Patent Grant
7095017
The present invention relates to systems and methods for delivering samples for mass spectrometric analysis. More specifically, the present invention relates to systems and methods for facilitating high throughput mass spectrometric analysis of generated electrospray ionized samples.
Generating electrospray ionized samples for mass spectrometric analysis generally requires samples to be delivered to electrospray ionization source at a low flow rate. To minimize contamination and improve the accuracy of the results of the mass spectrometric analysis, before another sample can be processed by a delivery system which delivers the samples to the electrospray ionization source, those portions of the delivery system which were exposed to the previous sample need to be thoroughly rinsed. Existing sample delivery systems accomplish this rinse cycle at the same flow rate at which the samples are delivered to the electrospray ionization source. Because the volume of rinsing agent that may be required to adequately rinse the delivery system can be quite large, existing delivery systems cannot support high throughput mass spectrometric analysis protocols since a significant amount of time for rinsing has to be expended between the introduction and analysis of each subsequent sample.
The present disclosure is directed at improved systems and methods for delivering samples for high-throughput mass spectrometric analysis to an atmospheric-pressure ionization source. In an exemplary embodiment in accordance with present disclosure, the system has a solvent reservoir for storing a solvent solution, a first valve which is coupled to the solvent reservoir, first and second pumps for delivering solvent solution and which are coupled to the first valve, an injection system having a sample injector and an second valve which is coupled to the first valve and which is capable of being coupled to an electrospray ionization source. In an exemplary embodiment, the delivery flow rate of the first pump is greater than the delivery flow rate of the second pump and, additionally, the injection system, which is coupled to the second valve, can deliver a sample to the second valve. The system can also include a controller to control the operations of the first valve, the first pump, the second pump, the second valve and the injection system.
In a preferred embodiment, the first and second pumps are highly accurate programmable syringe pumps and the second valve and the first valve are two position, multi-port fluid processors.
In another exemplary embodiment in accordance with the present disclosure, the system may further include an atmospheric-pressure ionization chamber, an atmospheric-pressure ionization sprayer coupled to the second valve, a nebulizer gas source having a nebulizer gas, which is in fluid communication with the atmospheric-pressure ionization sprayer, and a voltage supply source coupled to the atmospheric-pressure ionization sprayer. A distal end of the atmospheric-pressure ionization sprayer can be located within the atmospheric-pressure ionization chamber. The system may similarly have a controller to control the operations of the atmospheric-pressure ionization sprayer, the nebulizer gas source and the voltage supply source. The system may further include a transfer line connected to the second valve and the atmospheric-pressure ionization sprayer. In such an embodiment, the injection system may deliver a sample to the transfer line via the second valve.
In one exemplary embodiment, a delivery flow rate of the injection system is greater than the delivery flow rate of the second pump.
In one preferred embodiment, the system further includes a puffer valve that is coupled to the nebulizer gas source and the atmospheric-pressure ionization sprayer and a gas puffer that is coupled to the puffer valve. A distal end of the gas puffer may be located within the atmospheric-pressure ionization chamber and aligned with the distal end of the atmospheric-pressure ionization sprayer and the puffer valve may control the delivery of the nebulizer gas to the atmospheric-pressure ionization sprayer and the gas puffer.
In an exemplary embodiment in accordance with present disclosure, a method for facilitating high throughput mass spectrometric analysis of generated ionized samples can include the steps of (A) delivering a sample to a transfer line which can be coupled to an ionization sprayer of an atmospheric-pressure ionization source; (B) initiating a low flow delivery of a liquid to the transfer line containing the sample, wherein the low flow delivery of the liquid to the transfer line can cause the sample to be delivered to the atmospheric-pressure ionization source; (C) terminating the low flow delivery of the liquid to the transfer line; (D) rinsing the transfer line by directing a high flow delivery of a liquid to the transfer line, wherein the high flow delivery of the liquid is greater than the low flow delivery of the liquid; and then repeating steps A through D for the next sample to analyzed.
In an exemplary embodiment, the delivering of the sample to the transfer line is controlled by an injector system which rinses the sample injector and prepares the next sample for delivery after a first sample has been delivered to the transfer line. Additionally, in a preferred embodiment, the injector system delivers the sample to the transfer line at a flow rate which is greater than the rate of the low flow delivery of the solvent solution.
In another preferred embodiment, the method further includes the step of directing a gas at a distal end of the electrospray ionization sprayer to remove any droplets which may be present at the distal end before the electrospray ionization sprayer is energized.
Still other objects and advantages of the present invention will become readily apparent to those skilled in the art from the following detailed description wherein several embodiments are shown and described. As will be realized, the invention is capable of other and different embodiments, and its several details are capable of modifications in various respects, all without departing from the invention. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not in a restrictive or limiting sense, with the scope of the application being indicated in the claims.
For a fuller understanding of the nature and objects of the present invention, reference should be made to the following detailed description taken in connection with the accompanying drawings in which the same reference numerals are used to indicate the same or similar parts wherein:
The present disclosure is directed to systems and methods that utilize a parallel high-throughput screening (HTS) strategy to quickly identify, via mass spectrometric analysis, the presence of a small compound, or compounds, within prepared samples. The present disclosure may have broad applications in high throughput mass spectrometry as pertains to high throughput screening, automated analysis, and quality control. The present disclosure describes systems and methods for high throughput electrospray ionization (ESI) in which an ESI sprayer can be coupled to a control module in which sample flow rates, buffer flow rates, nebulizing gas, and ionization voltages can be synchronized (via the control module) to enable rapid sample transfer to the ionization source (e.g., an ESI sprayer).
The systems and methods described herein, therefore, may enable significant improvement in sample throughput, allowing a greater number of samples to be analyzed in a given period of time. Operation in a low flow sample delivery mode may provide improved sensitivity as the ESI process is generally more efficient at low flow rates, and because the analysis of a given volume of sample can then be signal-averaged for extended intervals, thereby providing improved signal-to-noise detections. In addition to enabling more rapid analysis of analytes (i.e., samples) or mixtures of analytes, operation of the ESI source in a voltage-gated mode, where a voltage is applied to the ESI source only when data acquisition is taking place, may result in reduced source fouling as the ESI plume is generated (and sampled) only during the period of data acquisition (by a MS/Data acquisition system). Reduced source fouling can translate to directly reducing instrument down time which results from the cleaning and maintenance of the ionization source components.
In accordance with the present disclosure,
The high flow pump 72 and low flow pump 74 each can deliver a highly accurate volume of solvents solution to solvent lines 172, 174 (and beyond), respectively. The high flow pump 72 has a volume discharge that is substantially greater over the same period of time than that of the low flow pump 74. In an exemplary embodiment in accordance with the present disclosure, the high flow pump 72 and low flow pump 74 are highly accurate syringe pumps and, in a preferred embodiment, the high flow pump 72 and low flow pump 74 are programmable syringe pumps, model number 74901-10 available from Cole-Parmer Instrument Company of Vernon Hills, Ill., having a 500 μL and 50 μL syringe reservoirs, respectively. The syringe body (not shown) of the high flow pump 72 acts as an internal solvent reservoir. The depression of a plunger (not shown) within the syringe body can cause the solvent solution contained within the syringe body to be pumped into solvent line 172 and, depending upon the configuration of the fluidics valve 80, into fluidics valve 80 and solvent line 154 and beyond. Thus, the high flow pump 172 can generate a positive pumping pressure, and the discharge volume and flow rate of the solvent solution can be accurately controlled by appropriately regulating the stroke of the high flow pump's plunger. As is discussed in greater detail below, the syringe body of the high flow pump 72 may then be refilled with solvent solution (for a later high flow use) from the solvent reservoir 70 by at least partially displacing the plunger from the high flow pump's syringe body. The withdrawing of the high flow pump's 72 plunger can cause some of the solvent solution that is present in the solvent reservoir 70 to flow, via solvent line 170, fluidics valve 80 and solvent line 172, into the syringe body of the high flow pump 72. The low flow pump 74 may be similarly controlled to pump solvent solution into solvent line 174 (and beyond) and to draw solvent solution from the solvent reservoir 70 via solvent line 170, fluidics valve 80 and solvent line 174.
As stated, the high flow pump 72 and the low flow pump 74 of system 10 can pump solvent solution by applying a positive pumping pressure to solvent lines 172 and 174, respectively, to the fluidics valve 80 and, depending upon the configuration of the fluidics valve 80, to the injection valve 90 (and beyond) via solvent line 154. The pumping pressures generated by the high flow pump 72 will generally be substantially greater than the pumping pressures generated by the low flow pump 74. Accordingly, the high flow pump 72 may generate a higher flow of solvent solution to the fluidics valve 80 (and beyond, depending upon the configuration of the fluidics valve 80) than the low flow pump 74.
In an exemplary embodiment, the fluidics valve 80 and the injection valve 90 are two-position multi-port fluid processors. Thus, each valve 80 and 90 can have two positions, the configuration of which may be controlled by the data acquisition/controller 20, as discussed below. In a preferred embodiment in accordance with the present disclosure, the fluidics valve 80 can be a two-position, six-port fluid processor, model number AVO-6084 available from Phenomenex of Torrance, Calif., while the injection valve 90 can be a two-position, ten-port fluid processor, model number 100.LC102H available from Leap Technologies, Inc. of Carrboro, N.C.
System 10 further includes an autosampler injection system 50, a high voltage supply 46, a nebulizer gas source 60, an electrospray ionization chamber 40 and an electrospray ionization (ESI) sprayer 42, which is at least partially located within an electrospray ionization chamber 40. The autosampler injection system 50 of system 10 can be a wide variety of commercially available conventional automated injection systems, such as the CTC HTS PAL autosampler available from LEAP Technologies, Inc. of Carrboro, N.C. Such an autosampler injection system 50 may have a programmable robotic injection sample delivery system having an internal reservoir of solvent solution/ESI buffer. It can extract samples from 96-well microtiter plates and inject a sample into a 10 μL sample loop integrated with an internal fluidics handling system which allows differential control of the sample/buffer flow rate.
In alternate exemplary embodiment of the systems and methods described herein, the electrospray ionization chamber 40 and an electrospray ionization (ESI) sprayer 42 of system 10 may be substituted with an atmospheric-pressure chemical ionization chamber and an atmospheric-pressure chemical ionization sprayer, respectively, without departing from the scope of the present disclosure.
As shown in
The high voltage supply 46 may provide an energy supply to the ESI sprayer 42 via electrical line 48. The distal end 420 of the ESI sprayer 42 is located within the electrospray ionization chamber 40. The electrospray plume (or bead) 460 [see
System 10 of
As shown in
The system 10 of
While the data acquisition/controller 20 and the mass spectrometer 30 are illustrated as separate components or devices, in practice they may be components of a single device or system. For example, one data acquisition/controller 20 combined with a mass spectrometer 30 is the Apex II 70e electrospray ionization Fourier transform ion cyclotron resonance (FTICR) mass spectrometer with an actively shielded seven telsa superconducting magnet, which is available from Bruker Daltonics, Inc. of Billerica, Mass. However, persons skilled in the art will readily recognize a wide variety of mass spectrometer and data acquisition/control systems may be used without departing from the scope of the present disclosure. As shown, the mass spectrometer 30 is coupled to the electrospray ionization chamber 40 so as to receive, process and detect the delivered ionized samples.
To emphasize different details,
Once step 207 has been completed (i.e., the ESI sprayer 42 is energized and receiving the nebulizer gas), electrospray ionization of the injected sample and data acquisition may begin, step 209. In a preferred embodiment, as illustrated in timeline 300, the accomplishment of steps 201, 205 and 207 and the initiation of step 203 (such that step 209 may begin) may require approximately nine seconds. In a preferred embodiment, step 209 may take approximately 25 seconds to complete. After step 209 has been completed, the ESI sprayer 42 may be de-energized and the delivery of the nebulizer gas to the ESI sprayer may be stopped, step 211. Once the ESI sprayer 42 is de-energized, step 211, the electrospray ionization low flow step 203 may then be completed (i.e., terminated) and the low flow refill of the low flow pump 74, step 213, and the high flow rinse, step 227, may be initiated. In a preferred embodiment, after data acquisition step 209 is completed, the ESI sprayer 42 is immediately de-energized, step 211, and the fluidics valve 80 is switched to high flow rinse, step 227. Additionally, the low flow pump 74 may continue to run for 1–2 seconds after the fluidics valve 80 switches to ensure that data acquisition, step 209, is complete—the low flow pump 74 may then immediately begins its re-fill cycle, step 213. While method 200 depicts steps 211 and 213 as occurring serially, in some embodiments in accordance with the present disclosure, steps 211 and 213 may occur in parallel. In a preferred embodiment, as depicted in
Concurrent with step 203 (and possibly preceding it), the high flow refill of the high flow pump 72, step 221, may be initiated and when completed, the rinsing of the sample injector (not shown) which is internal to the autosampler injection system 50 may then subsequently be initiated, step 223. Once the sample injector has been rinsed, step 223, the next sample to be tested can be obtained by the sample injector of the autosampler injection system 50, step 225, in anticipation of sample injection, step 210. and the high flow rinse, step 227, may be initiated. While method 200 of
Commensurate (or approximately commensurate) with the repositioning of injection valve 90, the low flow pump 74 may be commanded to pump a previously established volume of solvent solution (e.g., ESI buffer). The pumping forces from the low flow pump 74 cause a pre-determined volume of solvent solution to be pumped, via solvent line 174, the position 2 pathway 88 of the fluidics valve 80, solvent line 154 and the position 2 pathway 98 of the injection valve 90, toward and to transfer line 156. The volume of solvent solution that is to be pumped by the low flow pump 74 was pre-determined, based upon this pathway, so as to achieve a desired flowrate identified for optimal and efficient ESI performance of approximately 70 μL/Hr. However, the recitation of this low flow rate should not be construed as limiting the scope of the present disclosure; persons skilled in the art will readily recognize a wide range of low flow rates that are within the scope of the present disclosure and that are conducive to accurate and efficient electrospray ionization mass spectrometric analysis. Thus, the low flow pump 74 is responsible for delivering a regulated (low) flow of the sample from the transfer line 156 to the electrospray ionization source so as to facilitate the electrospray ionization of the sample.
In a preferred embodiment, the operations of step 221, the high flow refill of the high flow pump 72, overall, at least partially, with the operations of step 203, the low flow delivery of solvent solution from the low flow pump 74 to the transfer line 156. In some embodiments, step 221, or a portion thereof, may also be conducted concurrently with step 201 (or a portion thereof), the delivery of the sample from the autosampler injection system 50 to the transfer line 156. As depicted in
Commensurate with step 221, or alternatively, occurring thereafter, the injector (not shown) of the autosampler injection system 50 may be rinsed, step 223, in anticipation of loading the next sample within the autosampler injection system 50. Prior to preparing the next sample for delivery, the autosampler injection system 50 flushes the internal components that are exposed to the presence of a sample with a solvent solution (e.g., ESI buffer). The discharge of this solvent solution from the autosampler injection system 50 can be directed by the autosampler injection system 50, which may or may not be controlled by data acquisition/controller 20 to a waste receptacle (not shown), which may be reached via transfer line 152, the position 2 pathway 96 of the injection valve 90 and waste line 158.
The internal loading of the next sample to be tested/evaluated within the autosampler injection system 50, step 225, can then be conducted once step 223 has been completed.
Immediately after, or concurrent with, the initiation of the introduction of the ESI solvent solution/buffer low flow, step 203 (not shown in
After the completion of step 205, the puffer valve 66 may then be switched (e.g., commanded) to a second position to divert the delivery of the nebulizer gas to the ESI sprayer 42 and the ESI sprayer 42 may then be energized via the high voltage supply 46 (e.g., via a TTL pulse from the data acquisition/controller 20), step 207. Thus, in this exemplary embodiment as shown in
In a preferred embodiment in accordance with the present disclosure, by using known mass spectrometry (MS) technologies such as Fourier Transform Ion Cyclotron Resonance-Mass Spectrometry (FTICR-MS), for example, the HTS strategy can be used to identify the small molecule(s) that bind to a RNA target. Moreover, the HTS strategy disclosed herein can be a key component of a Multitarget Affinity/Specificity Screening (MASS) protocol. A MASS assay can take advantage of the “intrinsic mass” label of each compound and target RNA to screen large mixtures of small molecules against multiple RNA targets simultaneously such that the identity of the small molecule(s) which bind, the RNA target to which it binds, the compound-specific binding affinity, and the location of the binding site on the RNA can each be determined in one set of rapid experiments.
At the core of the MASS approach is the premise that in a solution containing multiple targets and multiple ligands (i.e., a sample), the molecular interaction between any given target-ligand combination is independent of the presence (or absence) of the other ligands and targets in solution. The applicants have demonstrated that in a mixture of 3 targets and 26 ligands, that a ligand binding a specific RNA will do so in the presence of the other ligands even at a significantly lower concentration than the total concentration of the other ligands. Accordingly, the present disclosure encompasses systems and methods for automating the MASS assay into a multiply-parallel high throughput format. For example, in accordance with the present disclosure, a sample (e.g., a solution containing at least one RNA target and at least one ligand), can be injected to a mass spectrometer and mass analyze every 39 seconds. During the 39 seconds, spectra can be co-added while the autosampler injection systems is rinsing its internal syringe, sample loop and injector and preparing to inject the next sample. Typically, 25 compounds at 50 uM each are screened against 3 targets at 5 uM each. In this mode 75 molecular interactions can be evaluated every 39 seconds which corresponding to approximately 0.52 seconds/analysis. In this way, in less than 7 hours, 6 microtiter plates can be analyzed which allows >40,000 molecular interactions to be evaluated. Therefore, a tremendous amount of mass spectrometry data can be generated in a short period of time in accordance with the present disclosure. In the gated automated approach of the present disclosure, tens of thousands of molecular interactions, for example, may be interrogated in a single day.
Since numerous embodiments may be used to achieve the above systems and methods without departing from the scope of the present invention, it is intended that all matter contained in the above description or depicted in the accompanying drawings shall be interpreted as merely illustrative and not limiting the scope of the invention, which is set forth in the following claims.
This application claims priority to U.S. Provisional Patent Application No. 60/295,588 filed Jun. 4, 2001, the entire contents of which are herein incorporated by reference.
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| Number | Date | Country | |
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| 20050029447 A1 | Feb 2005 | US |
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| Number | Date | Country | |
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| Child | 10939753 | US |
