The present invention provides systems and methods for optimizing the analysis of co-eluting precursor compounds during an analysis cycle of a tandem mass spectrometer system. For example, the present invention provides in different aspects: very fast switching between different MS/MS analyses (precursor ions) during a cycle; complex sampling patterns over a chromatographic peak using the fast switching capabilities; and collecting data for all compound ions of interest at different collision energies, but without having to vary the collision energy while a compound ion is investigated. One skilled in the art will appreciate that embodiments of the invention may be applied to different types of tandem spectrometers.
In one embodiment, collision cell 130 operates by sending compound ions 105 through a region containing a background gas, typically an inert gas, which causes compound ions 105 to fragment into smaller (fragment) ions 108, a process known in the art as collision-induced dissociation (CID). Other embodiments can use other collision cell types such as photoionization, surface ionization or electron impact. Collision cell 130 may have an energy setting that corresponds to the kinetic energy of compound ion 105. The kinetic energy may be controlled by varying a voltage, a pressure gradient, or other suitable environmental settings. A collision cell energy may also be varied by the pressure of the background gas.
Collision cell 130 may also focus the fragment ions 108 into the second mass analyzer MS-2. MS-2 is configured to filter out the fragment ions of interest, so that they may be detected by a detector 140. When mass analyzer MS-1 begins to analyze a new compound ion, the MS-1 setting is changed, for example a change to the mass-to-charge ratio (m/z) of the new compound ion to be filtered. The order of the compound ions measured is termed a sampling pattern. Herein, a compound ion may also be referred to as a precursor ion.
In one embodiment, MS-2 includes a time-of-flight (TOF) analyzer. MS-2 may alternatively include a magnetic sector device, quadrupole mass filter or other such means for obtaining a mass spectrum such that the operation is fast enough to allow sufficiently rapid sampling. In a tandem mass spectrometer, MS-1 and the collision cell typically includes one or more quadrupoles (such as in a QqTOF), but any other multipole or other suitable devices may be used. For example, some embodiments of collision cells may include ringstacks or other devices to confine and transmit ions in the presence of a collision gas. The collision cell also may only be run in an RF only mode, which only uses an AC potential, which is typically designated with the lower case “q”.
Control system 170 is provided to control overall operation of mass spectrometer device 100, including automatic tuning operations such as controlling focusing element 110, the energy of the collision cell 130, and controlling the operation of detector 140. For example, control system 170 automatically adjusts instrument control parameters, e.g. m/z settings, in one aspect. Control system 170 implements control logic that allows system 170 to receive user input and provide control signals to various system components. In certain aspects, control system 170 controls the sampling pattern of the compound ions 105.
In certain aspects, control system 170 includes a stand-alone computer system and/or an integrated intelligence module, such as a microprocessor, and associated interface circuitry for interfacing with the various systems and components of mass spectrometer device 100 as would be apparent to one skilled in the art. For example, control system 170 in one aspect includes interface circuitry for providing control signals to the different mass analyzers, and to the collision cell 130 for adjusting its energy. Control system 170 also typically includes circuitry for receiving data from the mass spectrometer system 100. The computer system (and/or the data-generation system) may include a computer readable medium, such as a hard disk drive or a device that reads a portable computer readable medium such as a CD or DVD reader, that is configured to store various computer code embodiments of the present invention. Control system 170 may be configured to run the computer code to execute various embodiment of the present invention. While control system 170 and mass spectrometer system 100 are shown as discrete systems, these systems may be an integrated system.
As mentioned above, in one embodiment, a QqTOF is used to implement embodiments of the present invention. In this embodiment, MS-2 of mass spectrometry system 100 is a TOF mass analyzer. For illustrative purposes, a QqTOF system will be used in the following discussion. However, it should be understood that aspects of the present invention also apply to other MS/MS spectrometry systems.
When used for MS-2, a TOF spectrometer differentiates among different fragment ions 108 based on the differences in time for the fragment ions to move from a starting point to detector 140. Ions with a higher mass arrive later than ions with a smaller mass. The ions are accelerated with a fixed electric field for a short period of time, thus creating a pulse of ions. For each pulse, the detector records a corresponding spectrum, called a transient. Typically, many transients are summed to create a mass spectrum.
Thus, during one analysis sub-cycle of a single QqTOF cycle, a specific compound (precursor) ion, such as type A, is chosen by MS-1. Compound ions A then move into the collision cell 130. In the collision cell, fragment ions are created from compound ions A. The fragment ions are then moved into the TOF analyzer at a steady rate to form a beam of fragment ions. A pulser applies an electric field at a set frequency, e.g. several kHz, which accelerates pulses of fragment ions that are each detected as a transient. Note that a reference of compound ion A corresponds to multiple ions of compound A.
As an illustration, assume that three compounds A, B, and C co-elude from a chromatograph during a single peak having a three second elution window. A single MS/MS analysis cycle that includes this elution window is used to analyze the compound ions A, B, and C. Thus, to perform an MS/MS analysis of A, B and C over the 3 second elution window, on average only one second is used to analyze each of the ions. The ions resulting from this elution window give an “ionic signal” that is analyzed by the mass analyzers. The remaining description will discuss the invention in terms of 3 precursors, however, it should be understood that the described embodiments and agents are applicable to analyzing 2, 3 or more compounds.
To improve coverage of the ionic signal, the above example is often changed using a different sampling pattern.
At times, additional information is required. For instance, using a range of collision cell energies during a sub-cycle may give more information. Varying the collision energy is particularly recommended when one analyzes unknown compounds for which one does not know the ideal collision energy. Varying the collision energies also provides a lower limit on the number of sub-cycles. Since the entire range of applicable collision cell energies is used during a single sub-cycle, a single sub-cycle must have a minimum number of transients in order to investigate every collision cell energy. Thus, as a species (compound ion) is filly investigated entirely before the next species are investigated, there are large contiguous time periods of the elution window that are not sampled for a particular compound ion. This poor coverage gives statistically inferior data.
For example, given the requirement of relatively long sub-cycles when multiple collision cell energies are investigated, the moment when a particular compound is present in the mass spectrum at high enough abundance could be missed. Thus, fully investigating a species before the next species is investigated can lead to the possibility that while species A was low and species B was high, A got sampled with varying collision energies until species B vanished. Such sampling patterns would cause a loss in sensitivity for carefully evaluating these particular compounds.
However with improved QqTOF systems that allow multi-channel rapid sampling, the chances to “catch” a compound when it is close to or at an apex value can be increased. For example, due to the increased sensitivity and increased speed of new Agilent QTOF systems, sophisticated sampling patterns can be handled over a narrow chromatographic peak (e.g., 2 to 3 seconds wide), which are, for instance, produced with Agilent's new HPLC-Chip cube. For example, switching the species rather than the collision energy offers much greater flexibility. In one embodiment, such complex sampling patterns are enabled by a very fast DSP-based sampling engine that allows not only to switch parameters, e.g., voltages for setting collision energy in the collision cell, during transient accumulation, but also to switch the compound ion to be investigated in MS/MS.
In a separate set of cycles, the compounds ions A, B, and C are analyzed a second time in a second set of sub-cycles. Returning to
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Compound ions analyzed in one set may not be analyzed in another set. For example, all of the compound ions may be analyzed in one set while only A and B are analyzed in a second set. Also, an additional compound ion may be measured only once in a cycle, such as for calibration purposes. Also, a set may have more than one sub-cycle that analyzes the same compound ion, as long as the full range of collision cell energies are not consecutively explored for that compound ion in that set.
In certain aspects, there is a benefit of only having to change the collision energy between two sub-cycles. For example, both sub-cycles 903 and 911 analyze precursor C. As these sub-cycles occur right after each other, then only the collision cell energy has to be changed to go from one sub-cycle to the other. This type of occurrence may even happen within the same set of sub-cycles, such as in the last set. In certain aspects, the sampling pattern can be highly complex and may need to be adjusted to the current situation. Factors that impact the choice of pattern include the signal strength of given ions at any given time and the knowledge of a reasonable range of collision energy.
One skilled in the art will recognize that there are other combinations of the number of transients collected for a sub-cycle, the order of the compound ions that are analyzed within a set of sub-cycles, the number of sub-cycles within a set and distribution of collision energies that are possible. In one aspect, the number of sub-cycles in a set of sub-cycles equals the number of precursor ions to be analyzed, e.g., the number of co-eluting precursor compounds.
To accomplish fast switching, a user sets up and starts an “Auto” or “Targeted” MS/MS cycle via the PC application. This information is sent to embedded processor 1010 via the interface which tells the firmware to start fast switching cycles. Fast MS/MS switching control of the mass spectrometer hardware is performed by DSP 1015, which is connected to embedded processor 1010 via a high speed interface, such as the peripheral component interconnect (PCI) interface. DSP 1015 controls the mass spectrometer by sending signals to a main board 1025. Main board 1025 may contain high voltage circuitry for the mass analyzers and the collision cell, as well as contain data acquisition circuitry for receiving data from a detector of the mass spectrometer. For example, in one aspect includes circuitry that creates voltages to steer and set the collision energy of the beam. It sends commands to power amplifier that drive the QTOF's quad mass analyzer (first mass filter in a QTOF). An “acquisition card” including detection element(s), or other detector devices, detects ions and creates spectra by summing “transients”.
For each fast switching cycle, the firmware on embedded processor 1010 creates a complete package of parameters needed by DSP 1015 to perform the fast MS/MS switching cycle. This complete package of data includes all cycle parameters, such as those from cycles 500-900, and all hardware parameters which need to be changed at each sub-cycle. In one embodiment, the firmware provides all the cycle data at once to DSP 1015. Since DSP gets all the cycle data at once, the latency between each sub-cycle can be minimized. Processor 1010 can receive mass spectrum data from DSP 1015 at every measurement step, e.g., cycle, and provide the results to the PC application software via the interface. The final results are displayed on a monitor and/or stored to memory, e.g., in a file, without missing a cycle.
In a “Targeted” mode, the user inputs the specific sampling pattern to be used. In the “Auto” mode, a pre-scan is made by the mass spectrometer. In one aspect, in this pre-scan, the mass spectrometer only analyzes the compound ions from the chromatograph, but does not use the collision cell to create fragment ions. Thus, this is an MS mode. The mass spectrometer is used as a tandem mass spectrometer when fragment ions are analyzed by a second mass analyzer, termed an MS/MS mode. The information obtained from the pre-scan is used, e.g. by embedded processor 1010, to create the sampling pattern parameters needed by DSP 1015 to perform the fast MS/MS switching cycle. For example, a pre-scan in certain aspects tells the firmware what ions are present in the sample; based on this input, decisions are made to influence any further cycles. Also, control system 1000 is capable of displaying “real time” waveform data as the mass spectrometer 1005 is switched between MS mode and MS/MS mode when running in “Auto” mode.
In “Auto” mode, a determination could be made of the number of co-eluting precursor compounds that are of interest; an appropriate number of collision energies for each co-eluting precursor compound of interest; the number of sets, the number of sub-cycles for each set, the compound ion to be analyzed for each sub-cycle, the number of transients for each sub-cycle, and/or the collision cell energy for each sub-cycle.
In step 1101, the DSP receives a sampling pattern from the embedded processor. In step 1102, the DSP allocates a memory slot 1040 in DRAM 1030 for each compound ion to be analyzed. DRAM 1030 may be any suitable memory device that is readable and writeable, such as SDRAM or flash memory. In step 1103, DSP implements the settings for a sub-cycle, e.g., “500 transients for ion A using a given fixed collision energy CE1(A)”, and then starts the transient accumulation sub-cycle. In step 1104, after the scan for the sub-cycle finishes, DSP 1015 moves data from data acquisition board 1030 to DRAM ion A slot.
In step 1105, the implementation of the other sub-cycles, such as 602 and 603, and the movement of the resulting data into the DRAM ion B slot and the DRAM ion C slot are respectively done. Accordingly, these steps include implementing the settings for “2,000 transients for ion B using a given fixed collision energy CE1(B)” and then starting a transient accumulation sub-cycle. After the scan finishes, DSP moves data from data acquisition board to DRAM ion B slot. DSP then implements “500 transients for C using a given fixed collision energy CE1(C)” and then starts a transient accumulation sub-cycle. After that scan finishes, the DSP moves data from data acquisition board 1030 to DRAM ion C slot.
In step 1106, DSP implements “500 transients for A using a given fixed collision energy CE2(A)” and starts a transient accumulation sub-cycle. In step 1107, after the scan finishes, DSP 1015 moves data from data acquisition board 1030 and sums this data with the data already in DRAM ion A slot. In step 1108, the implementation of the remaining sub-cycles is done and the resulting data is moved into and summed with the data already in the appropriate DRAM ion slot.
Code for implementing methods described herein, and other control logic, may be provided to control systems, such as systems 170 and 800, using any means of communicating such logic, e.g., via a computer network, via a keyboard, mouse, or other input device, on a portable medium such as a CD, DVD, or floppy disk, or on a hard-wired medium such as a RAM, ROM, ASIC or other similar device.
While the invention has been described by way of example and in terms of the specific embodiments, it is to be understood that the invention is not limited to the disclosed embodiments. To the contrary, it is intended to cover various modifications and similar arrangements as would be apparent to those skilled in the art. One skilled in the art will recognize the many ways that the aforementioned methods and systems may be combined to produce different embodiments of the present invention. Therefore, the scope of the appended claims should be accorded the broadest interpretation so as to encompass all such modifications and similar arrangements.