than 2nA (e.g., no more than 1nA, no more than 500pA, no more than 200pA, no more than 150pA, no more than 100pA, no more than 10pA, no more than 20pA, no more than 18pA, no more than 15pA, no more than 10pA, no more than 5pA, no more than 3pA, no more than 2pA, no more than 1pA, no more than 0.5pA, etc.). Any of the above ranges are possible (e.g., at least 0.1pA and no more than 2nA, or at least 3pA and no more than 20 pA). Other ranges are also possible.

In addition, as discussed, certain aspects relate to methods of ionizing a fluid using an ion source, for example, to produce individual ions or clusters of ions. Certain embodiments include passing a fluid into a capillary that defines an opening having a cross-sectional dimension of less than 125nm or 100nm, or the like, or other configurations such as those discussed herein.

In some embodiments, the fluid comprises a sample and a solvent. The sample may include any substance of interest that may ionize from the opening of the capillary. For example, according to certain embodiments, the substance of interest comprises a biopolymer (e.g., a nucleic acid such as DNA or RNA, a peptide or protein, etc.). Other examples include other types of polymers, such as nylon, polyethylene, etc., or other substances of interest that are not necessarily polymers, such as biomolecules. Non-limiting examples of biomolecules may include monomers, such as amino acids, nucleotides, and the like. In some cases, the substance of interest is unknown, and it is desirable to determine, at least in part, the structure of the substance, such as by ionizing the substance and detecting ion fragments, such as in mass spectrometry or other related techniques.

In some embodiments, the solvent may be any liquid that may be used to dissolve or suspend the sample or substance of interest. For example, according to certain embodiments, the solvent comprises 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 greater than or equal to 10mM, greater than or equal to 20mM, greater than or equal to 30mM, greater than or equal to 50mM, greater than or equal to 100mM, greater than or equal to 150mM, greater than or equal to 200mM, greater than or equal to 300mM, greater than or equal to 400mM, greater than or equal to 500mM, greater than or equal to 750mM, greater than or equal to 1M, greater than or equal to 2M, greater than or equal to 5M, or greater than or equal to 7.5M. In some embodiments, the aqueous solution may have a salt concentration of less than or equal to 10M, less than or equal to 7.5M, less than or equal to 5M, less than or equal to 2M, less than or equal to 1M, less than or equal to 750mM, less than or equal to 500mM, less than or equal to 400mM, less than or equal to 200mM, less than or equal to 150mM, less than or equal to 100mM, less than or equal to 50mM, less than or equal to 30mM, less than or equal to 20mM, less than or equal to 10mM, etc. Combinations of the above ranges are possible (e.g., greater than or equal to 100mM and less than or equal to 10M, or greater than or equal to 150mM and less than or equal to 1M).

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

In some embodiments, the fluid comprises a solvent having a relatively low pH. 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 no more than 4 (e.g., no more than 3.9, no more than 3.8, no more than 3.6, no more than 3.4, no more than 3.2, no more than 3.1, etc.). Combinations of the above ranges are possible (e.g., at least 3 and not more than 4). Other ranges are also possible.

Additionally, in some embodiments, the fluid comprises a solvent (e.g., water) having a relatively high volatility, e.g., to facilitate the generation of ions or ion clusters. For example, water has a boiling point of 100 c and may be considered volatile in some cases. In some embodiments, liquids having boiling points near room temperature may be used to facilitate the generation of ions or ion clusters. In some embodiments, the solvent that may be used to facilitate ion or ion cluster generation may have a boiling point of less than or equal to 100 ℃, less than or equal to 80 ℃, less than or equal to 60 ℃, less than or equal to 40 ℃, less than or equal to 20 ℃, and the like. In addition, the solvent may have a boiling point of 10 ℃ or more, 30 ℃ or more, 50 ℃ or more, 70 ℃ or more, 90 ℃ or more, or the like. Combinations of these are also possible; for example, the solvent may have a boiling point between 50 ℃ and 100 ℃. Other examples of solvents having relatively high volatility include, but are not limited to, acetone, isopropanol, hexane, and the like.

In some embodiments, the temperature of the capillary (in addition to the type of fluid it contains) may be varied to control the number of solvent molecules in the resulting ion clusters. In some embodiments, the temperature of the capillary is set such that the plurality of solvent molecules comprises less than or equal to a number of solvent molecules, e.g., such that, on average, the ion clusters generated by the ion source contain less than or equal to 7, 6, 5, 4, 3, or 2 solvent molecules. In some embodiments, the temperature is at least 20 ℃, at least 30 ℃, at least 40 ℃, at least 50 ℃, at least 60 ℃, or at least 70 ℃. In some embodiments, the temperature is no more than 80 ℃, no more than 70 ℃, no more than 60 ℃, no more than 50 ℃, no more than 40 ℃, no more than 30 ℃. Combinations of the above ranges are possible (e.g., greater than or equal to 20 ℃ and less than or equal to 80 ℃). In some cases, the temperature of the capillary is controlled by a resistive heater, a peltier junction, an infrared heater, or the like.

In some embodiments, a suitable electric field range and a suitable capillary opening size range may be selected to cause at least some molecules to leave as ions or clusters of ions, e.g., as discussed herein.

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

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 no more than 100 mPa. In certain embodiments, the environment may have a pressure of no more than 1000mPa, no more than 300mPa, no more than 100mPa, no more than 30mPa, no more than 10mPa, no more than 3mPa, no more than 1.5mPa, no more than 1mPa, no more than 0.3mPa, no more than 0.1mPa, etc. In some embodiments, ions or clusters of ions from the fluid are directed into the vacuum environment.

It should be appreciated that some embodiments provided herein focus on delivering ionized molecules directly from a fluid into an environment having a pressure of no more than 100mPa. However, it should be understood that the pressure within the environment is not limited to 100mPa. In some embodiments, the pressure may also be greater than or equal to 100mPa and less than or equal to 1Pa.

In some embodiments, the mass spectrometer comprises a pump. The pump may be used to create a reduced pressure or vacuum environment, for example as discussed herein. Non-limiting examples of pumps include diffusion pumps, molecular drag pumps, turbo-molecular pumps, and the like.

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

According to certain embodiments, the openings of the capillaries are sized such that solvents with relatively high volatility remain unfrozen at the openings of the capillaries when exposed to relatively low pressure. In some embodiments, the openings of the capillaries are small enough that the relatively high volatility solvent remains unfrozen when entering the surrounding environment. In some embodiments, the opening of the capillary is small enough that the fluid comprising the sample and solvent remains unfrozen as the substance of interest ionizes, such that at least some of the substance of interest ionizes to form ions (e.g., single ions) or clusters of ions.

Some aspects relate to a mass spectrometer comprising an ion source as described herein. However, ion sources such as described herein are not limited to mass spectrometers, but may be used in other applications such as lithography, sputtering, propulsion (e.g., spatial propulsion), and the like. As a non-limiting example, in lithography, a Focused Ion Beam (FIB) machine may be used to inspect and/or modify a lithography mask, and/or etch features into a material by sputtering. Sputtering is the process of removing atoms from a solid surface by ion impact with high kinetic energy. In some embodiments, the ion sources described herein are present in a Focused Ion Beam (FIB) machine, which can be used to deliver molecules to a substrate material in a patterned manner.

In certain embodiments, an ion source as described herein may be used with a liquid chromatography mass spectrometry system. For example, a liquid chromatograph may be coupled to the ion source to separate peptides or other molecules before they are ionized and delivered to the mass spectrometer. In some cases, mass spectrometers can be used to perform single or tandem (MS/MS) analysis to identify ionized peptides or molecules, such as proteomic experiments. Advantageously, the use of the ion source described herein (having a capillary with a nano-sized opening and/or tip) to deliver ions directly into a low pressure environment can increase the sensitivity of the instrument, ion transport efficiency in such systems, and eliminate the need for multiple pump stages.

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

As another example, in some embodiments, the ion sources described herein are used for propulsion. For example, when ions are ejected in a rearward direction, forward propulsion of the object may be generated. In some embodiments, an ion source such as described herein is used in a propulsion system. This may be used, for example, to transfer high thrust forces relative to the weight of the ion source, for example, due to the small size of the ion source. In addition, in some cases, the propulsion system may be made compact and consume relatively less fuel than conventional propulsion systems.

In addition, some aspects relate to mass spectrometers including the ion sources described herein. In some cases, a mass spectrometer may include components such as a vacuum chamber (e.g., capable of producing any reduced pressure described herein), ion optics (e.g., one or more lenses, e.g., einzel lenses, etc.), filters (e.g., quadrupole rods filters, sector magnetic field filters, etc.), detectors, ion benders, ion traps, or the like, in addition to ion sources such as described herein. Examples of specific detectors include, but are not limited to, faraday cups, electron multipliers, dynodes, charge Coupled Devices (CCDs), CMOS sensors, fluorescent screens, and the like. Other non-limiting examples of mass spectrometers are described in provisional application entitled "systems and methods for single ion mass spectrometry with time information" filed on day 4, month 23 of 2021, which is incorporated herein by reference in its entirety.

In addition to the ion source, various ion optical elements may be placed downstream of the ion source such that the exiting molecules (e.g., ions and ion clusters) may in some cases be transported along a path downstream of the ion source, e.g., the downstream direction is the direction in which the ions or ion clusters travel. Referring again to fig. 1A-1B, as a non-limiting example, ion optics 100 may be located downstream of ion source 20 such that exiting molecules 54 may travel along a path downstream of ion source 20. Those of ordinary skill in the art will be familiar with the various ion optics used in mass spectrometry. In some embodiments, the ion optical element includes one or more single lenses (e.g., a first single lens and a second single lens). When the ion optics transmit molecules (e.g., ions or ion clusters) to the filter, the mass-to-charge ratio (m/z) of the molecules (e.g., ions and ion clusters) can be analyzed by the filter. In some cases, the mass filter may be located downstream of the ion optical element. For example, as shown in fig. 1A-1B, a filter 100 (e.g., a magnetic filter) may be located downstream of the ion optical element 100. Examples of filters include, but are not limited to, quadrupole rod filters, sector magnetic filters, and the like.

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

In some embodiments, a mass spectrometer such as described herein may include an average or overall ion transmission rate (e.g., a ratio of detected ions and ion clusters to ions and ion clusters exiting from the fluid at the capillary opening) of greater than 0.01, and in some cases, a transmission rate of 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 transport rate is no more than 1, no more than 0.99, no more than 0.95, no more than 0.93, no more than 0.9, no more than 0.8, no more than 0.75, no more than 0.7, no more than 0.6, no more than 0.5, no more than 0.4, no more than 0.3, no more than 0.2, no more than 0.15, no more than 0.1, no more than 0.05, or no more than 0.02. Combinations of the above ranges are possible (e.g., at least 0.02 and no more than 0.9, or at least 0.1 and no more than 0.8, at least 0.9 and no more than 1), and the like. Other ranges are also possible.

In one set of embodiments, a mass spectrometer includes a tip having an opening with a cross-sectional dimension (e.g., less than 100nm, less than or equal to 65nm, less than or equal to 60nm, less than or equal to 50nm, less than or equal to 40nm, less than or equal to 30nm, etc.) as described herein that can have an ion transport 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 transport efficiency described above may have a deviation of +/-3%, +/-2%, or +/-1%.

In some embodiments, tips having an inner diameter less than or equal to 65nm (e.g., less than or equal to 60nm, less than or equal to 40nm, less than or equal to 20nm, etc.) may be employed to produce a high proportion (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, such as ions that do not include solvent molecules. In some embodiments, the tip produces only bare ions. In some cases, emitting bare ions may be particularly advantageous compared to ion clusters or charged droplets, as direct emission of bare ions may allow for improved determination of differential amino acids (e.g., including amino acid variants with post-translational modifications).

In some embodiments, when ions and/or ion clusters (if present) are emitted into a vacuum at the tip of a capillary, the ions and/or ion clusters experience little, if any, collisions with gas molecules (e.g., background gas molecules). For example, in some embodiments, the likelihood that the ions and/or ion clusters will undergo 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%, etc.).

Certain aspects relate to sequencing polymers (such as biopolymers) using an instrument (e.g., such as a mass spectrometer as described herein) that includes an ion source.

For example, in some embodiments, the polymer may be the substance of interest. The substance 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 substance of interest. Furthermore, it should be understood that other types of polymers, such as synthetic or artificial polymers, may also be sequenced in some cases. Furthermore, the structure of the non-polymeric substance of interest may similarly be determined.

In some cases, for example, the structure, sequence, and/or identity of a substance of interest (e.g., a polymer) can be determined by determining an ionizing fragment using a detector. The sequence of the substance of interest may be detected, for example, by monitoring the time of arrival of individual ionised fragments (e.g. ions or ion clusters) at the detector, for example by ionising the polymer and generating ions or ion clusters as described above. Without wishing to be bound by any theory, it is believed that, for example due to the size of the capillary openings, the substance of interest (e.g., polymer) may ionize in a substantially linear manner, and then the ions or ion clusters generated may be determined by the detector discussed herein, e.g., in the order in which the ions or ion clusters were generated from the substance of interest. In some embodiments, the capillary comprises carbon nanotubes or boron nitride nanotubes, wherein the cross-sectional dimension (e.g., inner diameter) of the nanotubes is sufficiently small, e.g., 1nm to 2nm, such that the polymer molecules can ionize in a sequence reflecting the primary structure of the polymer. Of course, in other embodiments, larger diameters or other materials are also possible, such as discussed herein. It should be noted that in some cases, such as when ions or ion clusters are transferred into a reduced pressure environment, the detector may be able to determine such order with relatively high fidelity, for example, because ions or ion clusters do not collide relatively with gas molecules as they pass through the detector. Thus, based on the determined order of ions or ion clusters, the structure or sequence of the substance of interest can be determined.

In some embodiments, a mass spectrometer described herein can include more than one ion source. For example, a mass spectrometer may include an ion source as described herein and one or more additional ion sources. For example, in some aspects of the disclosure, disclosed herein are multiplex mass spectrometers comprising a plurality of ion sources. The plurality of ion sources may be a plurality of identical (or different) ion sources. Some or all of the plurality of ion sources may include capillaries containing a substance of interest suspended in a fluid. In some embodiments, a multiplex mass spectrometer may allow for simultaneous detection and sequencing of multiple species of interest contained within capillaries of multiple ion sources.

In some embodiments, a multiplex mass spectrometer may include a plurality of ion sources, a magnetic filter downstream of the plurality of ion sources, and a detector array downstream of the magnetic filter. A non-limiting schematic representation of a multiplex mass spectrometer is shown in fig. 12. As shown, the multi-mass spectrometer 110 includes a plurality of ion sources 120, a filter 190 (e.g., a magnetic filter) downstream of the ion sources 120, and a detector array 170 (e.g., an imaging detector) downstream of the filter 190. Some or all of the plurality of ion sources shown in fig. 12 may be the same as the ion sources described in fig. 1A-1B. For example, some or all of the ion sources may include capillaries and electrodes near the capillary or capillary nanotip. The capillary tube may have any of the properties described elsewhere herein, such as having an open tip portion (e.g., nanotip), a body portion, and/or containing a substance of interest suspended or dissolved in a fluid, etc. In some cases, the plurality of ion sources may be arranged in a linear array. In some cases, some or all of the plurality of capillaries may include nanotips arranged in a linear array. For example, as shown in fig. 12, mass spectrometer 110 includes a plurality of capillaries 130a, 130b, and 130c, each having nanotips, arranged in a linear array. In some embodiments, some or all of the plurality of ion sources may contain a substance of interest within the capillary. The species of interest within the ion source may be the same or different.

The multiplex mass spectrometer may include any suitable number of ion sources. For example, a multiplex mass spectrometer may include at least 2 (e.g., at least 3, at least 5, at least 10, at least 25, at least 50) and/or up to 100 (e.g., up to 200, up to 500, or up to 1000) ion sources. Combinations of the above ranges are possible (e.g., at least 2 and up to 1000). Other ranges are also possible.

In some embodiments, the multiplex mass spectrometer may further comprise a light source directed at the plurality of ion sources. The light source may have any of the characteristics and/or configurations described elsewhere herein. A multiplex mass spectrometer such as that shown in fig. 12 may include the same light source (not shown) as that shown in fig. 1A-1B (e.g., a UV laser). As described elsewhere herein, the light source may be configured to fragment a substance of interest within a capillary (e.g., a tip portion of the capillary) of the ion source into individual components. The fragmented individual components may in turn be ionized from the capillary tip into a vacuum. For example, as shown in fig. 12, the light source may be configured to fragment a substance of interest within a capillary of the ion source 120 (e.g., a tip portion of the capillaries 130a, 130b, 130 c) into individual components, which may then be ionized to a vacuum 180. 154 shows trajectories of ionized individual components from the nanotip.

In some embodiments, the multiplex mass spectrometer comprises a magnetic mass filter capable of simultaneously separating ionized individual components exiting each ion source based on their mass-to-charge ratios. For example, the magnetic filter may be configured to transfer ionized fragmented individual components from each ion source to a detector array downstream of the magnetic filter. The detector array, in turn, may be configured to simultaneously detect ionized fragmented individual components from each of the plurality of ion sources. For example, as shown in fig. 12, a magnetic filter 190 (e.g., a magnetic sector) may be used to separate ionized individual components exiting each ion source based on mass-to-charge ratio.

According to some embodiments, a magnetic filter may be used to separate and focus the ionized individual components in lateral and transverse directions before they strike the imaging detector array. Referring to fig. 12, as a non-limiting example, a magnetic mass filter 190 may be used to separate and focus the ionized individual components 154 in lateral and transverse directions before the individual components 154 strike the detector array 170 (e.g., imaging detector). According to some embodiments, the detector array may be arranged in a two-dimensional array capable of detecting ionized individual components in lateral and transverse directions. A multiple mass spectrometer having the configuration described herein may allow for sequencing of various types of substances of interest simultaneously and with high throughput.

The multiplex mass spectrometer may include any suitable additional components described elsewhere herein. For example, the multiplex mass spectrometer may also include ion optics (e.g., optical lenses, etc.) located between the ion source and the mass filter. A non-limiting example of one embodiment of an ion optical element is shown in fig. 1A-1B, for example as shown by ion optical element 100.

U.S. provisional patent application 63/235,601, entitled "System and method for peptide photolysis analysis for Single molecule protein sequencing", filed by Stein et al at 2021, 8, 20, is incorporated herein by reference in its entirety. Furthermore, U.S. provisional patent application 63/341,992, filed by Stein et al at 5/13, 2022, "systems and methods for peptide photolysis analysis for single molecule protein sequencing," is also incorporated herein by reference in its entirety.

U.S. provisional patent application Ser. No. 63/015,407, entitled "nanotip ion Source and method," filed by Stein et al at 24, 4, 2020, is incorporated herein by reference in its entirety.

Furthermore, U.S. provisional patent application Ser. No. 63/179,064, entitled "System and method for Single ion Mass Spectrometry with time information", filed by Stein et al at month 4, 2021, is also incorporated herein by reference in its entirety. International patent application publication No. PCT/US2022/025902, entitled "System and method for Single ion Mass Spectrometry with time information" filed on month 22 of 2022, is also incorporated herein by reference in its entirety.

International patent application publication No. PCT/US2021/028954 filed on 4/23 at 2021 is incorporated herein by reference in its entirety.

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

Example 1

This example analyzes the feasibility of using light to fragment peptides into their constituent amino acids, which are then identified by Mass Spectrometry (MS) for single molecule protein sequencing purposes. Laser power considerations strongly favor photo-fragmenting the peptide in solution before it leaves the ion source, rather than in the gas phase. Ultraviolet (UV) wavelengths around 200nm are poorly absorbed in water and a single photon can selectively cleave peptide bonds linking amino acids together. These properties make UV fragmentation more promising than infrared or X-ray based methods. This example developed a simple model regarding the probability of complete release of an amino acid by cleavage of peptide bonds on both sides of the amino acid before photodamage to the amino acid itself. It is predicted that 193nm light can release a variety of amino acids with probabilities ranging from 0.65 to 0.92; however, the probability of intact release of the aromatic amino acids, histidine, methionine, arginine and lysine, which are relatively vulnerable to photodamage, ranges from 0.004 to 0.330. These findings indicate that UV fragmentation can reveal a large number of sequence information of individual proteins to a mass spectrometer.

Methods of sequencing individual proteins based on a nanocapillary ion source are described. The basic idea is shown in fig. 1A-1B. The voltage applied to the source drives the positively charged peptide to the nanometer Kong Jianduan, the tip being small enough to force the peptide chain to form a linear structure. The ion source sequentially emits the constituent amino acids from the liquid into a vacuum. The amino acid ions pass through a magnetic filter that separates them according to mass to charge ratio before striking the ion single ion detector array. The location of the impact reveals the identity of the amino acid, and the time of detection reveals the reproduction of its original sequence. The individual amino acids are separated from the parent peptide chain by an intermediate step prior to mass filtration. In proteomics, light is widely used for optical fragmentation of peptides. This example analyzes the feasibility of using a laser to photo fragment peptides in a single molecule protein sequencing method.

FIG. 1A is a schematic diagram of single molecule protein sequencing by nanopore mass spectrometry. The schematic shows trajectories of heavy amino acid ions and light amino acid ions emitted from the nano-capillary ion source. The ions pass through the ion optics and the magnetic mass filter and strike a single ion detector array. Ultraviolet laser is used to fragment the peptide. FIG. 1B is a schematic representation of the optical segmentation of an elongated peptide chain near the tip of a nanocapillary ion source.

In order to fragment the peptide before it passes through the mass filter, a laser beam may be directed into the ion path on the vacuum side of the ion source. The problem with this approach is that the peptide passes through the beam very quickly. It is envisaged that for example the arginine dipeptide has a mass of 330amu and a charge of +2e. If an extraction voltage of 300V is applied (approaching the lower limit required for the ion source), the dipeptide ion will acquire a kinetic energy of 600eV and travel the entire distance from the source to the detector of the instrument in less than 20 microseconds; it takes even less time in the beam path. This sets a lower limit on the illumination power required to achieve high probability fragmentation. For comparison, in previous studies, 50W CO ₂ laser irradiated peptides for 10ms or more to induce photofragmentation in vacuo. To deliver the same energy in 20 microseconds as a 50W laser delivers in 10ms requires 25,000W laser power. Thus, an impractical powerful laser is required to fragment the peptide on the vacuum side of the ion source.

Another possibility is to direct the laser to the peptide while it is still in solution within the nanocapillary ion source. There, the peptide moves at least seven orders of magnitude slower than in vacuum. The fastest transport process appears to be the convective flow mechanism.

The ion flow along the charged surface of the taylor cone causes the circulating fluid to flow, reaching a maximum velocity near the tip. The highest velocity in the nanocapillary is estimated to be about 10 ^-4 m/s. Another relevant transport mechanism is electrophoresis; however, even the largest electric field in the instrument of this example acts on the peptide with the highest electrophoretic mobility, which migrates an order of magnitude slower than the electroconvective flow. Brownian motion is also a relatively slow transmission process over a distance comparable to the radius of the laser beam. The most diffusive peptide takes approximately 3 minutes to diffuse a distance of 0.5mm—two orders of magnitude longer than would be required for the same distance of convective flow propagation. Thus, the peptides in the liquid should pass through the laser beam slowly enough so that the 0.1W laser can deliver them the same energy as the 50W laser within 10 ms. Clearly, targeting the peptide while still in solution substantially reduces the need for incident laser power, making liquid photofragmentation viable and safe.

The wavelength of the light affects the type of molecular fragments produced by fragmentation of the light. Fig. 2 shows the structure of the dipeptide, the structure of the common photo-fragmentation product, and the approximate frequencies at which lasers of different wavelength ranges induce specific transformations in vacuo. The parent dipeptide comprises two amino acids sharing a chemical backbone. The peptide bond of the backbone needs only about 4eV to break, one of the most labile bonds in the molecule. If one of skill in the art is able to selectively break this bond, the resulting amino acid can be readily identified by its mass. Or breaking different bonds in the backbone will transfer some mass from one amino acid to its neighboring amino acid, but one skilled in the art should be able to interpret this mass transfer and still identify each amino acid. However, if photons damage or pop up amino acid side chains (which are distinguishing features), this can significantly complicate the protein sequencing scheme.

FIG. 2 shows a comparison of peptide light fragmentation by IR, UV and soft X-ray. The thickness of the line indicates the relative abundance of important fragmentation products, similar to the study in vacuo.

The absorption of light by water further limits the properties of light that can be used for sequencing. The linear absorption coefficient (μ) of water at 10.6 microns is 10 ⁵m^-1, so the incident IR laser beam will be absorbed over a characteristic distance of only 10 μm35. This suggests that a very small fraction of the laser power can be absorbed by the peptide in solution and that the incident power required to induce the multiphoton dissociation process can be very high. The absorption of ultraviolet light in water is weaker by several orders of magnitude, with μ=10m ^-1 at 193nm and μ=1m ^-1 at 222nm, giving characteristic absorption distances of 10cm and 1m, respectively. The heating effect may set an upper limit on the incident power density at a given wavelength. It is believed that if the absorbed light heats the water in the nano-capillary to boiling point, it may interfere with the ion evaporation process.

The temperature rise that should be caused by absorption of light of different wavelengths in IR and UV is calculated by numerical calculation. The capillary was modeled as a truncated cone with a tip radius of 20nm, a length of 500 microns, and a cone aperture of 6 °. Assume that a 1mm wide laser beam centered on the capillary tip irradiates the cone from the side (light arrives perpendicular to the cone axis); this exposes the entire cone to a uniform incident power density ρ. For example, a 1W laser beam focused to a diameter of 1mm produces a power density of 1.3X10 ⁶W/m². Calculating steady-state temperature distribution by solving steady-state heat equation

Where k=0.6 Wm ^-1K^-1 is the thermal conductivity of water at 300K, T (r) is the temperature at the r position, and qv (r) is the volumetric heat generation (Wm ^-3) caused by light absorption. To simplify the calculation, it is assumed that qv (r) depends only on the axial distance z from the cone tip, and qv (z) is obtained by calculating the average power density absorbed by each thin circular cross section at the axial position z, ignoring the refraction of the light entering the cone, but considering the case where μ penetrates to different depths as light rays. Zero heat flux boundary conditions are applied to the tip and sides of the cone and the bottom is maintained at a constant temperature. Equation 1 for axisymmetric cones was solved by finite element analysis of a two-dimensional grid using the partial differential equation toolbox in Matlab.

Fig. 3A shows a normalized steady-state temperature profile along the cone axis. For all wavelengths studied, the temperature rose monotonically from bottom to tip, with the rise in UV wavelengths 193nm and 222nm being relatively steep compared to the rise in IR wavelength of 10.6 microns.

Fig. 3B plots maximum temperature rise at the tip as a function of ρ. It depends largely on the wavelength. Light of 10.6 microns can cause significant heating of the tip, with a power density of only about 4x 10 ⁴W/m², equivalent to a 32mW laser with a beam diameter of 1mm, required to reach the boiling point of water in room temperature experiments. In contrast, the UV wavelength generates minimal heat; even with an extremely high power density of 10 ⁷W/m², 193nm and 222nm light should heat the tip below 10K and 1K, respectively. These results indicate that the heating of water limits the use of IR light in this sequencing protocol.

FIG. 3A shows calculated heating profiles in a nanocapillary under stable illumination with light of 10.6 μm, 193nm and 222 nm. Fig. 3B shows the dependence of the maximum temperature increase in the nanocapillary on the incident laser power density for 10.6 microns (triangle), 193nm (square) and 222nm (dot) light. The symbols show the results of the finite element calculations, and the curve is a linear fit of the data.

Summarizing the comparison of wavelengths, UV light provides relatively specific peptide backbone cleavage, low power requirements, and low water absorption, all of which facilitate sequencing of individual proteins.

This example now estimates whether UV light can reliably separate amino acids from each other without damaging them to an unidentifiable extent. With UV exposure, the probability of backbone cleavage between two amino acids increases, but the probability of cleavage of other bonds also increases, which may complicate sequencing. To evaluate these tradeoffs, a simple light sheet segmentation process probability model was developed.

Table 1 shows the UV absorption and photodecomposition characteristics of peptides and amino acids.

TABLE 1

The peptide bond and amino acid act as separate UV absorbers in aqueous solution. The molar absorptivity ε _i is attributed to each at 193 nm. Thus, a particular peptide bond or amino acid absorbs a photon at an average rate of jσ _i, where J is the local photon flux and σ _i is the absorption cross section of absorber i, calculated from epsilon _i. After absorption of the photon, the increased energy may lead to fragmentation of the molecule and also to a vibration dissipation process, leaving the chemical bonds intact. The fraction Φ _i of absorbed photons that cause photolysis is also referred to as quantum efficiency. The combination of the absorption and dissociation processes gives the average photolysis rate of the particular species, jσ _iΦ_i. The cumulative probability of photolytic cleavage of a particular peptide or amino acid, P _di, increases over time t according to the following equation

Experimental values of epsilon, sigma _i and phi i for fifteen of the twenty different amino acids and peptide bonds and hydroxyproline (a commonly modified form of the amino acid proline) are summarized in table 1. Experimental values for the amino acids asparagine (asn), cysteine (cys), glutamine (gln), glutamic acid (glu) and isoleucine (ile) have not been obtained.

Fig. 4A compares the cumulative probability of dissociation of peptide bonds P _d,pep.P_d,pep at different values of ρ to rise and asymptotically agree on a characteristic time scale inversely proportional to ρ. When ρ=10,000 w/m ², the characteristic time scale is 0.7s, the power density corresponds approximately to a 10mW, 1mM wide laser beam.

It may also be helpful to estimate the number of amino acids that can be exposed to UV light without photolysis occurring. The cumulative retention probability P _si for amino acid i is

Fig. 4B plots P _s,i for 16 different amino acids at r=10,000 w/m ². The retention of amino acids, measured by the characteristic decomposition time (jσ _iΦ_i)^-1), is largely dependent on their type, aromatic amino acids tyrosine (Tyr), phenylalanine (Phe) and tryptophan (Trp) decay relatively fast, photodecomposition occurs on the time scales of 0.07s, 0.17s and 0.20s, respectively, histidine (His) decays relatively fast, characteristic decomposition time is 0.20s, compared to the amino acids valine (Val), threonine (Thr), leucine (Leu), serine (Ser), proline (Pro), hydroxyproline (Hyp), glycine (Gly), alanine (Ala) and aspartic acid (Asp) which have a long lifetime, and decomposition time scales of 7-56s methionine (Met), arginine (Arg) and lysine (Lys) which have a decomposition time in the range between the long-life group and the short-life group.

This example also examined the possibility of releasing a given amino acid from a protein without destroying the amino acid to an unrecognizable level. The most direct mechanism is to fragment the two peptide bonds connecting amino acid i and the peptide chain without causing photolysis of the amino acid itself; the cumulative probability of this selective cleavage, P _seli, is obtained by combining equations 2 and 3:

P_sel,i＝P_s,i(P_d,pep)². (4)

Fig. 4C plots the time evolution of P _sel,i of 16 amino acids, where ρ=10,000 w/m ². In all cases, P _sel,i increases over time, after which it peaks and then decays. For amino acids with longer characteristic dissociation times, the peak is higher and occurs after longer UV exposure. For aromatic amino acids and His, P _sel,i peaks in the range of 0.004-0.028 and within 0.25 s. After exposure times in the range of 2s-3s, the peak of long-lived amino acids is in the range of 0.65-0.92. The middle group (Met, arg and Lys) reached a peak P _sel, i in the range of 0.086-1.33 after an exposure time in the range of 0.5s-1.5 s.

FIG. 4A shows the cumulative probability of dissociation of the peptide bond obtained from equation 2 for different intensities of 193nm laser exposure, as shown. Fig. 4B shows the probability of amino acid non-decomposition as a function of 193nm laser exposure time for ρ=10,000 wm ^-2, calculated according to equation 3. Fig. 4C shows the probability of selective amino acid release (i.e., cleavage of two peptide bonds linking an amino acid and a peptide without damaging the amino acid) as a function of 193nm laser exposure time for ρ=10,000 wm ^-2. The probability is calculated according to equation 4. In fig. 4B and 4C, the different amino acids are represented by colors and ordered by probability.

It was found that UV light should be able to completely release intact amino acids from proteins with a relatively high efficiency. The released amino acid is defined as the amino acid resulting from cleavage of two flanking peptide bonds. Many amino acids can be released with a probability in the range of 65% -92%. If the possibility of liberating amino acids by cleavage of different (i.e. non-peptide) bonds along the backbone is also considered, or the amino acids can still be identified by their mass after further photolysis processes, the proportion of identifiable fragments should be increased. For example, breaking bonds in aromatic groups of amino acids may significantly alter their light absorption spectra (this change is considered photolytic in optical measurements) without altering their mass.

In summary, UV light provides a promising approach to fragment peptides into their constituent amino acids for single molecule analysis. The extremely high speed of ion travel from the ion source to the detector in vacuo makes it preferable to photo-fragmentation when the peptide is in solution prior to ion extraction. Ultraviolet wavelengths around 200nm are most promising for sequencing, thanks to their low absorptivity in water, their relatively high selectivity for fragmentation of peptide backbone bonds, and the moderate laser power requirements required for single photon bond cleavage processes. Calculation of the rate of competing photochemical processes suggests that it should be possible to cleave peptide bonds flanking many amino acids before the amino acids are damaged by the identified side chains. The accuracy of amino acid calls in sequencing can be over 90% for the most stable side chains, but for less and less stable side chains, a decrease is expected. Future measurements of the photo-fragmentation products and the relative selectivity of different wavelengths can be used to optimize UV light fragmentation to analyze the composition and sequence of individual proteins.

Example 2

Introduction to the invention

Mass Spectrometry (MS) is the mainstay of proteomics research because of its ability to distinguish amino acids from small peptides by mass. Its usefulness also stems to a great extent from the availability of light ionization techniques for the complete transfer of peptide ions into the gas phase. In particular, electrospray ionization (ESI) transfers analytes into a mass spectrometer by a plume of charged droplets emerging from a liquid cone jet at the end of a voltage-biased capillary, as shown in fig. 5A. The droplets pass through a background gas that initiates a series of evaporation and coulomb explosion cycles, ultimately releasing the analyte ions into the gas phase. However, the background gas required to release ions from the droplet is also one cause of serious loss of sample, limiting the sensitivity of MS.

The background gas and its resulting plume of charged droplets widely disperse ions, most of which collide with the transport capillary, which 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 emission tips of hundreds of microns in diameter, with only one ion reaching the mass analyzer in about 10 ⁴. Nano electrospray ionization (nano-ESI) increases ion transport efficiency in a typical measurement to about 1% (sometimes up to 12%) by using an emitter with a micron-sized tip to reduce the flow rate to a range of several nL/min. However, synergistically optimizing the efficiency of multiple analytes is fundamentally challenging because ESI involves the process of physically separating different ionic species within a plume. Most advanced mass spectrometry instruments still require thousands to millions of copies of a protein for their identification. This sensitivity does not reach the sensitivity expected for single cell proteomics and single molecule analysis. Achieving single molecule sensitivity requires an ion source that can avoid loss mechanisms from ejecting charged droplets into the background gas.

Presented in this example is a nanopore ion source that emits amino acid and small peptide ions directly into a high vacuum from its tip (fig. 5B). The ion source comprises a drawn quartz capillary having a tip with an inner diameter of less than 100nm. It is speculated that the small probability of the tip affects ion emission in a number of ways: first, the surface tension of water can maintain a stable liquid-vacuum interface that can support many atmospheres when stretched through nanoscale openings. Second, the fluid flow rate, which is proportional to the inverse cube of the tip diameter, may be too low to form a stable electrospray conical jet, and this may prevent the charged droplets from being completely emitted. Third, the electric field can be concentrated at a sharp conductive tip, such as a nano-capillary filled with electrolyte, reaching-1V/nm at the meniscus and extracting ions at a high rate through the ion evaporation process.

Described herein in this example is a characterization of ion emission from an aqueous solution through a nanopore ion source directly into a high vacuum. Mass spectra of amino acids and small peptides were obtained using a custom quadrupole mass spectrometer, wherein the nanopore ion source was operated at a pressure below 10 ^-6 torr (fig. 5C). In addition, greater than 93% current transmission was measured between the electrolyte-filled ion source and the downstream faraday cup. Furthermore, the use of a magnetic sector separates the charged droplets from the effect of ions on the tip current, demonstrating that the nanopore ion source can emit ions only. This example demonstrates the simplicity of the nanopore ion source described herein to efficiently transfer ions into a high vacuum without the complexity of traditional ESIs such as ion funnels, multi-pump stages, transfer capillaries, and droplet plumes.

Results

Emitting amino acid ions from a nanopore ion source

The emission of amino acids in aqueous solutions was characterized in a custom quadrupole mass spectrometer as shown in fig. 5C. In a typical experiment, ion emission from a nanopore ion source is initiated by applying an extraction voltage V _E in the range of +260V to +360V between the tip and the extraction electrode. This range of V _E may be significantly lower than the voltage typically required to initiate electrospray in conventional ESI or nano-ESI sources. The tip current I _T employed is typically in the range of 3-20 pA. The opening of the current may be abrupt and is typically accompanied by a measurement of ions striking the instrument detector. At these low tip currents, easily interpreted mass spectra can be collected within a few minutes.

FIG. 5D shows a mass spectrum of a 100mM arginine aqueous solution. This spectrum was obtained in positive ion mode using a nanopore ion source with a tip internal diameter of 41 nm. Five peaks are clearly visible. The peak at 175m/z corresponds to singly charged arginine ions (Arg ⁺). The higher m/z peaks are separated by 18m/z, which is the displacement caused by additional water molecules. Thus, the other peaks correspond to the solvation state of arginine (Arg ⁺(H₂O)_n), where the solvation number n ranges from 1 to 4.

Figure 6A illustrates how tip diameter affects arginine mass spectra. The spectra shown were obtained using nanocapillary tubes with tip inner diameters of 20nm, 125nm and 300 nm. The largest tip produced a broad spectrum of peaks including bare arginine ions, eight incrementally hydrated arginine ion clusters, and a peak at 349m/z corresponding to arginine dimer ion (Arg Arg+H) ⁺. A medium-sized tip produces a narrower spectrum that includes bare arginine ions, six incrementally hydrated arginine ion clusters, and relatively weakened arginine dimer ion peaks. The smallest tips produced predominantly bare arginine ions, but decaying peaks corresponding to the mono-and di-hydrated arginine ion clusters could also be seen in the spectra. Smaller tips tend to produce a relatively stronger signal and less noise spectrum than larger tips, as can be seen by comparing the baselines of the three spectra in fig. 6A. Some differences were observed in solvation state distribution between the nano-capillaries with similar tip sizes (e.g., when comparing the spectrum generated by the 41nm tip in fig. 5D with the spectrum generated by the 20nm tip from fig. 6A). However, only nanocapices with tip inside diameters less than about 65nm can produce spectra measured in the unsolvated state for most amino acid ions.

FIG. 6B shows the mass spectra obtained from 16 different aqueous amino acid solutions, with the concentration of all amino acids being 100mM except for tryptophan being 50 mM. These measurements used four different nanocapillaries, with tip inner diameters of 20, 25, 57 and 58nm, respectively. The most pronounced amino acid peak in each profile shown in fig. 6B corresponds to singly charged and unsolvated ions. The spectra of glycine, alanine, proline, valine, cysteine, glutamine and phenylalanine showed no additional peaks that could correspond to solvated amino acid ions. The spectra of serine, threonine, asparagine, lysine, methionine, histidine, arginine and tryptophan showed a sub-peak at 18m/z to the right of the unsolvated peak, corresponding to the amino acid ion monohydrate. Leucine showed a third and possibly a fourth peak corresponding to a higher solvation state. Tryptophan spectra showed peaks below 200m/z, which corresponds to the hydration state of hydronium ions and also appears in control measurements of aqueous solutions in the absence of amino acids. Tryptophan has a lower solubility than the other amino acids studied, producing a relatively weak signal. The absence of four protein amino acids in fig. 6B: because of their low isoelectric points, aspartic acid and glutamic acid were not attempted to be measured in positive ion mode; isoleucine was also ignored because it is indistinguishable from leucine according to m/z, and tyrosine has poor emission characteristics, possibly related to its low solubility in water.

Measurement of post-translationally modified peptides

FIG. 6C shows mass spectra of glutathione and two chemically modified variants, s-nitrosoglutathione and s-acetyl glutathione. Glutathione is a tripeptide that is present in high concentrations in most cells, and the variants studied herein originate from common post-translational modifications. An ion source having a tip internal diameter of 20nm generates peptide ions from a 100mM aqueous solution, the pH of which is between 3.1 and 3.9, adjusted by the addition of acetic acid. The glutathione spectra showed a single peak at 307m/z, corresponding to singly charged, unsolvated glutathione ions. The spectra of s-acetyl glutathione and s-nitrosoglutathione show major peaks at 349m/z and 336m/z, respectively, corresponding to singly charged, unsolvated peptide ions; each spectrum also shows two progressively smaller peaks 18 and 36m/z to the right of the main peak, corresponding to mono-and di-solvated peptide ions, respectively.

Ion transport efficiency

The efficiency of ion transfer from the nanopore source to the remote detector in a high vacuum environment was measured (fig. 7A). Ions from the source are focused into a 2cm opening of a faraday cup located about 50cm away. The ratio of the currents I _C and I _T collected by the faraday cup is the ion transport efficiency. FIG. 7B shows I _C、I_T and ion transport efficiency measured during a 17 minute long experiment using a tip with an inner diameter of 39nm filled with 100mM sodium iodide aqueous solution. The average ion transport efficiency was measured to be 93.4% +/-1.7%. Although I _T drifts between about 780pA to 840pA on a time scale of a few minutes, the slow rise and fall of I _T is reflected in I _C, resulting in a relatively stable transmission efficiency.

Separating ions and charged droplets

The possibility that the nanopore source emits charged droplets in addition to ions was investigated by adding a fan-shaped magnetic field to the flight path, as shown in fig. 7C. A magnetic sector of 6cm diameter and 0.54T deflects the charged species according to its mass to charge ratio. Droplets with diameters greater than 15nm deflect less than 2.7 deg. and enter the faraday cup even if charged to the rayleigh limit. The faraday cup is used to measure the current I _Drop of the charged droplet. At the same time, ions having m/z in the range of about 100 to about 350 are deflected onto a separate faraday plate and produce ion flow I _Ion. FIG. 7D shows the ion fraction of the total measured current and I _Ion、I_Drop of a 2 minute long measurement using a 28nm tip filled with 100mM NaI aqueous solutionI _Ion rose from about 60pA to about 80pA, whereas I _Drop was not observed. The nanopore source appears to emit ions only; however, this measurement cannot exclude the presence of highly charged droplets smaller than about 15 nm.

Calculating the probability of ion scattering

Calculations indicate that most ions follow a collision-free trajectory from the ion source to the detector. Fig. 8 shows the probability of collision of amino acid ions with a hydrated shell with gas molecules in this example based on the theory of gas dynamics. The ion is assumed to pass through the distribution of evaporating water molecules and the uniform low pressure background of N ₂. Fig. 8 depicts the physical situation and plots the number density of gas molecules and the cumulative collision probability as a function of distance from the meniscus. The cumulative probability of ion collisions with gas molecules over the entire 50cm trajectory from the source to the detector is only 2.1%, indicating that the vast majority of ions do not experience any collisions. Most collisions occur in the 200nm range of the liquid meniscus because the water molecules evaporated there have a very high density. A detailed description of these calculations can be found in the following supplemental information.

Discussion of the invention

Two lines of evidence led to the exclusion of the traditional droplet mediated electrospray mechanism (fig. 5A) as the primary source of measured ions. First, no droplets greater than 10nm were measured in the charged species delivered by the source (fig. 7D). Second, the instrument lacks background gas that typically maintains evaporation of water in droplets in electrospray. In high vacuum, nanoscale water droplets lose only a small portion of their mass before the evaporation process freezes due to latent heat loss. Thus, sustained release of ions from the droplet cannot be achieved in this instrument.

These findings can be explained by another ion emission mechanism: ions are evaporated directly from the liquid-vacuum interface at the nanopore as shown in fig. 5B. Ion evaporation is a thermal process in which ions escape from a liquid with the aid of a strong electric field at the surface. When the ratio of the conductivity K of the charged liquid to the flow rate Q is sufficiently large, a sufficiently strong electric field is usually generated. Thus, previous studies have observed ion evaporation in highly conductive liquids (e.g., liquid metals, ionic liquids, and concentrated electrolyte solutions in formamide). The measured amino acid solution has a relatively low conductivity (in the range of 0.01-0.5S/m), but the flow rate generated in the nano-capillary is very low (< 10 pL/min) at an applied pressure of 1atm, so the K/Q generated is still large. The K/Q value of this example is on the same order of magnitude as reported for ionic liquid EMI-BF ₄ while exhibiting ionic evaporation without droplet emission. Furthermore, the distribution of ion solvation states measured in fig. 5D and elsewhere is similar to that measured from sodium iodide in formamide, also due to ion evaporation.

It was observed that the majority measured in fig. 6B-6C were bare ions, not hydrated ion clusters. Experiments described herein were performed to determine if ions were either i) emitted in the bare state or ii) emitted in the hydrated state and then detached from their hydrated shell on their way to the detector. Since only about 2% of the emitted ions will experience even one collision (fig. 8), the mechanism of desolvation of collisions with gas molecules as ion clusters is excluded. Furthermore, tip size appears to affect hydration status (fig. 6A); this indicates that it is the local environment of the source that controls the hydration state, not the process that occurs in flight.

The high ion transport efficiency (fig. 7B) is a direct result of the nanopore ion source emission mechanism. Ion evaporation enables the individual ions to be transferred directly into a high vacuum environment where they neither collide with background gas molecules nor undergo coulomb explosions pushing charged species in random directions. The trajectory of each ion emitted from the source is primarily determined by the electric field formed by the ion optics.

In summary, presented herein is a nanopore ion source that can emit amino acids and small peptide ions directly into a high vacuum. The ability to emit bare ions helps identify different amino acids and post-translational modifications compared to solvated ion clusters or charged droplets. The ions obviously evaporate directly from the liquid meniscus at the tip, without the need for background gas to release ions from the droplet. By eliminating background gas collisions and the need to transfer ions from ambient pressure to high vacuum, the nanopore ion source can eliminate the dominant way of ion loss that is characteristic of electrospray ionization.

Method of

Preparation of nano-capillary

The nanocapillary was drawn from a quartz capillary (QF 100-70-7.5 from Sutter Instruments) 7.5cm long, 0.7mm inside diameter, 1mm outside diameter. The laser puller (P-2000,Sutter Instruments) pulled a nano-capillary with a tip below 100nm according to the following single line recipe: heat=650, speed=45, delay=175, pull=190. The nanocapillaries were coated with 5nm carbon and imaged by scanning electron microscopy (LEO 1530vp, zeiss) to measure tip size. The nanocapillary was plasma cleaned in air for two minutes using a plasma purifier (PLASMATIC SYSTEMS inc.) prior to filling with analyte solution.

Amino acid solution

Amino acid solutions were prepared by dissolving the amino acid of interest (Sigma-Aldrich) in DI water (Millipore) at a concentration of 100mM, except tryptophan, which was prepared at a concentration of 50mM. Glacial acetic acid (Sigma-Aldrich) between 0.1-0.5% v/v is added to the amino acid solution to reduce the pH below the isoelectric point of the amino acid. Glutathione, s-acetyl glutathione and s-nitrosoglutathione solutions were prepared by dissolving the peptides in DI water at a concentration of 100 mM. Glutathione and S-acetyl glutathione were purchased as powders (Sigma-Aldrich), and S-nitrosoglutathione was synthesized from glutathione in the laboratory according to the protocol of t.w.hart. 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.

Delivery of solutions to ion sources

The sample solution is delivered to the nano-capillary tip and rinsed out through the tube-in-tube system. The thin inner diameter PEEK tube (150 microns inner diameter, 360 microns outer diameter) (IDEX HEALTH AND SCIENCE) carries the sample solution, while the wider PEEK tube (0.04 microns inner diameter, 1/16 micron outer diameter) (IDEX HEALTH AND SCIENCE) carries the used solution away from the tip outside the inner tube. The inner tube was observed using a syringe pump (NE-300,New Era Pump Systems) to supply fresh solution to the ion source. VacuTight upchurch fitting (IDEX HEALTH AND SCIENCE) is used to form a seal around the bottom of the nanocapillary and the end of the outer tube to prevent leakage of solution into the vacuum. The tube-in-tube system was installed in a steel tube 1/4 inch in diameter that was passed through a KF-40 quick connect adapter (Lesker Vacuum) into the vacuum chamber of the mass spectrometer.

Quadrupole mass spectrometer

The instrument used in this example for all amino acid and peptide measurements was a custom quadrupole mass spectrometer. The instrument includes a custom single lens, a quadrupole mass filter (MAX-500, extrel), an ion bender (Extrel), and a channel electron multiplier detector with a conversion dynode (conversion dynode) (DeTech 413), which is sensitive to single ions. The instrument base pressure was about 10 ^-8 torr. When a nanopore ion source is introduced into a mass spectrometer, the pressure is typically raised to 10 ^-7-10^-6 torr.

Amino acid and glutathione measurements

Amino acid or peptide solutions were prefilled into the nanocapillary using a microfiltration flexible needle (microfil flexible needle) (World Precision Instruments). The filled nano-capillary is then mounted on a tube-in-tube system and inserted into a mass spectrometer. The solution at the tip was continuously refreshed by pumping the solution from a syringe pump (NE-300,New Era Pump Systems) into the inner tube at a rate of 0.4 mL/hour. A voltage of +100V was applied to the electrode within the capillary using a high voltage source meter (2657A,Keithley Instruments) while a negative voltage was slowly applied to the extraction electrode using a high voltage source (Burle) until ionization was observed. Emission typically occurs when the total extraction voltage is between 200 and 350V.

Ion transport efficiency measurement

The measurement of ion transport efficiency was performed in a custom vacuum chamber containing a set of ion optics and faraday cups (fig. 7A). The emission current is measured by 2410SourceMeter (Keithley Instruments), which also applies a high voltage to the tip through the Ag electrode. The leakage current through the BNC cable connecting sourcemeter to the tip is measured and subtracted from the measured emission current. The current impinging the faraday cup was measured using an SR570 current pre-amplifier (Stanford RESEARCH SYSTEMS) connected to NIPCIe-6251 DAQ card (National Instruments). An 8-channel high voltage power supply (CAEN DT 8033) was used to control the optical voltage. A custom Labview program was used to control the voltage applied to the tip and record the current emitted and transmitted.

Sector magnetic field measurement

A basic sector field mass spectrometer was constructed by adding a magnet and faraday plate in the vacuum chamber described above. The magnet consisted of a neodymium magnet with a yoke made of a low carbon magnet (ASTM a 848). The yoke concentrates the field in a flat circular region of 6cm diameter and 1cm height, which is located immediately downstream of the ion-optical element. The field strength in the flat circular area was measured using a magnetometer to be b=0.54 +/-0.02T. The faraday plate used was a stainless steel disk, 4cm in diameter and 0.02 inches thick, connected directly to the power feed via steel wire. The faraday plate is mounted at 45 ° to the faraday cup at the same distance from the center of the magnetic sector. The current emitted from the tip was measured using 2410Sourcemeter (Keithley) and the ion current and droplet current were measured using a separate SR570 current pre-amplifier (Stanford RESEARCH SYSTEMS).

Supplementary information single amino acid ion measurement condition

Fig. 6B presents a mass spectrum obtained from an aqueous amino acid solution. Table 2 shows the relevant experimental parameters for these data.

Table 2: experimental conditions for measuring the amino acid ion spectrum in fig. 6B.

The profile is measured in positive ion mode, for which the pH must be lowered below the isoelectric point of the dissolved amino acid. This is done by adding acetic acid. The pH and conductivity K of each solution are listed in Table 2. Ions are emitted from the nanopore ion source directly into the high vacuum. The tip inner diameter of the nano-capillary is in the range of 20nm-58nm, and a single tip is often used to measure a plurality of amino acid solutions. Table 2 reports the tip inner and outer diameters of each of the nano-capillaries used to obtain the data in fig. 6B, and measurements made from the same tip are indicated by the tip numbers. Table 2 also reports the time average pressure P, extraction voltage V _e, and emission current I _e of the vacuum chamber continuously monitored during the experiment.

Probability of collision of emitted ions with gas molecules

In fig. 6B, amino acid ions are detected predominantly in the unsolvated state. Collisions with gas molecules are the mechanism by which solvent molecules separate from ions in conventional electrospray ionization. However, the instrument operates under high vacuum conditions, collisions with gas molecules may be rare. Aerodynamic theory is used to calculate the probability of an emitted ion cluster colliding with at least one gas molecule. This solves the problem of whether ions are present in unsolvated solutions or whether they carry solvated shells that are knocked off by collisions with gas molecules on their way to the detector.

The gas distribution within the vacuum chamber is considered to be the sum of the two components, i.e. the uniform background of density n _b and the distribution of water molecules evaporated from the meniscus at the tip of the nanocapillary n _w. The background air pressure in this example is typically about 7X10 ^-8 torr (see Table 2). At this pressure, the mean free path of the water molecules in nitrogen exceeds 1km, indicating that the evaporated water molecules are far away from the meniscus along a ballistic trajectory. The meniscus is modeled as a hemisphere, with the evaporated water molecules travelling radially outward as shown in fig. 9A. The density decay of water molecules is n _w～r^-2, where r is the distance to the center of the hemisphere. Although the rate at which water evaporates from the liquid meniscus into the vacuum cannot be well established by experimentation, it is well known that the flux of water molecules evaporating from the liquid surface cannot exceed the flux of molecules entering at equilibrium. Thus, by subtracting the incoming molecules, the highest possible density that the (outgoing) evaporating water molecules can reach on the vacuum side of the liquid interface will be half the water vapor density at equilibrium. By assuming a water vapor density on the vacuum side of the meniscus ofThe maximum probability of collisions of the emitted ions with the gas molecules is found, where p ₀ = 17.5torr is the approximately equilibrium vapor pressure of water at 20 ℃, and k _B T is the thermal energy.

The cumulative probability of an ion cluster experiencing at least one collision before reaching r, C _p (r) is also determined. To calculate C _p (r), P _s(r)＝1-C_p (r) is more easily considered, the probability that the ions remain until the distance r is not subject to collision. P _s (r) and P _s (r-dr) are related by the probability of collision with gas molecules in the interval r-dr→r, expressed by the stochastic process P _c (r-dr→r),

P_s(r)＝P_s(r-dr)(1-P_c(r-dr→r)). (5)

Under the restriction of lean gas and infinitely small displacements,

Where σ _w is the cross section of ion clusters colliding with water molecules, σ _b is the cross section of ions with background gas molecules, n _w,0 is the number density of water on the vacuum side of the meniscus, r ₀ is the radius of the meniscus, and n _b is the number density of background gas molecules. Combining equations 5 and 6 and rearranging to arrive at the differential equation for P _s

Integral equation 7 from r ₀ to r applies the boundary condition P _s(r₀) =1 and solves for P _s (r) to give

Finally, the cumulative probability of an ion experiencing at least one collision before reaching r is given by

C_P(r)＝1-P_s(r). (9)

Fig. 9B plots C _p (r) in the present embodiment. The cross sections σ _w and σ _b are given by pi (a _i+a_w)² and pi (a _i+a_b)², respectivelyIs the kinetic radius of water,/>Is the kinetic radius of the background gas molecule,/>Is the approximate radius of an amino acid with a completely water solvated shell. N _b＝2.25×10¹⁵m^-3 and n _w,0＝6.44×10²³ are the number density of the background gas and the number density of the water molecules on the vacuum side of the meniscus, respectively. The meniscus radius r ₀＝30nm.C_p (r) rises rapidly in the first 100nm and then saturates at 1.8%. C _p (r) increases slowly over centimeter-scale distances due to the limited density of background gas molecules; c _p (r) reached 2.1% within 50cm distance from the ion source to the detector.

These results indicate that there are few collisions between gas particles and ion clusters emitted by the nanopore ion source compared to conventional electrospray. Since the instrument is operated under high vacuum conditions, most ions follow a collision-free trajectory from the source to the detector. The lack of collisions means that the ions are emitted in the same state as the detection, concluding that the nanopore ion source may be capable of emitting predominantly non-solvated amino acid ions.

The nanopore ion source operates below the minimum steady flow rate of the cone-jet electrospray

There may be a minimum flow rate Q _e below which stable cone-jet electrospray cannot exist. The expression Q _e is given herein for a polar liquid cone jet with a high or low reynolds number Re. Re >1 is expected if a cone jet of aqueous amino acid is formed, so that Q _e is suitably expressed as

Where ε ₀ and ε are the vacuum permittivity and the relative permittivity, respectively, γ is the surface tension, ρ is the density, and K is the conductivity. Using equation 10, for the measurement of amino acid solutions, Q _e＝5×10^-13m³/S, or 30nL/min, is predicted, given e=80, γ=0.073N/m, ρ=1000 kg/m ³ and k=0.1S/m.

The expected flow rate through the nanopore ion source is at least three orders of magnitude lower than Q _e. The flow rate through the nanocapillary was measured by blowing water droplets into the silicone grease under a fixed applied pressure and measuring the growth rate of the droplets. The nano-capillary is initially filled with water and its rear end is connected to a nitrogen cylinder by a pressure regulator. The capillary tips were immersed in a silicone disc under an optical microscope. The back pressure is slowly increased from the nitrogen cylinder until it can overcome the laplace pressure of the water-grease interface at the tip of the nano-capillary and begin to expand the droplet. Video of droplet growth at a fixed backpressure was recorded at an image rate of 10 Hz. Fig. 10 plots the fluid conductance of 14 nano-capillaries as a function of their tip inner diameters. The flow rate through the smallest tip with an inner diameter of 120nm measured in this way will be 30pL/min at an applied pressure of 1 atm. Thus, even a larger tip of 100nm diameter allows only three orders of magnitude lower flow rates than the minimum flow rates required to maintain a stable cone jet.

Fig. 10 also plots the theoretical flow rate through the nanocapillary. This calculation treats the nano-capillary geometry as a semi-infinite truncated cone with a tip inner diameter r ₀. The vertex half angle θ is used as a fitting parameter. Fluid conductance obtained by Poiseuille flow (Poiseuille flow) of the cone

Where ΔP is the pressure drop over the capillary and μ is the viscosity. The least squares fitting of equation 11 to the data in fig. 10 yields θ=2.5°. The data in fig. 7A is obtained by multiplying the fluid conductance in fig. 10 by the applied pressure of 1atm, and also shows the minimum flow rate Q _e calculated from equation 10.

Simulating ion and droplet trajectories through a magnetic sector

Simulations were performed using custom Python codes to determine the m/z range of particles expected to strike faraday cups and faraday plate detectors used in the sector field experiments. The particles are assigned an m/z ratio and an initial kinetic energy qV _T is imparted, where q is the charge on the particles and V _T is the tip voltage. Then, the trajectory of the particle through the magnetic sector is numerically calculated by solving the lorentz force law using the fourth-order range-Kutta equation. The magnetic field in the z direction in the range of the sector magnetic field (circle with a diameter of 6 cm) was assumed to be 0.54T, and the other places were all 0. Given the geometry of the instrument and the position of the detectors relative to the magnetic sector, the minimum/maximum deflection angle required for the particles to strike both detectors can be calculated. To strike the faraday cup and to help measure the droplet current I _Drop, the particles can be deflected between 0 ° and 3.15 °, and for faraday plate (I _Ion), the particles can be deflected between 30.96 ° and 59.04 °. It was found that particles of 75< m/z <315 would strike the faraday plate, corresponding to singly charged sodium ions with 3 to 16 additional water molecules attached. It was also found that particles with m/z >37000 hit the faraday cup, which corresponds to water droplets charged to the rayleigh limit with a radius >15 nm. It should be noted that the deflection of droplets charged below the rayleigh limit will be smaller.

Although several embodiments of the present disclosure have been described and illustrated herein, a variety of other means and/or structures for performing a function and/or obtaining a result and/or one or more of the advantages described herein will be readily apparent to those of ordinary skill in the art, and each such variation and/or modification is deemed to be within the scope of the present disclosure. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present disclosure are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the disclosure may be practiced otherwise than as specifically described and claimed. The present disclosure is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present disclosure.

In the event that the present specification and documents incorporated by reference include conflicting and/or inconsistent disclosure, the present specification controls. If two or more files incorporated by reference contain conflicting and/or inconsistent disclosure, the later-validated file is subject to validation.

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

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

The phrase "and/or" as used in the specification and claims should be understood to mean "either or both" of the elements so combined, i.e., elements that in some cases exist in combination and in other cases exist separately. The various elements listed as "and/or" should be interpreted in the same manner, i.e., as "one or more" elements so connected. In addition to the elements specifically identified by the "and/or" clause, other elements may optionally be present, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, references to "a and/or B" may refer to only a in one embodiment when used in conjunction with an open language such as "include"; in another embodiment, B (optionally including elements other than a) may be referred to only; in yet another embodiment, both a and B (optionally including other elements) may be referred to; etc.

As used herein in the 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" and/or "should be interpreted as inclusive, i.e., including at least one of the plurality of elements or list of elements, but also including more than one element of the plurality of elements or list of elements, and optionally other unlisted items. Only the terms explicitly indicated to the contrary, such as "only one" or "exactly one", or "consisting of" when used in the claims, shall mean that only one element of a plurality or list of elements is included. In general, the term "or" as used herein should be interpreted as referring to an exclusive alternative (i.e., "either or not both") only when preceded by the exclusive term "either," one, "" only one, "or" exactly one.

As used herein in the specification and claims, the phrase "at least one" in reference to a list of one or more elements is understood to mean at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one of each element specifically listed within the list of elements, and does not exclude any combination of elements in the list of elements. The definition also allows that elements other than the specifically identified elements within the list of elements to which the phrase "at least one" refers may optionally be present, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of a and B" (or equivalently, "at least one of a or B," or equivalently "at least one of a and/or B") may refer, in one embodiment, to at least one, optionally including more than one, a, while absent B (and optionally including elements other than B); in another embodiment, at least one, optionally including more than one, B, while no a is present (and optionally including elements other than a); in yet another embodiment, reference is made to at least one, optionally including more than one, a, and at least one, optionally including more than one, B (and optionally including other elements); etc.

When the word "about" is used herein to refer to a number, it is to be understood that yet another embodiment of the present disclosure includes a number that is not modified by the presence of the word "about".

It should also be understood that, in any method claimed herein that includes more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order of the steps or acts of the method as described, unless explicitly stated to the contrary.

In the claims and the above description, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "making up," and the like 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" should be closed or semi-closed transitional phrases, respectively, as described in section 2111.03 of the U.S. patent office patent review program handbook.

Claims

1. A method, comprising:

light is used in mass spectrometers to fragment proteins into individual molecules.

2. The method of claim 1, wherein the mass spectrometer is a nano Kong Zhipu meter.

3. The method of any one of claims 1 or 2, wherein the protein is contained in solution.

4. A mass spectrometer, comprising:

Nanotips that allow proteins to be aligned in a linear configuration; and

A laser positioned to direct light to dissociate the protein into fragments.

5. The mass spectrometer of claim 4, further comprising one or more detectors disposed downstream of the nanotip.

6. The mass spectrometer of claim 5, wherein the one or more detectors are single ion detectors.

7. The mass spectrometer of any of claims 4-6, further comprising a magnetic mass filter downstream of the nanotip.

8. The mass spectrometer of any of claims 4-7, wherein the nanotip comprises an opening having a cross-sectional dimension of less than 100 nm.

9. The mass spectrometer of any of claims 4-8, wherein the nanotip comprises an opening having a cross-sectional dimension less than or equal to 65 nm.

10. The mass spectrometer of any of claims 4-9, wherein the nanotip comprises an opening having a cross-sectional dimension less than or equal to 25 nm.

11. The mass spectrometer of any of claims 4-10, wherein the nanotip comprises an opening having a cross-sectional dimension less than or equal to 5 nm.

12. The mass spectrometer of any of claims 4-11, further comprising a vacuum chamber housing the nanotip.

13. The mass spectrometer of claim 12, wherein the vacuum chamber has a pressure of no more than 10 mPa.

14. The mass spectrometer of claim 13, wherein the vacuum chamber has a pressure of no more than 1.5 mPa.

15. The mass spectrometer of any of claims 4-14, wherein the nanotip is a tip portion of a capillary having an aspect ratio of length to cross-sectional dimension greater than or equal to 100.

16. The mass spectrometer of any of claims 4-15, wherein the nanotip is a tip portion of a capillary having an aspect ratio of length to cross-sectional dimension greater than or equal to 1,000.

17. The mass spectrometer of any of claims 4-16, further comprising an electrode adjacent to the nanotip.

18. The mass spectrometer of any of claims 4-17, wherein the nanotip comprises a nanotube.

19. The mass spectrometer of any of claims 4-18, wherein greater than 50% of light having a wavelength greater than or equal to 150nm and less than or equal to 213nm enters the nanotip.

20. The mass spectrometer of any of claims 4-19, wherein the nanotip has a maximum cross-sectional dimension greater than or equal to 1nm and less than 100 nm.

21. The mass spectrometer of any of claims 4-20, wherein the nanotip has a maximum cross-sectional dimension greater than or equal to 5nm, less than 80 nm.

22. The mass spectrometer of any of claims 4-21, further comprising one or more additional nanotips adjacent to the nanotip.

23. The mass spectrometer of claim 22, wherein the nanotip and the one or more additional nanotips are arranged in a linear array upstream of the magnetic filter.

24. The mass spectrometer of claim 23, further comprising an electrode adjacent at least one of the one or more additional nanotips.

25. The mass spectrometer of any of claims 4-24, wherein the one or more detectors comprise a detector array arranged in a two-dimensional array.

26. A method, comprising:

arranging the proteins in a substantially linear configuration in the nanotip;

Fragmenting the protein into amino acids by applying a laser to the protein;

Emitting the amino acid from the nanotip; and

Detecting the amino acid emitted from the nanotip.

27. The method of claim 26, wherein the laser is ultraviolet light.

28. The method of any one of claims 26-27, wherein the laser has a wavelength of at least 150nm and no greater than 222 nm.

29. The method of any one of claims 26-28, wherein the laser has a wavelength of at least 150nm and no greater than 213 nm.

30. The method of any one of claims 26-29, wherein the laser has a wavelength of at least 150nm and no greater than 193 nm.

31. The method of any one of claims 26-30, wherein the method has an average ion transport efficiency of at least 85%.

32. The method of any one of claims 26-31, wherein the method has an average ion transport efficiency of at least 90%.

33. The method of any one of claims 26-32, wherein the method has an average ion transport efficiency of at least 93%.

34. The method of any one of claims 26-33, wherein the nanotip comprises an opening having a cross-sectional dimension of less than 100 nm.

35. The method of any one of claims 26-34, wherein the nanotip comprises an opening having a cross-sectional dimension less than or equal to 65 nm.

36. The method of any one of claims 26-35, wherein the nanotip comprises an opening having a cross-sectional dimension less than or equal to 60 nm.

37. The method of any one of claims 26-36, wherein the nanotip comprises an opening having a cross-sectional dimension less than or equal to 25 nm.

38. The method of any one of claims 26-37, wherein the nanotip comprises an opening having a cross-sectional dimension less than or equal to 5 nm.

39. The method of any one of claims 26-38, wherein the nanotip comprises an opening having a cross-sectional dimension greater than or equal to 1 nm.

40. The method of any one of claims 26-39, wherein the emitted amino acids are in the form of bare ions and/or ion clusters.

41. The method of any one of claims 26-40, wherein at least 80% of the emitted amino acids are in the form of bare ions.

42. The method of any one of claims 26-41, wherein at least 90% of the emitted amino acids are in the form of bare ions.

43. The method of any one of claims 26-42, wherein the transmitted amino acids are transmitted in a sequential manner.

44. The method of any one of claims 26-43, wherein detecting the amino acid comprises detecting the amino acid in the order in which the amino acid is emitted from the nanotip.

45. The method of any one of claims 26-44, further comprising determining the sequence of the protein by determining the emitted amino acids with the detector.

46. The method of any one of claims 26-45, wherein the protein is contained in a solution.

47. The method of claim 46, wherein the solution comprises water.

48. The method of any one of claims 46-47, wherein the solution comprises formamide.

49. A method, comprising:

applying light having a wavelength greater than or equal to 150nm and less than or equal to 213nm to a protein to cleave fragments from the protein; and

The fragments were sequenced using mass spectrometry.

50. The method of claim 49, wherein the protein is contained in a solution.

51. The method of any one of claims 45-50, wherein the light is applied using a laser.

52. The method of any one of claims 45-51, wherein the light has a wavelength of 193nm +/-5 nm.

53. The method of any one of claims 45-52, wherein the light has a wavelength of 193nm +/-3 nm.

54. The method of any one of claims 45-53, wherein the light has a wavelength of 193nm +/-1 nm.

55. The method of any one of claims 45-54, wherein the fragment comprises an amino acid.

56. A method, comprising:

Applying light having a wavelength greater than or equal to 150nm and less than or equal to 222nm to a protein to cleave fragments from the protein; and

The fragments were sequenced using mass spectrometry.

57. The method of claim 56, wherein said protein is contained in a solution.

58. A method, comprising:

applying a laser to a protein to cleave an amino acid from the protein; and

The amino acids were sequenced using mass spectrometry.

59. The method of claim 58, wherein the protein is contained in a solution.

60. The method of any one of claims 58 or 59, wherein said fragment comprises an amino acid.

61. The method of any one of claims 58-60, wherein said laser is absorbed by a peptide bond of said protein to cleave said protein at said peptide bond.

62. A mass spectrometer, comprising:

An ion source comprising a capillary;

A light source directed to the ion source, wherein the light source is capable of producing light having a wavelength greater than or equal to 150nm and less than or equal to 213 nm;

A magnetic mass filter downstream of the ion source;

a detector array downstream of the magnetic filter.

63. The mass spectrometer of claim 62, wherein the light source is capable of producing light having a wavelength of 193nm +/-5 nm.

64. The mass spectrometer of claim 62 or 63, wherein the light source is a laser.

65. The mass spectrometer of any of claims 62-64, wherein the ion source comprises an electrode proximate the capillary.

66. The mass spectrometer of any of claims 62-65, wherein the capillary comprises a body portion and a tip portion fluidly connected to the body portion.

67. The mass spectrometer of claim 66, wherein greater than 50% of light having a wavelength greater than or equal to 150nm and less than or equal to 213nm is transmitted into the tip portion of the capillary.

68. The mass spectrometer of any of claims 66-67, wherein less than 50% of light having a wavelength greater than or equal to 150nm and less than or equal to 213nm is transmitted into the body portion of the capillary.

69. The mass spectrometer of any of claims 66-68, wherein the tip portion of the capillary has a cross-sectional dimension of less than 100 nm.

70. The mass spectrometer of any of claims 66-69, wherein the tip portion of the capillary has a cross-sectional dimension of less than 80 nm.

71. The mass spectrometer of claims 66-70, wherein the tip portion of the capillary has a cross-sectional dimension less than 65 nm.

72. The mass spectrometer of claims 66-71, wherein the tip portion of the capillary has a cross-sectional dimension of less than 25 nm.

73. The mass spectrometer of claims 66-72, wherein the tip portion of the capillary has a cross-sectional dimension of less than 5 nm.

74. The mass spectrometer of any of claims 62-73, further comprising one or more additional ion sources adjacent to the ion source.

75. The mass spectrometer of claim 74, wherein the ion source and the one or more additional ion sources are arranged in a linear array upstream of the magnetic filter.

76. The mass spectrometer of any of claims 74-75, wherein at least one of the one or more additional ion sources comprises a capillary.

77. The mass spectrometer of claim 76, wherein at least one of the one or more additional ion sources comprises an electrode proximate the capillary.

78. The mass spectrometer of any of claims 62-77, wherein the detector array downstream of the magnetic filter is arranged in a two-dimensional array.

79. A mass spectrometer, comprising:

An ion source comprising a capillary;

a laser positioned to direct light to the capillary; and

A detector downstream of the ion source.

80. A mass spectrometer, comprising:

An ion source comprising a capillary and an electrode proximate the capillary, wherein the capillary comprises an opening having a cross-sectional dimension of less than 125 nm;

A magnetic mass filter downstream of the ion source;

a detector array downstream of the magnetic filter; and

A light source directed to the ion source, wherein the light source is capable of generating light having a wavelength greater than or equal to 150nm and less than or equal to 213 nm.

81. The mass spectrometer of claim 80, wherein the light has a wavelength of 193nm +/-5 nm.

82. A method of sequencing a protein comprising:

fragmenting a protein by applying light having a wavelength greater than or equal to 150nm and less than or equal to 213nm to the protein to produce fragments;

passing the fragments through a magnetic mass filter;

directing the fragments to a detector array; and

Determining the sequence of the protein by determining the fragments with the detector array.

83. The mass spectrometer of claim 82, wherein the light has a wavelength of 193nm +/-5 nm.

84. A method of sequencing a protein comprising:

delivering a fluid comprising a protein into a capillary defining an opening;

Applying light having a wavelength greater than or equal to 150nm and less than or equal to 213nm to the protein adjacent to the opening to produce fragments;

Delivering the fragments directly into an environment having a pressure of no more than 100 mPa;

passing the fragments through a magnetic mass filter;

directing the fragments to a detector array; and

85. The mass spectrometer of claim 84, wherein the light has a wavelength of 193nm +/-5 nm.

86. A method, comprising:

Applying light having a wavelength greater than or equal to 150nm and less than or equal to 213nm to a peptide to cleave fragments from the peptide;

Passing at least 50% of the fragments through a magnetic filter; and

The fragments are directed to a detector.

87. The mass spectrometer of claim 86, wherein the light has a wavelength of 193nm +/-5 nm.