Simplification of Method or System Using Scout MRM

Abstract

Each sample of a series of samples is ejected at an ejection time and according to a sample order. Each ejected sample of the series is ionized, producing ion beam. A list of different sets of MRM transitions is received. Each set of the list corresponds to a different sample. A group of one or more different sets is selected from the list. Initially, each set selected for the group corresponds to a different sample of one or more first samples of the series. A mass spectrometer is instructed to execute each transition of each set of the group on the ion beam until a transition of a set of the group is detected, upon which, one or more next sets are selected from the list to be monitored using the set of the detected transition and the sample order.

Claims

1. A system for selecting the next multiple reaction monitoring (MRM) transitions to be monitored in a high-throughput sample introduction coupled mass spectrometry experiment based on the transition detected and the order of the samples, comprising: a sample introduction system that ejects each sample of a series of samples at an ejection time and according to a sample order, producing a plurality of ejection times corresponding to the series, and ionizes each ejected sample of the series, producing an ion beam; a tandem mass spectrometer that receives the ion beam; and a processor that receives a list of different sets of one or more MRM precursor ion to product ion transitions, wherein each set of the list corresponds to a different sample of the series, selects a group of one or more sets from the list, wherein initially each of one or more sets selected for the group corresponds to a different sample of one or more first samples of the series, instructs the tandem mass spectrometer to execute each transition of each set of the group on the ion beam during each cycle of a plurality of cycles until a transition of a set of the group is detected, and when a transition of a set of the group is detected, selects one or more next sets from the list to be monitored using the detected transition and the sample order.

2. The system of claim 1, wherein the list is ordered according to the sample order, wherein each transition of each set of the list is a scout transition that identifies one or more sets immediately following the each set on the list, and wherein, when a transition of a set of the group is detected, the processor selects the one or more next sets by selecting one or more sets of the list identified by the detected transition as immediately following the set of the detected transition.

3. The system of claim 2, wherein the processor further adds the selected one or more sets to the group if any are not already part of the group and instructs the tandem mass spectrometer to execute each transition of each set of the groupeach transition of each set of the group on the ion beam during each cycle of a plurality of cycles until a transition of a different set of the group is detected.

4. The system of claim 3, wherein the processor further removes any set of the group that precedes a set of the detected transition on the list before the processor instructs the tandem mass spectrometer to execute each transition of each set of the groupeach transition of each set of the group on the ion beam during each cycle of a plurality of cycles until a transition of a different set of the group is detected.

5. The system of claim 1, wherein the list is ordered according to the sample order, wherein a first set of the list and every mth set following the first set are marker sets for samples that include marker ions, wherein every marker set includes one or more scout transitions that identify m sets immediately following every marker set on the list, wherein the processor initially selects only the first set for the group, wherein, when a transition of a set of the group is detected, the processor selects the one or more next sets by selecting m sets identified by the detected marker transition.

6. The system of claim 5, wherein the processor further (a) removes all sets from the group, (b) adds m sets identified by the detected marker transition to the group, (c) instructs the tandem mass spectrometer to execute each transition of each set of the group on the ion beam during each cycle of a plurality of cycles until a marker transition of a set of the group is detected, and (d) repeats steps (a)-(c) until all sets of the list have been added to the group.

7. The system of claim 1, wherein, when a transition of a set of the group is detected, the processor selects one or more next sets from the list to be monitored by identifying a corresponding sample from the set of the detected transition and identifying a plurality of next samples of the series following the identified corresponding sample using the sample order, and selecting a plurality of sets of the list corresponding to the plurality of next samples as the one or more next sets and wherein the processor further instructs the tandem mass spectrometer to schedule execution of each transition of each set of the plurality of sets based on an ejection time of a sample corresponding to the each set, an ejection time of the corresponding sample from the detected transition, a detection time of the detected transition, and the sample order.

8. The system of claim 1, wherein the sample introduction system comprises a surface analysis system.

9. The system of claim 8, wherein the surface analysis system comprises one of a matrix-assisted laser desorption/ionization (MALDI) device or a laser diode thermal desorption (LDTD) device.

10. The system of claim 1, wherein the sample introduction system comprises a flow injection device and an ion source device.

11. The system of claim 10, wherein the flow injection device comprises a timed valve device that injects sample into a flowing stream through a valve at each ejection time of the plurality of ejection times and wherein the ion source device ionizes samples of the flowing stream, producing the ion beam.

12. The system of claim 10, wherein the flow injection device comprises a droplet dispenser that ejects the series of samples as droplets into a flowing stream at each ejection time of the plurality of ejection times and wherein the ion source device ionizes samples of the flowing stream, producing the ion beam.

13. The system of claim 12, wherein the droplet dispenser comprises an acoustic droplet ejection (ADE) device that ejects the series of samples as droplets into an inlet of a tube of an open port interface (OPI), wherein the OPI mixes the droplets of the series of samples with a solvent in the tube to form a series of analyte-solvent dilutions and transfers the series of dilutions to an outlet of the tube of the OPI, and wherein the ion source device receives the series of dilutions and ionizes samples of the series of dilutions, producing the ion beam.

14. A method for selecting the next multiple reaction monitoring (MRM) transitions to be monitored in a high-throughput sample introduction coupled mass spectrometry experiment based on the transition detected and the order of the samples, comprising: ejecting each sample of a series of samples at an ejection time and according to a sample order, producing a plurality of ejection times corresponding to the series, and ionizing each ejected sample of the series, producing an ion beam, using a sample introduction system; receiving the ion beam using a tandem mass spectrometer; receiving a list of different sets of one or more MRM precursor ion to product ion transitions using a processor, wherein each set of the list corresponds to a different sample of the series; selecting a group of one or more sets from the list using the processor, wherein initially each of one or more sets selected for the group corresponds to a different sample of one or more first samples of the series; instructing the tandem mass spectrometer to execute each transition of each set of the group on the ion beam during each cycle of a plurality of cycles until a transition of a set of the group is detected using the processor; and when a transition of a set of the group is detected, selecting one or more next sets from the list to be monitored using the detected transition and the sample order using the processor.

15. A computer program product, comprising a non-transitory and tangible computer-readable storage medium whose contents include a program with instructions being executed on a processor so as to perform a method for selecting the next multiple reaction monitoring (MRM) transitions to be monitored in a high-throughput sample introduction coupled mass spectrometry experiment based on the transition detected and the order of the samples, the method comprising: providing a system, wherein the system comprises one or more distinct software modules, and wherein the distinct software modules comprise a control module; instructing a sample introduction system to eject each sample of a series of samples at an ejection time and according to a sample order, producing a plurality of ejection times corresponding to the series, and ionize each ejected sample of the series, producing an ion beam, using the control module; instructing a tandem mass spectrometer to receive the ion beam using the control module; receiving a list of different sets of one or more MRM precursor ion to product ion transitions using the control module, wherein each set of the list corresponds to a different sample of the series; selecting a group of one or more sets from the list using the control module, wherein initially each of one or more sets selected for the group corresponds to a different sample of one or more first samples of the series; instructing the tandem mass spectrometer to execute each transition of each set of the group on the ion beam during each cycle of a plurality of cycles until a transition of a set of the group is detected using the control module; and when a transition of a set of the group is detected, selecting one or more next sets from the list to be monitored using the detected transition and the sample order using the control module.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0058] FIG. 1A is an exemplary system combining an acoustic droplet ejection (ADE) with an open port interface (OPI) sampling interface, as described in the '667 Application.

[0059] FIG. 1B is an exemplary system for ionizing and mass analyzing analytes received within an open end of a sampling OPI, as described in the '667 Application.

[0060] FIG. 2 is a block diagram that illustrates a computer system, upon which embodiments of the present teachings may be implemented.

[0061] FIG. 3 is an exemplary plot showing the proper alignment of detected peaks with ejection times from an ADE device timing file.

[0062] FIG. 4 is an exemplary plot showing three peaks detected over time by a mass spectrometer for analytes from four different samples sequentially provided to the mass spectrometer by an ADE device and an OPI.

[0063] FIG. 5 is an exemplary plot showing how the mismatch in the number of peaks detected and the number of ejection times in FIG. 4 results in different alignments of the four different sample ejections with the three peaks of FIG. 4.

[0064] FIG. 6 is an exemplary plot showing detected peaks misaligned with ejection times due to a missing peak and a low-intensity peak.

[0065] FIG. 7 is an exemplary diagram showing how n precursor ion to product ion MRM transitions correspond to n samples of an AEMS experiment, in accordance with various embodiments.

[0066] FIG. 8 is an exemplary diagram showing how precursor ion to product ion MRM transitions are added to and removed from a group of transitions as transitions are detected, in accordance with various embodiments.

[0067] FIG. 9 is an exemplary diagram showing how MRM transitions that include a first marker transition and then another marker transition every m transitions after the first transition correspond to samples including corresponding marker samples of an AEMS experiment, in accordance with various embodiments.

[0068] FIG. 10 is an exemplary diagram showing how MRM transitions that include marker transitions are added to and removed from a group of transitions as marker transitions are detected, in accordance with various embodiments.

[0069] FIG. 11 is an exemplary diagram showing how a group of MRM transitions corresponding to the first few samples can trigger scheduled MRM of the remaining transitions corresponding to the remaining samples, in accordance with various embodiments.

[0070] FIG. 12 is an exemplary diagram showing how scheduled MRM is triggered by a transition of a group of MRM transitions corresponding to the first few samples and how scheduled MRM proceeds on the remaining transitions corresponding to the remaining samples, in accordance with various embodiments.

[0071] FIG. 13 is a schematic diagram of a system for selecting the next MRM transitions to be monitored in a high-throughput sample introduction coupled mass spectrometry experiment based on the transition detected and the order of the samples, in accordance with various embodiments.

[0072] FIG. 14 is a flowchart showing a method for selecting the next MRM transitions to be monitored in a high-throughput sample introduction coupled mass spectrometry experiment based on the transition detected and the order of the samples, in accordance with various embodiments.

[0073] FIG. 15 is a schematic diagram of a system that includes one or more distinct software modules that performs a method for selecting the next MRM transitions to be monitored in a high-throughput sample introduction coupled mass spectrometry experiment based on the transition detected and the order of the samples, in accordance with various embodiments.

[0074] Before one or more embodiments of the present teachings are described in detail, one skilled in the art will appreciate that the present teachings are not limited in their application to the details of construction, the arrangements of components, and the arrangement of steps set forth in the following detailed description or illustrated in the drawings. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

DESCRIPTION OF VARIOUS EMBODIMENTS

Computer-Implemented System

[0075] FIG. 2 is a block diagram that illustrates a computer system 200, upon which embodiments of the present teachings may be implemented. Computer system 200 includes a bus 202 or other communication mechanism for communicating information, and a processor 204 coupled with bus 202 for processing information. Computer system 200 also includes a memory 206, which can be a random-access memory (RAM) or other dynamic storage device, coupled to bus 202 for storing instructions to be executed by processor 204. Memory 206 also may be used for storing temporary variables or other intermediate information during execution of instructions to be executed by processor 204. Computer system 200 further includes a read only memory (ROM) 208 or other static storage device coupled to bus 202 for storing static information and instructions for processor 204. A storage device 210, such as a magnetic disk or optical disk, is provided and coupled to bus 202 for storing information and instructions.

[0076] Computer system 200 may be coupled via bus 202 to a display 212, such as a cathode ray tube (CRT) or liquid crystal display (LCD), for displaying information to a computer user. An input device 214, including alphanumeric and other keys, is coupled to bus 202 for communicating information and command selections to processor 204. Another type of user input device is cursor control 216, such as a mouse, a trackball or cursor direction keys for communicating direction information and command selections to processor 204 and for controlling cursor movement on display 212. This input device typically has two degrees of freedom in two axes, a first axis (i.e., x) and a second axis (i.e., y), that allows the device to specify positions in a plane.

[0077] A computer system 200 can perform the present teachings. Consistent with certain implementations of the present teachings, results are provided by computer system 200 in response to processor 204 executing one or more sequences of one or more instructions contained in memory 206. Such instructions may be read into memory 206 from another computer-readable medium, such as storage device 210. Execution of the sequences of instructions contained in memory 206 causes processor 204 to perform the process described herein. Alternatively, hard-wired circuitry may be used in place of or in combination with software instructions to implement the present teachings. Thus, implementations of the present teachings are not limited to any specific combination of hardware circuitry and software.

[0078] In various embodiments, computer system 200 can be connected to one or more other computer systems, like computer system 200, across a network to form a networked system. The network can include a private network or a public network such as the Internet. In the networked system, one or more computer systems can store and serve the data to other computer systems. The one or more computer systems that store and serve the data can be referred to as servers or the cloud, in a cloud computing scenario. The one or more computer systems can include one or more web servers, for example. The other computer systems that send and receive data to and from the servers or the cloud can be referred to as client or cloud devices, for example.

[0079] The term “computer-readable medium” as used herein refers to any media that participates in providing instructions to processor 204 for execution. Such a medium may take many forms, including but not limited to, non-volatile media, volatile media, and transmission media. Non-volatile media includes, for example, optical or magnetic disks, such as storage device 210. Volatile media includes dynamic memory, such as memory 206. Transmission media includes coaxial cables, copper wire, and fiber optics, including the wires that comprise bus 202.

[0080] Common forms of computer-readable media or computer program products include, for example, a floppy disk, a flexible disk, hard disk, magnetic tape, or any other magnetic medium, a CD-ROM, digital video disc (DVD), a Blu-ray Disc, any other optical medium, a thumb drive, a memory card, a RAM, PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge, or any other tangible medium from which a computer can read.

[0081] Various forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to processor 204 for execution. For example, the instructions may initially be carried on the magnetic disk of a remote computer. The remote computer can load the instructions into its dynamic memory and send the instructions over a telephone line using a modem. A modem local to computer system 200 can receive the data on the telephone line and use an infra-red transmitter to convert the data to an infra-red signal. An infra-red detector coupled to bus 202 can receive the data carried in the infra-red signal and place the data on bus 202. Bus 202 carries the data to memory 206, from which processor 204 retrieves and executes the instructions. The instructions received by memory 206 may optionally be stored on storage device 210 either before or after execution by processor 204.

[0082] In accordance with various embodiments, instructions configured to be executed by a processor to perform a method are stored on a computer-readable medium. The computer-readable medium can be a device that stores digital information. For example, a computer-readable medium includes a compact disc read-only memory (CD-ROM) as is known in the art for storing software. The computer-readable medium is accessed by a processor suitable for executing instructions configured to be executed.

[0083] The following descriptions of various implementations of the present teachings have been presented for purposes of illustration and description. It is not exhaustive and does not limit the present teachings to the precise form disclosed. Modifications and variations are possible in light of the above teachings or may be acquired from practicing of the present teachings. Additionally, the described implementation includes software, but the present teachings may be implemented as a combination of hardware and software or in hardware alone. The present teachings may be implemented with both object-oriented and non-object-oriented programming systems.

Selecting the Next MRM Transitions Based on Sample Order

[0084] As described above, AEMS systems can eject samples at high-speed ejection rates (1 sample every second). However, in AEMS, there is a delay from when the acoustic ejection occurs to when the signal from the analyte is detected (several seconds). Even though an AEMS system can deliver samples from distinct sample wells to a detector of a mass spectrometer at a rate of more than one per second, the exact time that the sample arrives at the detector is difficult to calculate ahead of time.

[0085] For MRM assays that require different mass spectrometer/detector settings per sample well, it can be challenging to get the timing of changing these settings correct. Scheduled MRM is one solution to switch between different MRM transition per well, but it conventionally requires an upfront calculation of when the MRM should be changed. Increasing the number of MRM transitions to be monitored simultaneously can also expand the tolerance to the time setting. However, the dwell time and, therefore, the number of points acquired across an intensity versus time mass peak are sacrificed due to the sharp peak shape created by AEMS.

[0086] As a result, additional systems and methods are needed to reduce the number of MRM transitions monitored at any one time in an AEMS experiment in order to increase the number of measurements that can be made across an intensity versus time mass peak.

[0087] FIGS. 3-6 illustrate how not being able to calculate in advance the delay time from when an acoustic ejection occurs to when a signal from an analyte is detected in AEMS affects the alignment of MS measurements with samples.

[0088] FIG. 3 is an exemplary plot 300 showing the proper alignment of detected peaks with ejection times from an ADE device timing file. In plot 300, for example, intensity versus time peaks 311, 312, 313, 314, and 315 are properly aligned with ejection times depicted by arrows 321, 322, 323, 324 and 325, respectively.

[0089] However, if some peaks, particularly the first one or more peaks, are missing, this alignment may be confounded.

[0090] FIG. 4 is an exemplary plot 400 showing three peaks detected over time by a mass spectrometer for analytes from four different samples sequentially provided to the mass spectrometer by an ADE device and an OPI. In plot 400, intensity versus time peaks 412, 413, and 414 are detected by the mass spectrometer for three different samples ejected into the OPI by the ADE device. At time 411, a peak is missing for a fourth sample, which was the first sample that the ADE device attempted to eject into the OPI.

[0091] Arrows 421, 422, 423, and 424 show the ejection times of the four different samples relative to time peaks 412, 413, and 414. In other words, the position of arrows 421, 422, 423, and 424 relative to time peaks 412, 413, and 414 shows the time delay between sample ejection by the ADE device and sample analysis by the mass spectrometer. Plot 400 shows that, because of the time delay between sample ejection and analysis, arrows 421, 422, 423, and 424 must be aligned with peaks 412, 413, and 414 in order to determine the sample corresponding to each peak. The ejection times depicted by arrows 421, 422, 423, and 424 are stored in the timing file by the ADE device, for example.

[0092] FIG. 5 is an exemplary plot 500 showing how the mismatch in the number of peaks detected and the number of ejection times in FIG. 4 results in different alignments of the four different sample ejections with the three peaks of FIG. 4. In alignment 510, ejection times depicted by arrows 422, 423, and 424 are aligned with peaks 412, 413, and 414, respectively. In alignment 520, however, ejection times depicted by arrows 421, 422, and 423 are aligned with peaks 412, 413, and 414, respectively. Both alignments are possible.

[0093] As a result, plot 500 shows that the determination of the samples corresponding to peaks 412, 413, and 414 is confounded by the missing peak at time 411. In other words, a missing peak at time 411 makes it difficult to determine the identity of peaks 412, 413, and 414, potentially resulting in incorrect results for the four samples and all samples following those four samples.

[0094] FIG. 6 is an exemplary plot 600 showing detected peaks misaligned with ejection times due to a missing peak and a low-intensity peak. In plot 600, there is a missing peak at time 610 and there is a low-intensity peak at time 620 relative to the other peaks detected. As a result, the alignment of the ejection times represented by arrows 630 is off or shifted by two samples or wells. Consequently, the data reported for all 24 wells is incorrect due to the misalignment which leads to the incorrect MRM being measured for a given well.

[0095] FIGS. 4-6 show that additional AEMS systems and methods are needed to calculate the delay time from when an acoustic ejection occurs to when a signal from an analyte is detected in AEMS.

[0096] In various embodiments, the delay time from when an acoustic ejection occurs to when a signal from an analyte is detected is calculated in real-time using multiple reaction monitoring (MRM) transitions. More specifically, the next MRM transitions to be monitored in an AEMS experiment are selected in real-time based on the last transition detected and the order in which the samples are ejected. At least three different embodiments are possible for selecting the next MRM transitions to be monitored using the sample order.

[0097] In a first embodiment, each MRM transition corresponding to each sample ejected is a scout transition. A scout transition is a transition that identifies or triggers the next one or more transitions to be executed. In other words, a scout transition identifies additional transitions that should be monitored together.

[0098] Conventionally, MRM transitions have been triggered based on a retention time or retention time range. This is referred to as scheduled MRM. In order to make MRM methods less sensitive to changes in the retention time for compounds eluting from a liquid chromatography (LC) column, scout transitions were developed. Scout transitions simply require knowing what transitions should be turned on together rather than knowing the exact time of when a transition needs to be turned on.

[0099] AEMS experiments are very different from LC-MS experiments. There is no column. Compounds do not have an elution time. The samples come out of the transfer tube in the same order that they were acoustically injected into the OPI port. As a result, scout MRM transitions are well suited for use in AEMS experiments.

[0100] For example, since the samples come out in the same order they were injected in AEMS experiments, a scout transition A1 that is used to detect a compound of sample 1 can be used to turn on or trigger a scout transition A2 that is used to detect a compound of sample 2. Similarly, scout transition A2 can be used to trigger a scout transition A3 that is used to detect a compound of sample 3 and so forth. As a result, there is a rolling use of scout transitions.

[0101] As described above, however, some sample wells may be empty or the compounds of some sample wells may not be detected. Therefore, in various embodiments, a group of scout transitions is executed during each cycle of the mass spectrometer to handle missing samples. For example, a detection of scout transition A1 can trigger the addition of two or more transitions to the group, such as transitions A2, A3, and A4.

[0102] The detection of a scout transition can also trigger the removal of another scout transition from the group. For example, the detection of transition A2 results in the removal of transition A1 from the group. If for transition A2 a sample is missing from the sample well, then detection of transition A3 triggers the removal of both transition A1 and transition A2 from the group. Also, the detection of transition A3 triggers transitions A4, A5, and A6. If transition A4 is already a member of the group, then only transitions A5 and A6 are added to the group.

[0103] FIG. 7 is an exemplary diagram 700 showing how n precursor ion to product ion MRM transitions correspond to n samples of an AEMS experiment, in accordance with various embodiments. Specifically, transitions A1 to An correspond to samples S1 to Sn. In other words, transitions A1 to An are developed specifically to detect compounds in samples

[0104] S1 to Sn, respectively. Each transition of transitions A1 to An is a scout transition and identifies three additional transitions to be triggered by the scout transition.

[0105] For example, if the product ion of transition A3 is detected above a certain intensity level, then transition A3 is detected. If transition A3 is detected, then transitions A4, A5, and A6 are triggered. Triggered means, for example, adding the identified transitions to the group of transitions being executed. Also, the detection of transition A3 can trigger the removal of all transitions preceding A3 from the group of transitions being executed.

[0106] FIG. 8 is an exemplary diagram 800 showing how precursor ion to product ion MRM transitions are added to and removed from a group of transitions as transitions are detected, in accordance with various embodiments. FIG. 8 shows a series of cycles for a mass spectrometer. During each cycle, each of the transitions in the group of transitions is executed or monitored. The group of transitions is enclosed in the ellipse shown in FIG. 8.

[0107] The group of transitions includes four transitions. Initially, the first four transitions corresponding to the first four samples ejected by the AEMS system are selected for the group of transitions. Therefore, in cycle 1, the group of transitions includes transitions A1, A2, A3, and A4.

[0108] At cycle 5, the product ion of transition A3 is detected above a certain threshold intensity. Transitions A1 and A2 are removed from the group. Each of the transitions of the group is a scout transition. As a result, transition A3 triggers transitions A4, A5, and A6. Transition A4 is already in the group, so only transitions A5 and A6 are added to the group replacing transitions A1 and A2.

[0109] Because transition A3 was detected before transitions A1 and A2, the samples corresponding to these transitions were either missing from their sample wells or their intensities were too low to be recorded. Using a group of transitions allows transition A3 to be detected even though transitions A1 and A2 are missing.

[0110] At cycle 15, the product ion of transition A4 is detected above a certain threshold intensity. Transition A3 is removed from the group. Transition A4 triggers transitions A5, A6, and A7. Transitions A5 and A6 are already in the group, so only transition A7 is added to the group replacing transition A3.

[0111] For the ten cycles between cycle 5 and cycle 15, as many as ten measurements for transition A3 may have been obtained. As a result, as many as 10 points may have been collected across an intensity versus time peak for the sample corresponding to transition A3, for example.

[0112] At cycle 25, the product ion of transition A5 is detected above a certain threshold intensity. Transition A4 is removed from the group. Transition A5 triggers transitions A6, A7, and A8. Transitions A6 and A7 are already in the group, so only transition A8 is added to the group replacing transition A4. Again, between cycle 15 and cycle 25, as many as 10 points may have been collected across an intensity versus time peak for the sample corresponding to transition A4, for example.

[0113] At cycle 35, the product ion of transition A8 is detected above a certain threshold intensity. Transitions A5, A6, and A7 are removed from the group. Transition A8 triggers transitions A9, A10, and A11, which are all added to the group replacing transitions A5, A6, and A7. Again, because transition A8 was detected before transitions A6 and A7, the samples corresponding to these transitions were either missing from their sample wells or their intensities were too low to be recorded.

[0114] In a second embodiment, a first sample and every mth sample following the first sample include marker compounds. Marker compounds are compounds provided at a concentration high enough to produce a known measured intensity. In other words, samples are provided with marker compounds to ensure that the samples are detected. As a result, scout transitions are only provided for the marker samples. Each marker transition then triggers the next m transitions.

[0115] FIG. 9 is an exemplary diagram 900 showing how MRM transitions that include a first marker transition and then another marker transition every m transitions after the first transition correspond to samples including corresponding marker samples of an AEMS experiment, in accordance with various embodiments. Specifically, transitions M1 to An correspond to samples Y1 to Sn. The first sample, Y1, and every mth sample following the first sample (every 4.sup.th sample following Y1) include marker compounds, such as sample Y5. The first marker transition, M1, and then every m transitions after the first transition (every 4 transitions after M1) correspond to samples with one or more marker compounds.

[0116] Each marker transition, M1, M5, . . . , is a scout transition. Each marker transition triggers the following m transitions corresponding to the following samples in the sample order. For example, transition M1 triggers transitions A2, A3, A4, and M5 in FIG. 9. Transitions that are not marker transitions are not scout transitions. As a result, transitions A2, A3, and A4 do not trigger other transitions. Only marker transitions are scout transitions because they are known to produce a reliable measured signal.

[0117] FIG. 9 shows that if the product ion of marker transition M1 is detected above a certain intensity level, then marker transition M1 is detected. If marker transition M1 is detected, then transitions A2, A3, A4 and M5 are triggered. Note that each marker transition triggers another marker transition.

[0118] Again, triggered means, for example, adding the identified transitions to the group of transitions being executed. The detection of marker transition M1, for example, triggers its removal from the group of transitions being executed. After being detected above a certain intensity threshold, marker transitions, such as M1, can continue to be monitored but do not necessarily need continued monitoring since their primary function is to “mark” the location in the series of samples.

[0119] FIG. 10 is an exemplary diagram 1000 showing how MRM transitions that include marker transitions are added to and removed from a group of transitions as marker transitions are detected, in accordance with various embodiments. FIG. 10 shows a series of cycles for a mass spectrometer. During each cycle, each of the transitions in the group of transitions is executed or monitored. The group of transitions is enclosed in the ellipse shown in FIG. 10.

[0120] The group of transitions can include four transitions, for example. Initially, only the first marker transition corresponding to the first sample, which includes one or more marker compounds, is selected for the group of transitions. In FIG. 10, therefore, the group of transitions initially only includes marker transition M1 at cycle 1.

[0121] At cycle 5, the product ion of marker transition M1 is detected above a certain threshold intensity. Marker transition M1 is removed from the group. Transition M1 is a scout transition. As a result, the detection of transition M1 triggers transitions A2, A3, A4, and M5. These transitions are, therefore, added to the group.

[0122] For the 30 cycles between cycle 5 and cycle 35, as many as ten measurements may have been obtained for each of transitions A2, A3, and A4. As a result, as many as 10 points may have been collected across an intensity versus time peak for the samples corresponding to transitions A2, A3, and A4, for example.

[0123] At cycle 35, the product ion of marker transition M5 is detected above a certain threshold intensity. Again, marker transition M5 is removed from the group. The detection of marker transition M5 triggers transitions A6, A7, A8, and M9. These transitions are, therefore, added to the group.

[0124] FIGS. 7-10 show different ways scout transitions and the sample order can be used to make sure that intensity versus time peaks are properly aligned with their corresponding samples. Scout transitions have been used before to eliminate the need for scheduled MRM transitions.

[0125] For example, U.S. Pat. No. 10,566,178 (hereinafter the “'178 Patent”) describes using sentinel transitions to overcome the limitations of scheduled MRM. The '178 Patent provides that in scheduled MRM, each MRM transition defined in the workflow has a retention time associated it. Consequently, each MRM transition is monitored only around its retention time. Therefore, by scheduling the MRM transitions, the maximum number of transitions that are monitored at any point in time during an acquisition is optimized. In other words, not all MRM transitions need to be monitored for the entire acquisition time. This approach provides more data points across an elution peak and, therefore, better precision, sensitivity, and accuracy.

[0126] However, scheduled MRM has an important limitation. It is dependent on the accuracy and absolute value of the retention time used for each transition. Whenever the separation device changes or the gradient of separation changes, the retention time for each transition must be recomputed. This becomes particularly cumbersome when workflows include thousands of MRM transitions. This also makes it difficult to use scheduled MRM workflows across separation devices produced by different manufacturers that have different elution rates.

[0127] The '178 Patent provides systems and methods to limit the number of MRM transitions monitored at any one time without requiring the re-computation of retention time for each MRM transition, whenever the separation device changes or the gradient of separation changes. In these systems and methods, the MRM transitions to be used for an entire acquisition are ordered according to an expected retention time. The ordered MRM transitions are then divided into contiguous groups with different expected retention time ranges. In each group, at least one transition is selected as a sentinel transition. The sentinel transition in each group is used to identify the next group and trigger it for monitoring.

[0128] During acquisition, a first group of transitions is selected for monitoring. This is, for example, the group with the earliest expected retention time. When at least one sentinel transition in the first group is detected by the tandem mass spectrometer, the next group of transitions identified by the at least one sentinel transition is added to the list of transitions monitored by the tandem mass spectrometer. In other words, at least one sentinel transition in each group is used to trigger the transitions in the next contiguous group.

[0129] A group of transitions can also be removed from monitoring. For example, once at least one sentinel transition in the next contiguous group is detected, the transitions in the first group can be removed from monitoring.

[0130] As a result, by using sentinel transitions to trigger the addition and subtraction of MRM transitions from monitoring the overall number of MRM transitions being monitored at any one time is reduced. In addition, because the groups of transitions are not dependent on a specific retention time, workflows based on these systems and methods can be used without modification whenever the separation device changes or the gradient of separation changes.

[0131] In various embodiments, the systems and methods described herein provide a significant improvement over the '178 Patent. For example, these systems and methods are directed to using the sample order rather than retention time ranges to reduce the number of MRM transitions monitored at any one time and to align intensity versus time mass peaks with samples.

[0132] In addition, the use of the sample order is such an important improvement that it also allows a modified form of scheduled MRM to be used with a different type of scout or sentinel transition that simply triggers scheduled MRM. In the third embodiment mentioned above, a group of transitions corresponding to the first few samples is monitored. Once a transition of the group is detected, the time between sample ejection and mass analysis is known from the detection time of the transition and the ejection time of its corresponding sample.

[0133] In an AEMS system, the time between sample ejections is extremely precise and, once it is known, the time between sample ejection and mass analysis does not significantly vary. As a result, after the detection of the time between sample ejection and mass analysis, the experiment can proceed using a single MRM transition corresponding to each next sample in the sample order. The scheduled time for each scheduled transition is then the sum of the time between sample ejection and mass analysis and the ejection time of each next sample.

[0134] FIG. 11 is an exemplary diagram 1100 showing how a group of MRM transitions corresponding to the first few samples can trigger scheduled MRM of the remaining transitions corresponding to the remaining samples, in accordance with various embodiments. Again, transitions A1 to An correspond to samples S1 to Sn. To begin, a group of m transitions corresponding to the first m samples of the sample order is selected. For example, in FIG. 11, four transitions, A1 to A4, corresponding to samples S1 to S4, are selected for the group of transitions.

[0135] The group of m transitions is then monitored. When a transition from the group is detected, the sample of the transition detected is identified. For example, in FIG. 11, if transition A3 is detected, then sample S3 is identified. The ejection time of sample S3 is known from the timing file, as described above. From the detection time of detected transition A3 and the ejection time of sample S3, the time between the ejection of a sample and the mass analysis of a product ion, ΔT, is calculated.

[0136] Once ΔT is determined, scheduled MRM for the remaining transitions corresponding to the remaining samples is triggered. For example, in FIG. 11, when transition A3 is detected, scheduled MRM is triggered for the remaining transitions A4 to An. The scheduled time for each scheduled transition is then the sum of ΔT and the ejection time of each next sample. For example, in FIG. 11, transition A4 is scheduled at a time equal to the sum of ΔT and the ejection time of sample S4.

[0137] FIG. 12 is an exemplary diagram 1200 showing how scheduled MRM is triggered by a transition of a group of MRM transitions corresponding to the first few samples and how scheduled MRM proceeds on the remaining transitions corresponding to the remaining samples, in accordance with various embodiments. Again, FIG. 12 shows a series of cycles for a mass spectrometer. During each initial cycle, each of the transitions in an initial group of transitions is executed or monitored. The group of transitions is enclosed in the ellipse shown in FIG. 12.

[0138] The group of transitions can include four transitions, for example. In this case, transitions A1 to A4 corresponding to the first four samples, S1 to S4, respectively, are

[0139] selected for the group. As a result, at cycle 1, the group includes transitions A1 to A4. Each of these transitions is monitored at each cycle until a transition is detected.

[0140] At cycle 5, the product ion of transition A3 is detected above a certain threshold intensity. Like all transitions of the group, transition A3 triggers scheduled MRM for transitions corresponding to the remaining samples. For example, transition A4 is scheduled to be monitored between cycles 15 and 24, transition A5 is scheduled to be monitored between cycles 25 and 34, and transition A6 is scheduled to be monitored between cycles 35 and 44 as shown in FIG. 11.

[0141] Detected transition A3 continues to be monitored between cycles 6 and 14 in order to collect points across an intensity versus time peak for the sample corresponding to transition A3. In various embodiments, transition A3 may continue to be monitored along with other members of the group. In various alternative embodiments, the detection of transition A3 also triggers its scheduled monitoring. In other words, once transition A3 is detected, only transition A3 is monitored for a certain time period or number of cycles.

[0142] Note that the time period or number of cycles during which the transition is monitored using scheduled MRM is determined from the time between sample ejections. Again, this time is known from the timing file provided by the ADE device.

[0143] Although the above embodiments have been described in relation to AEMS, these embodiments are not limited to AEMS. For example, these embodiments can be equally applied to any system or method for selecting MRM transitions using any sample introduction system coupled to a mass spectrometer that ejects samples in a known sample order, records the sample ejection times of the ejections performed by the sample introduction system, and has a consistent delay time from ejection to mass analysis.

System for Selecting the Next MRM Transitions

[0144] FIG. 13 is a schematic diagram 1300 of a system for selecting the next MRM transitions to be monitored in a high-throughput sample introduction coupled mass spectrometry experiment based on the transition detected and the order of the samples, in accordance with various embodiments. The system of FIG. 13 includes sample introduction system 1301, tandem mass spectrometer 1302 (e.g. triple quadrupole mass spectrometer), and processor 1303.

[0145] Sample introduction system 1301 ejects each sample of a series of samples 1311 at an ejection time and according to a sample order. A plurality of ejection times 1312 corresponding to series of samples 1311 is produced. Sample introduction system 1301 also ionizes each ejected sample of series of samples 1311, producing an ion beam 1331. Tandem mass spectrometer 1302 receives ion beam 1331.

[0146] Processor 1303 receives a list 1313 of different sets of one or more MRM precursor ion to product ion transitions. For example, Set1 of list 1313 includes two MRM transitions, Set2 includes three MRM transitions, and Set3 includes one MRM transition. Each set of list 1313 corresponds to a different sample of series 1311. Processor 1303 selects a group of one or more different sets from list 1313. Initially, each set transition selected for the group corresponds to a different sample of one or more first samples of series 1311.

[0147] Processor 1303 instructs tandem mass spectrometer 1302 to execute each transition of each set of the group on ion beam 1331 during each cycle of a plurality of cycles until a transition of the group is detected. For each transition of each set of the group, tandem mass spectrometer 1302 selects and fragments a precursor ion of each transition and mass analyzes a small mass-to-charge ratio (m/z) range around the m/z of a product ion of each transition to determine if the product ion of each transition is detected. When a transition 1340 of a set of the group is detected, processor 1303 selects one or more next sets 1350 from list 1313 to be monitored using detected transition 1340 and the sample order of series 1311.

[0148] In various embodiment, each set of list 1313 includes a single transition. In other words, each set is equivalent to one transition as is shown in FIGS. 7-12.

[0149] In various embodiments, the sample order is the order in which samples are ejected ejected from their sample wells into sample introduction system 1301.

[0150] In various embodiments, one or more next sets 1350 are selected from list 1313 using scout transitions, such as those described in regard to FIGS. 7-8. Specifically, list 1313 is ordered according to the sample order of series 1311. Each transition of each set of list 1313 is a scout transition that identifies one or more sets immediately following the set of the scout transition on list 1313. When a transition 1340 of a set of the group is detected, processor 1303 selects one or more next sets 1350 by selecting one or more sets identified by detected transition 1340 as immediately following the set of detected transition 1340 in list 1313.

[0151] In various embodiments, processor 1303 further adds the selected one or more sets to the group if any are not already part of the group. In addition, processor 1303 instructs tandem mass spectrometer 1302 to execute each transition of each set of the group on ion beam 1331 during each cycle of a plurality of cycles until a different transition of the group is detected.

[0152] In various embodiments, processor 1303 further removes any set of the group that precedes the set of detected transition 1340 on list 1313. This removal takes place before processor 1303 instructs tandem mass spectrometer 1302 to execute each transition of each set of the group on ion beam 1331 during each cycle of a plurality of cycles until a transition of a different set of the group is detected.

[0153] In various embodiments, one or more next sets 1350 are selected from list 1313 using marker transitions that are scout transitions, such as those described in regard to FIGS. 9-10. List 1313 is ordered according to the sample order of series 1311. A first set of list 1313 and every mth set following the first set are marker sets for samples that include marker ions. Every marker set includes one or more scout transitions that identify m set immediately following every marker set on list 1313. Processor 1303 initially selects only the first set for the group. When a transition 1340 of a set of the group is detected, processor 1303 selects one or more next set 1350 by selecting m sets identified by the detected marker transition.

[0154] In various embodiments, processor 1303 further performs a number of steps. A. Processor 1303 removes all sets from the group. B. Processor 1303 adds m sets identified by the detected marker transition to the group. C. Processor 1303 further instructs tandem mass spectrometer 1302 to execute each transition of each set of the group on ion beam 1331 during each cycle of a plurality of cycles until a marker transition of a set of the group is detected. Processor 1303 repeats steps A-C until all sets of list 1313 have been added to the group.

[0155] In various embodiments, one or more next sets 1350 are selected from list 1313 using transitions that trigger scheduled MRM, such as those described in regard to FIGS. 11-12. When a transition 1340 of a set of the group is detected, processor 1303 selects one or more next set 1350 from list 1313 to be monitored by first identifying a corresponding sample from the set of detected transition 1340. Next, processor 1303 identifies a plurality of next samples of series 1311 following the identified corresponding sample using the sample order of series 1311. Finally, processor 1303 selects a plurality of sets of list 1313 corresponding to the plurality of next samples as one or more next sets 1350.

[0156] In various embodiments, processor 1303 further instructs tandem mass spectrometer 1302 to schedule execution of each transition of each set of the plurality of sets based on an ejection time of a sample corresponding to each set, an ejection time of the corresponding sample from detected transition 1340, a detection time of detected transition 1340, and the sample order of series 1311.

[0157] In various embodiments, sample introduction system 1301 includes a surface analysis system. In various embodiments, the surface analysis system can be, but is not limited to, a matrix-assisted laser desorption/ionization WALDO device or a laser diode thermal desorption (LDTD) device.

[0158] In various embodiments, sample introduction system 1301 includes a flow injection device and an ion source device. For example, the flow injection device can be a timed valve device that injects sample into a flowing stream through a valve at each ejection time of plurality of ejection times 1312 and the ion source device ionizes samples of the flowing stream, producing ion beam 1331.

[0159] In various embodiments, the flow injection device can be a droplet dispenser that ejects series of samples 1311 as droplets into a flowing stream at each ejection time of plurality of ejection times 1312 and the ion source device ionizes samples of the flowing stream, producing ion beam 1331.

[0160] In various embodiments, and as shown in FIG. 13, the droplet dispenser includes ADE device 1310 that ejects series of samples 1311 as droplets into inlet 1321 of tube 1322 of OPI 1320. OPI 1320 OPI mixes the droplets of series of samples 1311 with a solvent in tube 1322 to form a series of analyte-solvent dilutions. OPI 1320 transfers the series of dilutions to outlet 1323 of tube 1322 of OPI 1320. Ion source device 1330 receives the series of dilutions and ionizes samples of the series of dilutions, producing ion beam 1331. Ion source device 1330 can be an electrospray ion source (ESI) device, for example. Ion source device 1330 is shown as part of tandem mass spectrometer 1302 in FIG. 13 but can be a separate device also.

[0161] Tandem mass spectrometer 1302 can be any type of mass spectrometer. Tandem mass spectrometer 1302 is shown as being a triple quadrupole mass analyzer, but tandem mass spectrometer 1302 can include any type of mass analyzer including for example a time-of-flight (ToF) mass analyzer.

[0162] In various embodiments, processor 1303 is used to send and receive instructions, control signals, and data to and from sample introduction system 1301 and tandem mass spectrometer 1302. Processor 1303 controls or provides instructions by, for example, controlling one or more voltage, current, or pressure sources (not shown). Processor 1303 can be a separate device as shown in FIG. 13 or can be a processor or controller of sample introduction system 1301 or tandem mass spectrometer 1302. Processor 1303 can be, but is not limited to, a controller, a computer, a microprocessor, the computer system of FIG. 2, or any device capable of sending and receiving control signals and data and analyzing data.

[0163] Note that terms “eject,” “ejection,” “ejection times,” and the like are used throughout this written description in reference to a sample introduction system. One of ordinary skill in the art can appreciate that other terms can also be used to describe the movement of sample from the sample introduction system, such as, but not limited to, terms like “inject,” “injection,” and “injection times.”

Method for Selecting the Next MRM Transitions

[0164] FIG. 14 is a flowchart showing a method 1400 for selecting the next MRM transitions to be monitored in a high-throughput sample introduction coupled mass spectrometry experiment based on the transition detected and the order of the samples, in accordance with various embodiments.

[0165] In step 1410 of method 1400, each sample of a series of samples is ejected at an ejection time and according to a sample order using a sample introduction system. A plurality of ejection times corresponding to the series is produced. Each ejected sample of the series is ionized using the sample introduction system, producing an ion beam.

[0166] In step 1420, the ion beam is received using a tandem mass spectrometer.

[0167] In step 1430, a list of different sets of one or more MRM precursor ion to product ion transitions is received using a processor. Each transition of the list corresponds to a different sample of the series.

[0168] In step 1440, a group of one or more different sets is selected from the list using the processor. Initially, each set transition selected for the group corresponds to a different sample of one or more first samples of the series.

[0169] In step 1450, the tandem mass spectrometer is instructed to execute each transition of each set of the group on the ion beam during each cycle of a plurality of cycles until a transition of a set of the group is detected using the processor.

[0170] In step 1460, when a transition of a set of the group is detected, one or more next sets are selected from the list to be monitored using the detected transition and the sample order using the processor.

Computer Program Product for Selecting the Next MRM Transitions

[0171] In various embodiments, computer program products include a tangible computer-readable storage medium whose contents include a program with instructions being executed on a processor so as to perform a method for selecting the next MRM transitions to be monitored in a high-throughput sample introduction coupled mass spectrometry experiment based on the transition detected and the order of the samples. This method is performed by a system that includes one or more distinct software modules.

[0172] FIG. 15 is a schematic diagram of a system 1500 that includes one or more distinct software modules that performs a method for selecting the next MRM transitions to be monitored in a high-throughput sample introduction coupled mass spectrometry experiment based on the transition detected and the order of the samples, in accordance with various embodiments. System 1500 includes control module 1510.

[0173] Control module 1510 instructs a sample introduction system to eject each sample of a series of samples at an ejection time and according to a sample order. A plurality of ejection times corresponding to the series is produced. Control module 1510 also instructs a sample introduction system to ionize each ejected sample of the series, producing an ion beam. Control module 1510 instructs a tandem mass spectrometer to receive the ion beam.

[0174] Control module 1510 receives a list of different sets of one or more MRM precursor ion to product ion transitions. Each transition of the list corresponds to a different sample of the series. Control module 1510 selects a group of one or more sets from the list. Initially, each set transition selected for the group corresponds to a different sample of one or more first samples of the series.

[0175] Control module 1510 instructs the tandem mass spectrometer to execute each transition of each set of the group on the ion beam during each cycle of a plurality of cycles until a transition of a set of the group is detected. When a transition of a set of the group is detected, control module 1510 selects one or more next sets from the list to be monitored using the detected transition and the sample order.

[0176] Further, in describing various embodiments, the specification may have presented a method and/or process as a particular sequence of steps. However, to the extent that the method or process does not rely on the particular order of steps set forth herein, the method or process should not be limited to the particular sequence of steps described. As one of ordinary skill in the art would appreciate, other sequences of steps may be possible. Therefore, the particular order of the steps set forth in the specification should not be construed as limitations on the claims. In addition, the claims directed to the method and/or process should not be limited to the performance of their steps in the order written, and one skilled in the art can readily appreciate that the sequences may be varied and still remain within the spirit and scope of the various embodiments.