Solid-phase affinity selection in quality analysis

Abstract

In a system for affinity selection by mass spectrometry, a method is provided in which a plurality of drug candidates in a solution are separated based on affinity, the method including: introducing a solid-phase element having binding affinity for a selected protein into the solution; binding at least one of the plurality of drug candidates to the solid-phase element as the selected drug candidate; washing the solid-phase element and the selected drug candidate to separate any unbound material; sampling the selected drug candidate in a capture fluid flowing through a sampling region of an open-port sampling interface; and directing the sampled selected drug candidate and capture fluid to an ionization source.

Description

[Technical Field]

[0001] (Related U.S. Application) This application claims the benefit of priority from U.S. Provisional Application No. 63 / 029,028, filed May 22, 2020, the entire contents of which are incorporated herein by reference.

[0002] (Field) The present invention is directed to processing fluids, and more particularly to methods and systems for identifying and separating compounds based on selected affinities. [Background technology]

[0003] (background) Affinity selection by mass spectrometry (ASMS) involves binding of candidate molecules to immobilized or soluble receptors and has been used to screen large compound libraries in a time- and cost-effective manner. Traditional ASMS workflows are based on solution-phase incubation, in which a target protein in solution is added to a mixture of drug molecules. The unbound drug molecules are then separated from the drug-protein conjugates by mechanisms such as ultrafiltration, spin columns, and size-exclusion chromatography. After molecular weight separation, the protein-drug conjugates and unbound proteins are injected into a reverse-phase LC / MS for analysis. Drug molecules detected by MS (released in the LC) are identified using their binding affinity to the target protein. However, the analysis speed of traditional methodologies is limited due to the time-consuming separation (i.e., elution) of the free drug from the protein-drug conjugates using LC.

[0004] In solid-phase ASMS, an enzyme on the surface of a solid-phase element can be inserted into a drug mixture in solution to capture drug molecules using its affinity for the solid-phase surface of the solid-phase element. Examples of such solid-phase elements include magnetic particles and solid-phase microextraction (SPME) fibers; however, compared to other solid-phase elements such as SPME fibers, magnetic particles have a much larger surface area, which improves capture sensitivity. In one such approach, MagMASS (J. Nat. Prod. 2016, 79, 2898-2902), magnetic particles are used to "remove" drug molecules using protein binding affinity, leaving the unbound drug in solution. If necessary, the magnetic particles can be washed before elution of the drug molecules into the liquid phase and injection into LC-MS / MS.

[0005] It is also known to use an open port sampling interface (OPI) for direct sampling of solid-phase substrates with bound drug molecules, such as SPME fibers (see US10103015B2, the contents of which are incorporated herein by reference), where the solid-phase element is a magnetic particle, for which a magnet (e.g., an electromagnet) is used to transfer the magnetic particles between and / or from the sample wells to the OPI (see PCT / IB2018 / 089146, the contents of which are incorporated herein by reference). Care must be taken when using an OPI to transfer magnetic particles to the MS port to avoid the magnetic particles being entrapped in the MS.

[0006] The following references are included: "Solid Phase Microextraction and Related Techniques for Drugs in Biological Samples" by Moen et al. (J. Anal. Methods Chem, 2014), published February 13, 2014; "Direct Dynamic Protein-Affinity Selection Mass-Spectrometry" by Niels Jonker et al. (Chromatographia, July 2010), 72(1-2), 7-13; "Solution-Based Indirect Affinity Selection Mass Spectrometry - A General Tool for High-Throughput Screening of Pharmaceutical Compound Libraries" by O'Connell et al. (Anal. Chem., 2014, 96, pp. 7413-7420); and "Pulsed Ultrafiltration Mass Spectrometry" by Richard B. van Breemen et al. al. (Anal. Chem., 1997, 69, pp. 2159-2164), "A New Method for Screening Combinatorial Libraries," Michael D Rush, et al. (J. Nat. Prod. 2016, 79, pp. 2898-2902), "Magnetic Microbead Affinity Selection Screening (MagMASS) of Botanical Extracts for Inhibitors of 15-Lipoxygenase," Benjamin M. Johnson (Mass Spectrometry Reviews, 2002, 21, pp. 76-86), "APPLICATIONS OF PULSED ULTRAFILTRATION-MASS SPECTROMETRY," WO2017 / 093896 A1 (Don W. Arnold, et al.), and WO2019 / 102355 A1 (Don W. Arnold, et al.) are relevant background art. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent No. 10,103,015 [Patent Document 2] International Publication No. 2017 / 093896 [Patent Document 3] International Publication No. 2019 / 102355 Summary of the Invention [Means for solving the problem]

[0008] (summary) One aspect of the present invention is to provide improved methods and devices for transferring candidate molecules into the open port sampling interface OPI.

[0009] In one aspect, the candidate molecule is isolated from the solid phase element in a preparation step (sample well) and then introduced into the OPI (without the solid phase element) using a process that does not require the sample to be aspirated and removed using suction forces.

[0010] In another aspect, bound or unbound candidate molecules are introduced into the OPI following a process that filters out solid-state elements prior to introduction of ions into the MS. In one embodiment, a preparation step is performed in the sample well, and then the isolated solid-state elements are injected into the OPI, where the sample is separated from the solid-state elements using a solvent-based trapping fluid. The solid-state elements are then captured before entering the MS. In some embodiments, an external magnetic field captures the solid-state elements before delivering the sample to the MS ion source. In another embodiment, a trap may be provided in the OPI prior to electrospray ionization or in series with the transfer conduit.

[0011] In yet a further aspect, some preparation steps may be performed in the OPI and transfer conduit, with fewer steps performed in the sample well. For example, a first capture fluid may be used to capture the sample and solid-state elements, providing a wash action as the solid-state elements are captured with the sample, and a second separation fluid (i.e., solvent) may then be used to separate the sample from the captured solid-state elements. In certain embodiments, the second separation fluid may flow with a variable concentration gradient, increasing from 0 to 100% according to a predefined gradient or sequence of increasing concentrations. Also, in certain embodiments, an MS signal may be used to trigger a switch from the first capture fluid to the second separation fluid. In this embodiment, the first capture fluid is directed to the MS, which is useful if the wash component is MS-compatible. In another embodiment, the trapping fluid can be directed to a waste conduit and a timer is used to trigger a switch from a first trapping fluid to a second separation fluid and direct the separation fluid to the ion source; this can be useful if the wash chemistry is not MS-compatible.

[0012] Also, according to aspects described herein, OPI may be used to simplify the use of magnetic beads in solid-phase ASMS. According to other aspects, the solid-phase elements need not be magnetic, and drug molecule candidates may be sequestered based on size.

[0013] In other aspects, the solid phase elements may be uniformly suspended in a solution, may operate to capture selected candidates, may acoustically eject the candidates from the solution, may be trapped in a trapping fluid flowing through an OPI, and may be captured from the trapping fluid by a magnetic trap to allow the candidates to flow to the MS ionization source. In some aspects, the trap may comprise a magnetic trap or a size-based trap.

[0014] The above aspects can be achieved by a method for identifying and separating compounds based on a selected affinity, including introducing a plurality of compounds together into a solution; inserting a probe having a surface treatment operative to bind one or more compounds based on a selected affinity; binding one or more compounds from the plurality of compounds to the probe; removing the probe and the bound one or more compounds from the solution; separating the one or more compounds from the probe; capturing the separated one or more compounds with a flowing solvent at an open end of an open-port sampling interface; transporting the solvent and the captured one or more compounds to an ionization element; and ionizing the one or more compounds.

[0015] In certain embodiments, the method may further include analyzing the ionized compound or compounds in a mass spectrometer.

[0016] In certain embodiments, the method may further include separating the ionized one or more compounds based on ion mobility in a differential mobility spectrometer after ionizing the one or more compounds but before analyzing them.

[0017] In certain embodiments, the probe is selected from the group consisting of a solid phase microextraction (SPME) fiber, a REED (as described in U.S. Provisional Patent Application No. 62 / 692,274, the contents of which are incorporated herein), and a magnetic bead.

[0018] In certain embodiments, separating the one or more compounds from the probe may include inserting the probe and the bound one or more compounds into a debinding solvent in a separation vessel to debind the one or more compounds from the probe, and injecting the debinding solvent and the debound one or more compounds into the flowing solvent at the open end of the open-port sampling interface.

[0019] In some embodiments, the injecting may include aspirating the debinding solvent and the debound one or more compounds from a separation vessel and injecting the aspirated debinding solvent and the debound one or more compounds into a solvent stream that is pumped to the ionization element.

[0020] In some embodiments, injecting may include ejecting droplets of the debinding solvent and the debound one or more compounds from a separation vessel into a flowing solvent at an open end of the open port sampling interface.

[0021] In some embodiments, injecting may include acoustically or pneumatically ejecting the droplets.

[0022] Another aspect can be achieved in a system for affinity selection by mass spectrometry, in which a plurality of drug candidates in a solution are separated based on affinity by a method including: introducing a solid-phase element into the solution having binding affinity for a selected protein; binding at least one of the plurality of drug candidates to the solid-phase element as the selected drug candidate; washing the solid-phase element and the selected drug candidate to separate any unbound material; sampling the selected drug candidate in a capture fluid flowing through a sampling region of an open port interface (OPI); and directing the sampled selected drug candidate and capture fluid to an ionization source.

[0023] In some embodiments, the method may further include treating the Si-OH on the surface with an aminosilane reagent, followed by immobilizing the protein on the surface of the solid-phase element by reaction with glutaraldehyde (GA), the free end of which can react with the amino group of lysine, or through streptavidin-biotin interaction, or histidine tag, to capture the protein.

[0024] In certain embodiments, the method may further include sampling the selected drug candidate by acoustically ejecting the selected drug candidate from the sample well into the capture fluid.

[0025] In some embodiments, the method may further include ejecting the selected drug candidate from the sample well after washing.

[0026] In certain embodiments, the method may further include releasing the selected drug candidate from the solid phase element before the selected drug candidate is ejected from the sample well, isolating the selected drug candidate from the solid phase element, and ejecting the selected drug candidate without the solid phase element into the capture fluid.

[0027] In some embodiments, the selected drug candidates are injected while bound to a solid phase element.

[0028] In certain embodiments, the selected drug candidate is debound by the capture fluid.

[0029] In certain embodiments, the selected drug candidate and solid phase element are ejected from the sample well, and the system further comprises a trap prior to the ionization source for capturing the solid phase element.

[0030] In certain embodiments, the drug candidate is released from the captured solid phase element by introducing a solvent into the capture fluid.

[0031] In some embodiments, the trap comprises a magnetic trap.

[0032] In some embodiments, the trap comprises a filter or a size trap.

[0033] In some embodiments, the solid state element is ejected with elements separated from the candidate, while in other embodiments, the solid state element is ejected with elements bound to the candidate.

[0034] In some embodiments, the drug molecule candidates are isolated from the solid phase elements by a capture fluid, while in other embodiments, the drug molecule candidates are isolated by a release agent (e.g., a solvent) after the solid phase elements have been captured from the capture fluid.

[0035] These, together with other aspects and advantages which will become apparent subsequently, reside in details of structure and operation more fully described and claimed hereinafter and which form a part hereof, reference is made to the accompanying drawings, in which like numerals refer to like parts throughout. The present invention provides, for example, the following items. (Item 1) 1. A method for identifying and isolating compounds based on a selected affinity, comprising: introducing the compounds together into a solution; inserting a probe comprising a surface treatment operative to bind one or more compounds based on said selected affinity; binding one or more compounds from the plurality of compounds to the probe; removing the probe and the bound compound or compounds from the solution; separating the one or more compounds from the probe; and capturing the separated one or more compounds with a flowing solvent at the open end of an open port sampling interface; transporting the solvent and the trapped one or more compounds to an ionization element; ionizing said one or more compounds; A method comprising: (Item 2) 2. The method of claim 1, further comprising analyzing the ionized compound or compounds in a mass spectrometer. (Item 3) 3. The method of claim 2, wherein after ionizing the one or more compounds but before analyzing, the method further comprises separating the ionized one or more compounds based on ion mobility in a differential mobility spectrometer. (Item 4) The probe is a solid phase microextraction (SPME) fiber; REED and Magnetic particles and 4. The method according to any one of items 1-3, selected from the group consisting of: (Item 5) Separating the one or more compounds from the probe comprises: inserting the probe and the bound one or more compounds into a debinding solvent in a separation vessel to debind the one or more compounds from the probe; injecting the debinding solvent and the debound one or more compounds into the flowing solvent at the open end of the open port sampling interface; 4. The method according to any one of items 1 to 3, comprising: (Item 6) The injecting step comprises: aspirating the debinding solvent and the debound one or more compounds from the separation vessel; injecting the aspirated decoupled solvent and the decoupled one or more compounds into a solvent stream that is pumped through the ionization element; Item 6. The method according to item 5, comprising: (Item 7) 6. The method of claim 5, wherein the injecting comprises ejecting droplets of the debinding solvent and the debound one or more compounds from the separation vessel into the flowing solvent at the open end of the open port sampling interface. (Item 8) 8. The method of claim 7, wherein the injecting comprises acoustically or pneumatically ejecting the droplets. (Item 9) 1. A system for affinity selection by mass spectrometry, wherein a plurality of drug candidates in solution are separated based on affinity, comprising: introducing into said solution a solid phase element having binding affinity for a selected protein; Binding at least one of the plurality of drug candidates to the solid phase element as a selected drug candidate; washing the solid phase element and selected drug candidate to separate unbound material; sampling the selected drug candidate in a capture fluid flowing through a sampling region of an open port sampling interface; directing the sampled selected drug candidate and capture fluid to an ionization source; A method comprising: (Item 10) 10. The method of claim 9, wherein the solid-phase element is selected from the group consisting of solid-phase microextraction fibers, REEDs, and magnetic particles. (Item 11) 11. The method according to item 9 or 10, wherein the protein is immobilized on the surface of the solid-phase element by treating the Si—OH on the surface with an aminosilane reagent and then reacting with glutaraldehyde (GA), the free end of which can react with the amino group of lysine to capture the protein. (Item 12) 12. The method of any one of items 9-11, wherein the selected drug candidate is sampled by acoustically ejecting the selected drug candidate from a sample well into the capture fluid. (Item 13) Item 13. The method of item 12, wherein the selected drug candidate is ejected from the sample well after washing. (Item 14) Before the selected drug candidate is ejected from the sample well, the method further comprises: Releasing the selected drug candidates from the solid phase elements; isolating the selected drug candidates from the solid phase element; and injecting the selected drug candidate into the capture fluid without the solid phase element; Item 13. The method according to item 12, comprising: (Item 15) Item 13. The method according to item 12, wherein the selected drug candidate is injected in a state bound to the solid phase element. (Item 16) 16. The method of claim 15, wherein the selected drug candidate is debound by the capture fluid. (Item 17) 13. The method of claim 12, wherein the selected drug candidate and solid phase element are ejected from the sample well, and the system further comprises a trap for capturing the solid phase element before the ionization source. (Item 18) 18. The method of claim 17, wherein the drug candidate is released from the captured solid phase element by introducing a solvent into the capture fluid. (Item 19) Item 18. The method of item 17, wherein the trap comprises a magnetic trap. (Item 20) Item 18. The method of item 17, wherein the trap comprises a filter or a size trap. [Brief explanation of the drawings]

[0036] [Figure 1] FIG. 1 shows the steps in the MagMASS method, which uses magnetic particles that are used to capture drug molecules using protein binding affinity.

[0037] [Figure 2] FIG. 2 is a schematic diagram of an open port sampling interface (OPI) used in embodiments.

[0038] [Figure 3] FIG. 3 depicts a method for identifying and isolating compounds based on selected affinities.

[0039] [Figure 4] FIG. 4 depicts a method for identifying and isolating compounds based on selected affinities, according to one embodiment.

[0040] [Figure 5] FIG. 5 depicts a possible system for implementing the method of FIG.

[0041] [Figure 6] FIG. 6 depicts a method for identifying and separating compounds based on selected affinities, according to a further embodiment.

[0042] [Figure 7] FIG. 7 depicts a possible system for implementing the method of FIG.

[0043] [Figure 8]FIG. 8 depicts a method for identifying and separating compounds based on selected affinities, according to an additional embodiment.

[0044] [Figure 9] FIG. 9 depicts a possible system for implementing the method of FIG.

[0045] [Figure 10] FIG. 10 depicts a possible variation of the system of FIG. DETAILED DESCRIPTION OF THE INVENTION

[0046] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The inventors have discovered that the prior art MagMASS method uses magnetic particles to capture drug molecules using protein binding affinity, as shown in FIG. 1 . First, magnetic beads (B) are introduced into a sample container 100 containing drug molecule candidates (U and D) in solution. The drug molecule candidate (D) with affinity is then bound to the magnetic beads. The unbound drug molecule (U) is then removed in a wash container 110, while the beads (B) and the bound drug molecule candidate (D) are retained in the container via a magnetic field from a magnet 115. The washed beads are removed from the wash container and introduced into a separation container 120, where the drug molecule candidate (D) is sequestered from the beads using a solvent. The sequestered drug molecule candidate (d) is then aspirated from the separation container 120, while the magnetic beads are held in place via a magnetic field from a magnet 125. The aspirated drug molecule candidates are then eluted over time into an LC-MS / MS 130 for analysis. The magnetic beads can then be magnetically removed from the separation vessel 120 .

[0047] As discussed above, aspects of the present invention include improved methods and devices for transferring candidate molecules using OPIs with magnetic beads as solid-phase elements, and acoustic droplet ejection technology for non-contact introduction of samples into OPIs in a precise and controlled manner.

[0048] 2, an OPI 200 is shown comprising a first cylindrical member 205 disposed within a second cylindrical member 210 arranged in a coaxial arrangement, and an open tip 215. Additional details of the OPI 200 are provided below with reference to various embodiments.

[0049] Generally, as shown in FIG. 3, a method for identifying and separating compounds based on a selected affinity is provided. At 300, multiple compounds are introduced together in a solution. At 310, a probe is inserted into the solution, and the probe includes a surface treatment that operates to bind one or more compounds based on the selected affinity. One or more of the compounds are then bound to the probe at 320. In certain embodiments, the substrate surface may comprise a solid-phase microextraction (SPME) fiber that may contain embedded proteins with binding affinity. The substrate surface may be any material configured to retain proteins, and may include various examples such as a mesh material or a surface or a REED-like blade. In other embodiments, as discussed below, the surface treatment may include a magnetic material such as beads.

[0050] The probe with the bound compound(s) is then removed from the solution at 330. At 340, the compound(s) is / are separated from the probe. At 350, the separated compound(s) is / are captured at the open tip 215 of the OPI 200 using a flowing organic solvent. At 360, at the open tip 215 of the OPI 200, the solvent and the captured compound(s) are transported to an ionization element, such as an LC-MS / MS 130. Then, at 370, the compound(s) is / are ionized within the LC-MS / MS 130, as known in the art.

[0051] In one embodiment, a method for identifying and separating compounds based on selected affinities is provided, as described in FIG. 4 with reference to the system shown in FIG. 5. At 400, a plurality of drug molecule candidates (U and D) in solution and magnetic beads (B) are introduced into a sample container 100 using, for example, an electromagnetic sampling element or probe to which the beads are magnetically attached, such that the drug molecule candidate (D) with an affinity is bound to the magnetic beads. At 410, the beads (B) and bound drug molecule candidates (D) are transferred from the sample container 100 to a wash container 110 using, for example, an electromagnetic sampling element or probe; accordingly, the unbound drug molecule (U) is removed via washing, while the beads (B) and bound drug molecule candidate (D) are retained in the container via a magnetic field from a magnet 115. In 420, the washed beads with bound drug molecule candidates are removed from the washing vessel and introduced into the separation vessel 120, for example, using an electromagnetic sampling element or probe, and the drug molecule candidates (D) are released from the beads using an organic solvent. In 430, the drug molecule candidates (D) are isolated from the magnetic beads (B) via a magnet 125. In 440, the drug molecule candidates (D) are acoustically ejected from the separation vessel 120 into the OPI 200. Within the OPI 200, a capture fluid travels through the annular space 220 between the two cylindrical members toward the tip 215 and then away from the tip through the inner cylinder, as depicted by the arrow in the figure, which defines the fluid path. The capture fluid effectively eliminates the need for sample cleanup. In 450, the solvent and ejected drug candidates (D) flow from the tip 215 to the MS ionization source 530. Optionally, or if necessary, the drug molecule candidate (D) can be separated from the unbound drug molecule (U) using differential mobility spectroscopy (DMS) or MS techniques (e.g., fragmentation patterns in MS-MS, etc.).

[0052] In a further embodiment, a method for identifying and separating compounds based on selected affinities is provided, as described in Figure 6 with reference to the system shown in Figure 7. At 600, a plurality of drug molecule candidates (U and D) in solution and magnetic beads (B) are introduced into a sample container 100 using, for example, an electromagnetic sampling element or probe to which the beads are magnetically attached, such that the drug molecule candidate (D) with an affinity is bound to the magnetic beads. At 610, the beads (B) and bound drug molecule candidates (D) are transferred from the sample container 100 to a wash container 110 using, for example, an electromagnetic sampling element or probe; accordingly, the unbound drug molecule (U) is removed via washing, while the beads (B) and bound drug molecule candidate (D) are retained in the container via a magnetic field from a magnet 115. In 620, the washed beads with bound drug molecule candidates are removed from the washing vessel and introduced into the separation vessel 120, for example, using an electromagnetic sampling element or probe, and the drug molecule candidates (D) are released from the beads using an organic solvent. In 630, the drug molecule candidates (D) and beads (B) are acoustically ejected from the separation vessel 120 into the OPI 200. Within the OPI 200, the capture fluid travels through the annular space 220 between the two cylindrical members toward the tip 215 and then away from the tip through the inner cylinder, as depicted by the arrow in the figure, which defines the fluid path. The capture fluid effectively eliminates the need for sample cleanup. In 640, the solvent, beads (B), and drug candidates (D) flow from the tip 215 to the in-line trap 730, where the beads (B) are captured (640). At 650, the solvent and ejected drug candidate (D) flow from the trap 730 to the MS ionization source 530. Alternatively, rather than separating the drug molecule candidate (D) from the beads in the separation vessel 120, the drug molecule candidate (D) can be separated from the beads in the OPI 200, with the trapping fluid being the solvent.

[0053] Optionally, or if necessary, the drug molecule candidate (D) can be separated from the unbound drug molecule (U) using differential mobility spectroscopy (DMS) or MS techniques (e.g., fragmentation patterns in MS-MS, etc.).

[0054] For acoustic ejection at 630, it is preferred that the drug molecule candidate (D) is uniformly suspended in the sample solution in the separation container 120, for example, by mechanically agitating the separation container 120 prior to dispensing or by integrating an electromagnetic mixer within the acoustic dispensing system.

[0055] In an additional embodiment, a method for identifying and separating compounds based on selected affinities is provided, as described in FIG. 8 with reference to the system shown in FIG. 9. At 800, multiple drug molecule candidates (U and D) in solution and magnetic beads (B) are introduced into a sample container 100 using an electromagnetic sampling element or probe, e.g., to which the beads are magnetically attached, such that drug molecule candidates with affinity (D) are bound to the magnetic beads. At 810, unwashed drug molecule candidates (D) and beads (B) are acoustically ejected from the sample container 100 into an OPI 200. Within the OPI 200, a capture fluid travels through an annular space 220 between two cylindrical members toward a tip 215 and then travels away from the tip through an inner cylinder, as depicted by the arrows in the figure, that defines a fluid path. The capture fluid (e.g., water) effectively eliminates the need for sample cleaning. At 820, the solvent, beads (B), and unwashed drug candidates (D) flow (640) from the tip 215 to the in-line trap 730 where the beads (B) are captured and the drug candidates (D) are washed to remove unbound drug molecules (U). At 830, the flow of the trapping fluid (water) is switched to an organic solvent flow via valve 900 to separate the drug molecule candidates (D) from the beads (B). At 840, the solvent and selected drug candidates (D) flow from the trap 730 to the MS ionization source 530 via transport line 910.

[0056] Optionally, or if necessary, the drug molecule candidate (D) can be separated from the unbound drug molecule (U) using differential mobility spectroscopy (DMS) or MS techniques (e.g., fragmentation patterns in MS-MS, etc.).

[0057] Different embodiments of the trap 730 are envisioned, including a filter or size trap, or a permanent magnet that can be replaced at any time, or an electromagnet that can be energized to capture the magnetic beads (B) and then deenergized, for example, during a cleaning cycle to release any captured magnetic beads. As shown in Figure 10, the transfer line 900 can include a valve 920 for redirecting the flow of the capture fluid to a waste container, thereby avoiding the release of magnetic beads into the ionization source 530 during a cleaning cycle when the electromagnet is deenergized to release the captured beads.

[0058] In the system of Figure 7, the trap 730 may be a magnetic trap (i.e., an electromagnet surrounding one or both of the first cylindrical member 205 and / or the second cylindrical member 210) at the tip 215 of the OPI 200, and a cleaning cycle may be performed using a solvent-based trapping fluid to release the beads from the trap after the washed drug candidate is transported to the MS ionization source 530.

[0059] In another embodiment, the trap 730 can be placed in the ionization source 530 and the bead trajectories can be separated from the ions at the entrance to the MS ionization source 530 due to the beads being much heavier than the ions, for use with the systems shown in Figures 5 and 9.

[0060] In a further embodiment, trap 730 may be an in-line magnetic trap on transport line 900 of the system shown in Figure 9. It is envisioned that the in-line magnetic trap may be a replaceable section of transport line 900 that has a magnetic field sufficient to capture magnetic beads (B) within the transport line.

[0061] In the system of Figure 5, which employs acoustic ejection of drug molecule candidates (D) isolated from beads (B), it is also envisioned that a permanent magnetic protective trap may be included to protect the ionization source 530 and unintentional ejection of MS forms of magnetic beads from the container 120.

[0062] Although the systems depicted in Figures 5 and 7 discuss the use of separate sample, wash and separation vessels 100, 110, and 120, it is envisioned that sample preparation may be performed in a single vessel or multiple vessels.

[0063] In each of the embodiments described in Figures 4-10, as an alternative to introducing a compound to a drug molecule using its affinity for the solid surface of magnetic particles (B), it is envisioned that rather than pre-immobilizing the protein on magnetic particles (B), particles (B) can be added after protein-drug integration into free solution (e.g., after 400, 600, 800) and used to extract the protein-drug complex.

[0064] The many features and advantages of the present invention are apparent from the detailed description, and thus, it is intended by the appended claims to cover all such features and advantages of the present invention that fall within the scope of the present invention. Further, because numerous modifications and changes may readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and operation shown and described, and therefore, all suitable modifications and equivalents may be deemed to fall within the scope of the present invention.

Claims

1. 1. A method for identifying and isolating compounds based on a selected affinity, comprising: introducing the compounds together into a solution; inserting a probe comprising a surface treatment operative to bind one or more compounds based on said selected affinity; binding one or more compounds from the plurality of compounds to the probe; removing the probe and the bound compound or compounds from the solution; separating the one or more compounds from the probe; and capturing the separated one or more compounds with a flowing solvent at the open end of an open port sampling interface; transporting the solvent and the trapped one or more compounds to an ionization element; ionizing said one or more compounds; analyzing the ionized compound or compounds in a mass spectrometer; wherein the probe is a magnetic particle.

2. 10. The method of claim 1, wherein after ionizing the one or more compounds but before analyzing, the method further comprises separating the ionized one or more compounds based on ion mobility in a differential mobility spectrometer.

3. Separating the one or more compounds from the probe comprises: inserting the probe and the bound compound(s) into a debinding solvent in a separation vessel to debind the compound(s) from the probe; injecting the debinding solvent and the debound one or more compounds into the flowing solvent at the open end of the open port sampling interface; The method according to any one of claims 1-2, comprising:

4. The injecting step comprises: aspirating the debinding solvent and the debound one or more compounds from the separation vessel; injecting the aspirated decoupled solvent and the decoupled one or more compounds into a solvent stream that is pumped through the ionization element; The method of claim 3, comprising:

5. 4. The method of claim 3, wherein the injecting comprises ejecting droplets of the debinding solvent and the debound one or more compounds from the separation vessel into the flowing solvent at the open end of the open port sampling interface.

6. The method of claim 5 , wherein the injecting comprises acoustically or pneumatically ejecting the droplets.

7. 1. A system for affinity selection by mass spectrometry, wherein a plurality of drug candidates in solution are separated based on affinity, comprising: introducing into said solution a solid phase element having binding affinity for a selected protein; Binding at least one of the plurality of drug candidates to the solid phase element as a selected drug candidate; washing the solid phase element and selected drug candidate to separate unbound material; sampling the selected drug candidate in a capture fluid flowing through a sampling region of an open port sampling interface; directing the sampled selected drug candidate and capture fluid to an ionization source; wherein the solid phase elements are magnetic particles.

8. 8. The method of claim 7, wherein the protein is immobilized on the surface by treating Si-OH on the surface of the solid-phase element with an aminosilane reagent, followed by reaction with glutaraldehyde (GA), the free end of which can react with the amino group of lysine to capture the protein.

9. 9. The method of claim 7 or claim 8, wherein the selected drug candidate is sampled by acoustically ejecting the selected drug candidate from a sample well into the capture fluid.

10. The method of claim 9 , wherein the selected drug candidate is ejected from the sample well after washing.

11. Before the selected drug candidate is ejected from the sample well, the method further comprises: Releasing the selected drug candidates from the solid phase elements; isolating the selected drug candidates from the solid phase element; and injecting the selected drug candidate into the capture fluid without the solid phase element; 10. The method of claim 9, comprising:

12. The method of claim 9, wherein the selected drug candidate is injected while bound to the solid phase element.

13. The method of claim 12 , wherein the selected drug candidate is debound by the capture fluid.

14. 10. The method of claim 9, wherein the selected drug candidate and solid phase element are ejected from the sample well, and the system further comprises a trap for capturing the solid phase element before the ionization source.

15. 15. The method of claim 14, wherein the selected drug candidates are released from the captured solid phase elements by introducing a solvent into the capture fluid.

16. The method of claim 14 , wherein the trap comprises a magnetic trap.

17. The method of claim 14 , wherein the trap comprises a filter or a size trap.

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