PRECURSORS

GOVERNMENT LICENSE RIGHTS

[0001] This invention was made with government support under Grant No. CA201225 awarded by National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

[0002] Tandem mass spectrometry (MS/MS) is a powerful tool for structural identification of ions and is often used in untargeted mass spectrometry “-omics” methods (e.g., proteomics, metabolomics), as MS/MS can provide the information needed to correlate a signal at a given mass-to- charge ratio to a specific molecule within a sample.

[0003] Conventional tandem mass spectrometry data dependent precursor selection methods use total abundance as their sole criteria for selection. In such conventionally known methods, a survey scan is performed for all precursor ions, with the most abundant precursor ions being selected for MS/MS. This mode of operation is commonly referred to as “TopN”, where “N” represents the number of precursor ions selected from a survey scan.

[0004] However, in a so-called “-omics” analysis, the most abundant precursor ions, which would be those selected using the TopN approach, are not necessarily indicative of the presence of an abnormal (e.g., disease) condition in the sample, or specimen. Changes in lipid metabolism have been found to often be indicative of disease states within the host. Examples include increased abundance of triglycerides and cholesteryl esters in cardiovascular disease and aberrant phospholipid metabolism in multiple cancers. Lipid metabolism is also involved in Alzheimer’s, Parkinson’s, and Niemann-Pick diseases. Highlighting the need for further understanding of the role of each lipid species in disease and metabolism, a decrease in CE (22:6) trends closely with progression Alzheimer's disease, but is also considered to be indicative as a risk factor for cardiovascular disease when elevated. Conventional lipidomics methods allow for identification of aberrant lipid metabolism in diseased samples, but require that healthy, or control, samples be analyzed to serve as a reference set of values for differential measurements. Alternatively, threshold concentrations for diagnosis can be determined for a given condition and corresponding instrument responses validated by calibration methods. [0005] When considering new applications of such conventional lipidomics methods, the process of determining a clinically significant threshold concentration of a molecule (e.g., a precursor ion) for a given medical condition requires a lengthy validation period when compared to the use of a control sample, especially when the large number of potentially relevant lipid species is considered. As such, a need exists for identifying the presence of specific precursor ions, or a difference in precursor ions between two samples, in order to select the relevant precursor ions in order to perform the relevant “-omics” analysis. SUMMARY

[0006] According to a first example aspect, a method for performing differential abundance analysis comprises providing a multi-emitter ionization source comprising a first emitter, which is provided with a first specimen, and a second emitter, which is provided with a second specimen, arranging the first and second emitters adjacent to an inlet of a device for determining a quantity of analytes in a specimen introduced therein, connecting a power supply to the inlet of the device to apply a substantially constant voltage to the inlet, connecting a first power supply to the first emitter, connecting a second power supply to the second emitter, applying a first voltage to the first emitter and a second voltage to the second emitter, which causes an emission spray of only the first specimen from the first emitter towards the inlet of the device, applying the first voltage to the second emitter and the second voltage to the first emitter, which causes an emission spray of only the second specimen from the second emitter towards the inlet of the device, and determining a differential abundance for analytes contained in the first and/or second specimens.

[0007] In some embodiments, a system for performing differential abundance analysis comprises a multi-emitter ionization source comprising a first emitter, which is provided with a first specimen, and a second emitter, which is provided with a second specimen, a device for determining a quantity of analytes in a specimen introduced therein, wherein the first and second emitters are arranged adjacent to an inlet of the device, a power supply connected to the inlet of the system, the power supply being configured to apply a voltage to the inlet, a first power supply connected to the first emitter, and a second power supply connected to the second emitter, wherein the first power supply is configured to apply a first voltage to the first emitter and the second power supply is configured to apply a second voltage to the second emitter, which causes an emission spray of only the first specimen from the first emitter towards the inlet of the device, wherein the second power supply is configured to apply the first voltage to the second emitter and the first power supply is configured to apply a second voltage to the first emitter, which causes an emission spray of only the second specimen from the second emitter towards the inlet of the device, and wherein the device is configured to determine a differential abundance for analytes contained in the first and/or second specimens.

[0008] In some embodiments of the method, the first power supply is operable independent of both the power supply connected to the inlet of the device and the second power supply.

[0009] In some embodiments of the method, the device comprises a tandem mass spectrometer, the method comprising: when the activation voltage is applied to the first emitter, acquiring a mass spectrum of the first specimen; and when the activation voltage is applied to the second emitter, acquiring a mass spectrum of the second specimen. [0010] In some embodiments of the method, the activation voltage is applied to the first emitter at different times from when the activation voltage is applied to the second emitter.

[0011] In some embodiments, the method comprises calculating a ratio spectrum by dividing the mass spectrum of the first specimen by the mass spectrum of the second specimen for each analyte.

[0012] In some embodiments, the method comprises, for values in the ratio spectrum that are less than 1 , changing each value thereof to a reciprocal value to determine a DiffN spectrum.

[0013] In some embodiments of the method, each value in the ratio spectrum corresponds to an analyte present in the first specimen and/or the second specimen.

[0014] In some embodiments, the method comprises selecting N largest values in the DiffN spectrum, wherein N is a value between 1 and a maximum number of peaks in the DiffN spectrum.

[0015] In some embodiments, the method comprises acquiring a tandem mass spectrum for each of the N largest values in the DiffN spectrum.

[0016] In some embodiments, the method comprises waiting for the tandem mass spectrometer to acquire a number of mass spectra before switching the first power supply from the activation voltage to the inactivation voltage and the second power supply from the inactivation voltage to the activation voltage or before switching the first power supply from the inactivation voltage to the activation voltage and the second power supply form the activation voltage to the inactivation voltage. [0017] In some embodiments, the method comprises averaging results of the tandem mass spectrometer over the number of mass spectra accumulations that occur between when voltage of the first and second power supplies are switched.

[0018] In some embodiments of the method, the first specimen comprises a control sample and the second specimen comprises a sample suspected of having an abnormal abundance of one or more analytes.

[0019] In some embodiments of the method, the abnormal abundance of one or more analytes is indicative of the presence of a disease in the subject from which the sample for the second specimen was obtained.

[0020] In some embodiments of the method, the subject is a human being.

[0021] In some embodiments, the method comprises: determining an abundance for analytes contained within the first specimen; determining an abundance for analytes contained within the second specimen; comparing a relative abundance for a plurality of analytes between the first and second specimens; and selecting a quantity of the plurality of analytes between the first and second specimens based on the relative abundance for the plurality of analytes.

[0022] In some embodiments of the method, comparing the relative abundance between the first and second specimens for the quantity of analytes selected is performed for the quantity of analytes having a greatest differential abundance between the first and second specimens.

[0023] In some embodiments of the method, the multi-emitter ionization source comprises a nanospray or electrospray source.

[0024] In some embodiments of the method, the multi-emitter ionization source generates alternating emissions of the first specimen from the first emitter and the second specimen from the second emitter using a constant pressure for the first and second specimens within the first and second emitters, respectively.

[0025] In some embodiments of the method, emission of the first specimen from the first emitter and emission of the second specimen from the second emitter do not interfere with each other.

[0026] According to a second example aspect, a system for performing differential abundance analysis is provided, the system comprising: a multi emitter ionization source comprising a first emitter having a first specimen provided therein and a second emitter having a second specimen provided therein; a device for determining a quantity of analytes in a specimen introduced therein, wherein the first and second emitters are arranged adjacent to an inlet of the device; a power supply connected to the inlet of the system, the power supply being configured to apply a voltage to the inlet; a first power supply connected to the first emitter; and a second power supply connected to the second emitter; wherein the first power supply is configured to apply an activation voltage to the first emitter, such that the first specimen is emitted from the first emitter into the inlet of the device; wherein the second power supply is configured to apply an activation voltage to the second emitter, such that the second specimen is emitted from the second emitter into the inlet of the device; and wherein the device is configured to determine a differential abundance for one or more analytes contained within the first and/or second specimens.

[0027] In some embodiments of the system, the first power supply is configured to switch between applying the activation voltage and an inhibiting voltage to the first emitter in an alternating pattern, wherein, when the activation voltage is applied to the first emitter, the first specimen is emitted from the first emitter into the inlet of the system and, when the inhibiting voltage is applied to the first emitter, emission of the first specimen from the first emitter is inhibited and/or stopped; the second power supply is configured to switch between applying the activation voltage and the inhibiting voltage to the second emitter in an alternating pattern, such that, when the activation voltage is applied to the first emitter, the first specimen is emitted from the first emitter into the inlet of the system and, when the activation voltage is applied to the second emitter, the second specimen is emitted from the second emitter into the inlet of the system; and, when the activation voltage is not applied to the first or second emitters, the first or second emitters do not emit any of the first or second specimens, respectively.

[0028] In some embodiments of the system, the first power supply is operable independent of both the power supply connected to the inlet of the device and the second power supply.

[0029] In some embodiments of the system, the device comprises a tandem mass spectrometer; when the activation voltage is applied to the first emitter, the tandem mass spectrometer is configured to acquire a mass spectrum of the first specimen; and, when the activation voltage is applied to the second emitter, the tandem mass spectrometer is configured to acquire a mass spectrum of the second specimen.

[0030] In some embodiments, the system comprises a controller configured to control the first and second power supplies, such that the activation voltage is applied to the first emitter at different times from when the activation voltage is applied to the second emitter.

[0031] In some embodiments of the system, the system is configured to calculate a ratio spectrum by dividing the mass spectrum of the first specimen by the mass spectrum of the second specimen for each analyte.

[0032] In some embodiments of the system, the system is configured, for values in the ratio spectrum that are less than 1 , to change each value thereof to a reciprocal value to determine a DiffN spectrum. [0033] In some embodiments of the system, each value in the ratio spectrum corresponds to an analyte present in the first specimen and/or the second specimen.

[0034] In some embodiments of the system, using N largest values in the DiffN spectrum, the tandem mass spectrometer is configured to acquire a tandem mass spectrum for each of the N largest values in the DiffN spectrum, N being a value between 1 and a maximum number of peaks in the DiffN spectrum.

[0035] In some embodiments of the system, the tandem mass spectrometer is configured for acquiring a tandem mass spectrum for each of the N largest values in the DiffN spectrum.

[0036] In some embodiments of the system, the controller is configured to wait for the tandem mass spectrometer to acquire a number of mass spectra before switching the first power supply from the activation voltage to the inactivation voltage and the second power supply from the inactivation voltage to the activation voltage or before switching the first power supply from the inactivation voltage to the activation voltage and the second power supply form the activation voltage to the inactivation voltage

[0037] In some embodiments of the system, the system is configured to average results of the tandem mass spectrometer over the number of mass spectra accumulations that occur between when voltage of the first and second power supplies are switched.

[0038] In some embodiments of the system, the first specimen comprises a control sample and the second specimen comprises a sample suspected of having an abnormal abundance of one or more analytes. [0039] In some embodiments of the system, the abnormal abundance of one or more analytes is indicative of the presence of a disease in the subject from which the sample for the second specimen was obtained.

[0040] In some embodiments of the system, the subject is a human being.

[0041] In some embodiments of the system, the device is configured to determine an abundance for analytes contained within the first specimen; the device is configured to determine an abundance for analytes contained within the second specimen; and the system is configured to compare a relative abundance of a plurality of analytes between the first and second specimens and to select a quantity of the plurality of analytes to be compared between the first and second specimens based on the relative abundance of the plurality of analytes.

[0042] In some embodiments of the system, comparing the relative abundance between the first and second specimens for the quantity of analytes selected is performed for the quantity of analytes having a greatest differential abundance between the first and second specimens.

[0043] In some embodiments of the system, the multi-emitter ionization source comprises a nanospray or electrospray source.

[0044] In some embodiments of the system, the multi-emitter ionization source is configured to generate alternating emissions of the first specimen from the first emitter and the second specimen from the second emitter using a constant pressure for the first and second specimens within the first and second emitters, respectively.

[0045] In some embodiments of the system, emission of the first specimen from the first emitter and emission of the second specimen from the second emitter do not interfere with each other. BRIEF DESCRIPTION OF THE DRAWINGS [0046] The presently disclosed subject matter can be better understood by referring to the following figures. The components in the figures are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the presently disclosed subject matter (often schematically). In the figures, like reference numerals designate corresponding parts throughout the different views. A further understanding of the presently disclosed subject matter can be obtained by reference to an embodiment set forth in the illustrations of the accompanying drawings. Although the illustrated embodiment is merely exemplary of systems for carrying out the presently disclosed subject matter, both the organization and method of operation of the presently disclosed subject matter, in general, together with further objectives and advantages thereof, may be more easily understood by reference to the drawings and the following description. The drawings are not intended to limit the scope of this presently disclosed subject matter, which is set forth with particularity in the claims as appended or as subsequently amended, but merely to clarify and exemplify the presently disclosed subject matter.

[0047] For a more complete understanding of the presently disclosed subject matter, reference is now made to the following drawings in which:

[0048] FIG. 1A is a schematic illustration of an example embodiment of a system for producing an ionized nanospray emission to an inlet capillary of, for example, a mass spectrometer, in which a first emitter is active and a second emitter is inactive.

[0049] FIG. 1 B is a schematic illustration of the example embodiment of the system shown in FIG. 1A, with the first emitter being inactive and the second emitter being active.

[0050] FIG. 2A is an example illustration of results produced using a TopN approach in selecting MS/MS target species in a pair of mass spectra. [0051] FIG. 2B is an example illustration of results produced using a DiffN approach in selecting MS/MS target species in the same pair of mass spectra shown in FIG. 2A, in which the TopN approach is used.

[0052] FIG. 3A is an example illustration of an extracted ion chronogram.

[0053] FIG. 3B is an enlarged view of the extracted ion chronogram shown in FIG. 3A.

[0054] FIG. 3C is an example illustration of mass spectra obtained during scans 23 and 24, as indicated in FIG. 3B.

[0055] FIG. 4 is an example illustration of control waveforms used in setting the timing of nanospray emissions from the emitters of the system shown in FIGS. 1 A and 1 B.

[0056] FIGS. 5A-5D are example illustrations of respective extracted ion chronograms collected at differing scan speeds during pulsed activation of a single emitter.

[0057] FIG. 6 is an example illustration in which multiple datapoints collected during a single activation of an emitter are averaged and presented as a single datapoint for each pulse number.

[0058] FIG. 7 is an example illustration of a box plot showing extracted ion intensities and associated error measurements from five replicated pulses, or activations, of an emitter.

[0059] FIG. 8A is an example illustration showing overlap between total ion chronograms of pulsed emission spray and constant emission spray.

[0060] FIG. 8B is an example illustration comparing extracted ion current values for pulsed emission spray and constant emission spray. [0061] FIG. 9A is an example illustration of a mass spectrum of bovine heart tissue extract containing lipid internal standards.

[0062] FIG. 9B is an example illustration of a mass spectrum of bovine heart tissue extract without lipid internal standards.

[0063] FIG. 9C is a graphical plot of extracted ion chronograms for lipid internal standards from pulsed dual emitters.

[0064] FIGS. 10A-10D are respective graphical plots for a DiffN approach for analysis of analytes contained within two sample specimens in different concentrations.

[0065] FIG. 11 is a flowchart for an example embodiment of a method of using the DiffN approach.

DETAILED DESCRIPTION

[0066] Conventional tandem mass spectrometry data dependent precursor selection methods use total abundance as their sole criteria for selection. In such conventionally known methods, a survey scan is performed for all precursor ions, with the most abundant precursor ions being selected for tandem mass spectrometry (MS/MS). This mode of operation is commonly referred to as the “TopN” method, where “N” represents the number of precursor ions selected from a survey scan.

[0067] A novel method for use in such “-omics” analysis is disclosed herein, such novel method being referred to hereinafter as the “DiffN” method. According to the DiffN method, a differential abundance between two samples is used to select precursor ions for MS/MS. While an example embodiment of the DiffN method is disclosed herein with respect to an example “-omics” analysis, the “DiffN’ method disclosed herein is not limited to use associated with only such “-omics” analyses and, in fact, can be used in conjunction with any suitable compositional analysis of constituent components of a specimen, including, for example, in an analysis of an environmental specimen. According to the example application for the DiffN method discussed herein, in such “-omics” analysis, the use of differential abundance of precursor ions is advantageous because, for example, it is samples between two conditions (e.g., healthy and diseased) that are typically being compared; additionally, it is commonly the case that the most highly concentrated precursor ions, which would be those that would be selected for analysis using the conventional TopN approach, are often ubiquitous and are therefore not informative regarding the analysis being performed (e.g., are not indicative of any abnormal, or diseased, state in the sample(s) being analyzed). As such, among the advantages provided by using the DiffN method rather than the conventional TopN method is that the analysis performed is not biased to select predominantly, or solely, the most concentrated species (e.g., of precursor ion) in a sample. As such, the DiffN method disclosed herein is well- suited to providing structural information on the species (e.g., precursor ions) in a sample that are changing the most as a result of a change in the sample, or sample conditions (e.g., upon initial occurrence, or detection, of an abnormal, or diseased, state), regardless of the abundance, or concentration, of any particular species in question within the sample.

[0068] As mass spectrometer detection limits continue to improve, more can be learned regarding the importance of very low-abundance species (e.g., of precursor ions) in biological systems, which would virtually never be selected using the conventional TopN method. The DiffN method disclosed herein is well-suited to providing structural information on the species that are changing the most when a change in the sample, or sample conditions, occurs or is otherwise detected, regardless of the abundance, or concentration, of the species in question within the sample.

[0069] While the DiffN method is disclosed herein in an example embodiment as being performed using multiple nanospray ionization sources, the DiffN method is not limited to such embodiments and includes all suitable types of analysis, including, for example, parallel chromatography.

[0070] When considering new applications of conventional lipidomics methods (i.e., using “TopN”), the process of determining a clinically significant threshold concentration of a molecule (e.g., a precursor ion) for a given medical condition requires a lengthy validation period, at least when compared to using a control sample, especially when the large number of potentially relevant lipid species within the sample is considered. By using control samples, direct comparisons of instrument response can be made between healthy/control conditions and diseased/experimental conditions, whether experimental conditions include known or suspected disease states, environmental exposure, and/or therapeutic intervention. As the data of interest is a comparison of lipid abundance between samples, an ionization source that allows for real-time, simultaneous comparison of two or more samples is advantageous, as such an ionization source allows for an increase in throughput, while also accounting for biases inherent to performing separate analyses.

[0071] Leveraging the ability to control nanospray activity by application of voltage alone (e.g., by not requiring a differential pressure), a pulsing dual emitter nanospray ion source is suitable for use in such lipidomics experiments. Multi-emitter electrokinetically-pulsed nanospray are shown herein to provides a suitable framework for lipidomics applications. In some alternative example embodiments of the DiffN method, physical, or mechanical, barriers can be moved relative to the emitters (e.g., to physically block a path between an emitter and the inlet capillary) to prevent the emission spray for one or more electrospray emitters from accessing the inlet of the mass spectrometer, such as may occur when the emitters constantly generate the emission spray. However, the use of electrokinetic pulsing to control activation of emitters advantageously reduces sample consumption, especially when compared against the use of physical barriers, since the electrospray emission is only generated during relevant acquisition periods (e.g., when a voltage above a certain threshold is applied to a specified emitter, or set or emitters). Unlike in embodiments in which physical barriers are used to control sample induction into the mass spectrometer, in which case the emission spray continues (e.g., is continuous) irrespective of the location and/or orientation of the physical barrier(s) relative to the emitter(s) of the system. Multi-emitter electrospray ionization (ESI) source designs, including multi-emitter ESI source designs controlled electrokinetically and/or by any other suitable control methodology, can also be used for improving mass accuracy by, for example, providing reference signals for use (e.g., by a mass spectrometer) as one or more internal calibrants.

[0072] According to an example embodiment for a system, generally designated 10, which performs differential abundance analysis on samples, is disclosed herein and illustrated in FIGS. 1 A and 1 B. The system 10 comprises a plurality of emitters 20A, 20B. The emitters 20A, 20B can be electrospray emitters, nanospray emitters, or a combination of nanospray emitters and electrospray emitters. While the system 10 can have any suitable quantity of emitters, in the example embodiment shown in FIGS. 1 A and 1 B, the system 10 comprises a first emitter 20A and a second emitter 20B. Each of the emitters (e.g., first and second emitters 20A, 20B) contains and/or emits a sample (e.g., a lipid extract) obtained from and/or during, or comprising, a different experimental condition. In the following illustrative examples, in which the system 10 is used to perform the DiffN method, a sample corresponding to a disease state (e.g., having a composition indicating the presence of a disease state) is compared to a wild-type, or control, sample. FIGS. 1A and 1 B schematically illustrate various aspects of the example embodiment of the ion source of the system 10 and the example values for voltages suitable for use in implementing nanospray control are shown in FIGS. 1 A and 1 B as well.

[0073] As shown in FIGS 1A and 1 B, the system 10 comprises a dual nanospray ionization source, generally designated 20, which comprises the first and second emitters 20A, 20B. The first and second emitters 20A, 20B are connected, respectively, to first and second power supplies, generally designated 30A, 30B. The first and second power supplies 30A, 30B can be controlled independent of each other. As shown, the first and second power supplies 30A, 30B are electrically connected to the first and second emitters 20A, 20B, respectively, and are configured to apply, or transmit, an electric potential (e.g., voltage) at, on, and/or to the first or second emitter 20A, 20B to which such first or second power supply 30A, 30B is electrically connected. FIGS. 1 A and 1 B show example values for the activation voltages that can be applied by the first and second power supplies 30A, 30B to control (e.g., start and stop) the nanospray emission from the first or second emitter 20A, 20B, to which the first or second power supply 30A, 30B is electrically connected. These example voltages applied to the first and second emitters 20A, 20B have been determined to be suitable for use in the example DiffN methods disclosed herein, but activation voltages that are different from those shown in FIGS. 1 A and 1 B are included as well, and the scope of the subject matter disclosed herein should not be limited to the example values unless otherwise stated elsewhere herein.

[0074] In the example embodiment of the system 10 shown in FIGS. 1A and 1 B, each of the first and second emitters 20A, 20B is positioned in front of (e.g., adjacent to, and capable of nanospray emission generally towards) the inlet of a mass spectrometer 1. In the example embodiment shown, the inlet of the mass spectrometer 1 is an inlet capillary 3 at standard atmospheric pressure (e.g., at the prevailing, ambient, unaltered atmospheric pressure). Each of the first and second emitters 20A, 20B are connected to one of the first and second power supplies 30A, 30B, which are independently-controlled high voltage power supplies in this example embodiment. In some embodiments, one or more of (e.g., a plurality of) the emitters may be connected to a same power supply to form a group 20 (e.g., a first group) of emitters and one or more others of the emitters may be connected to another power supply to form another group 20 (e.g., a second group) of emitters. Any suitable quantity of groups 20 of emitters may be provided. Any suitable quantity of emitters may be selected for each group 20 of emitters. In some embodiments, one or more (e.g., all) of the groups 20 of emitters may have the same or different quantities of emitters. In some embodiments, the system 10 can comprise a quantity of emitters that can be any of 2, 3, 4, 5, 6, and the like, without limitation. In some embodiments, some or all groups 20 of emitters may comprise a single emitter and/or other groups 20 may comprise multiple emitters. In some embodiments, all groups 20 of emitters may comprise a plurality of emitters.

[0075] The inlet capillary 3 of the mass spectrometer 1 is electrically connected to a third power supply, generally designated 30C, which provides, or applies, an electrical potential (e.g., voltage) to the inlet capillary 3. The third power supply 30C is able to be controlled independent of the first and/or second power supplies 30A, 30B. In the example embodiment shown, the third power supply 30C is an independently-controlled high voltage power supply. As shown in FIGS. 1A and 1 B, the inlet capillary 3 has, via the third power supply 30C, a negative electrical potential of about -1.5 kV applied thereto. In FIG. 1 A, the first emitter 20A (or first group of emitters) is activated by the application of a first voltage (e.g., including a voltage of 0 V) to the first emitter 20A by the first power supply 30A, while a second voltage (e.g., a voltage having a same polarity as the voltage applied to the inlet capillary 3 by the third power supply 30C) is applied to the second emitter 20B (or second group of emitters) by the second power supply 30B to inhibit, or prevent substantially entirely, emission spray from the second emitter 20B. In FIG. 1 B, the second emitter 20B (or second group of emitters) is activated by the application of the first voltage to the second emitter 20B by the second power supply 30B, while the second voltage, which has, for example, the same polarity as the voltage applied to the inlet capillary 3, is applied to the first emitter 20A (or first group of emitters) by the first power supply 30A to inhibit emission spray from the first emitter 20A. In the example embodiment shown, the first voltage is 0 V and the second voltage is negative 500 V. Flowever, in some embodiments the first voltage may have an opposite polarity (e.g., a positive polarity) or a same polarity (e.g., a negative polarity) from the voltage applied to the inlet capillary 3. The magnitude of the second voltage, which is selected such that nanospray emission from the non-selected emitter is inhibited, or substantially entirely prevented, can be selected empirically based on, for example, the distance between the non-selected emitter and the inlet capillary 3, the magnitude and polarity of the voltage applied to the inlet capillary 3, and the like. During operation, the first and second voltages applied to the first and second sprayers are rapidly switched, or pulsed.

[0076] Under normal operation in a positive ion mode, the third power supply 30C applies a substantially constant negative voltage (e.g., from about -1 kV to about -3 kV, inclusive, for nanospray, and from about -3 kV to about -5 kV, inclusive, for electrospray) to the inlet capillary 3 and the active emitter 20A, 20B is grounded (e.g., has 0 V applied thereto, which is also defined for this example embodiment as the first voltage) relative to the inlet capillary 3. This increase in voltage difference between the inlet capillary 3 and the active emitter 20A, 20B, at least relative to the voltage difference between the inlet capillary 3 and the inactive emitter 20A, 20B, produces an activating electric field within an emission region, generally designated 5, defined between the tip of the active emitter 20A, 20B and the inlet capillary 3, this activating electric field being of sufficient magnitude to generate electrospray or emission spray from the active emitter. This is shown with respect to the first emitter 20A in FIG. 1 A and with respect to the second emitter 20B in FIG. 1 B.

[0077] Thus, in FIG. 1A, the inlet capillary 3 is energized at an electrical potential of about -1 .5 kV by the third power supply 30C, the first emitter 20A is energized at the first voltage (e.g., connected to ground, at about 0 V) by the first power supply 30A, and the second emitter 20B is energized at the second voltage (e.g., about -500 V) by the second power supply 30B, such that the first emitter 20A generates an emission spray 22A that is directed generally towards the inlet of the inlet capillary 3. Thus, as shown in FIG. 1 A, the voltage differential of about -1 .5 kV between the first emitter 22A and the inlet capillary 3 generates an electric field within the emission region 5, this electric field having sufficient magnitude to induce the emission spray 22A, while the voltage differential of about -1 .0 kV between the second emitter 22B and the inlet capillary 3 generates an electric field within the emission region 5 that is not of sufficient magnitude to induce an emission spray (e.g., 22B, see FIG. 1 B) from the second emitter 20B, such that emission of the sample from the second emitter 20B is inhibited, or substantially entirely prevented.

[0078] In FIG. 1 B, the inlet capillary 3 is energized at an electrical potential of about -1 .5 kV by the third power supply 30C, the second emitter 20B is energized at the first voltage (e.g., connected to ground, at about 0 V) by the second power supply 30B, and the first emitter 20A is energized at the second voltage (e.g., about -500 V) by the first power supply 30A, such that the second emitter 20B generates an emission spray 22B that is directed generally towards the inlet of the inlet capillary 3. Thus, as shown in FIG. 1 B, the voltage differential of about -1 .5 kV between the second emitter 22B and the inlet capillary 3 generates an electric field within the emission region 5, this electric field having sufficient magnitude to induce the emission spray 22B, while the voltage differential of about -1 .0 kV between the first emitter 22A and the inlet capillary 3 generates an electric field within the emission region 5 that is not of sufficient magnitude to induce an emission spray (e.g., 22A, see FIG. 1 A) from the first emitter 20A, such that emission of the sample from the first emitter 20A is inhibited, or substantially entirely prevented.

[0079] Applying, to the emitter 20A, 20B that is intended to be inactive, an external high voltage of the same polarity as the voltage applied to the inlet capillary 3 reduces the electric field present within the emission region 5, between the inactive emitter (e.g., 20B in FIG. 1A and 20A in FIG. 1 B) and the inlet capillary 3, thereby inhibiting, or preventing, electrospray or nanospray emission spray of the sample from this inactive emitter. This combination of first and second voltages applied (e.g., in an alternating pattern) to the first and second emitters 20A, 20B allow for a pulsed emission spray (e.g., 22A in FIG. 1 A, 22B in FIG. 1 B) between different sprayers (e.g., between the first and second emitters 20A, 20B), or groups of such emitters, by switching the respective voltage provided by the first and second power supplies 30A, 30B connected to the first and second emitters 20A, 20B between a negative voltage (e.g., the second voltage) and a zero voltage (e.g., the first voltage) while the third power supply 30B provides a substantially constant voltage (e.g., a third voltage, different in magnitude and/or polarity from the first and/or second voltages) to the inlet capillary 3.

[0080] The voltage needed to stop an emission spray (e.g., 22A, 22B) from any of the emitters 20A, 20B using the first or second power supply 30A, 30B, respectively, has a lower magnitude than the voltage applied by the third power supply 30C to the inlet capillary 3, since the requisite electric field between the emitter 20A, 20B that is intended to be inactive only needs to be lower than the threshold electric field required to initiate emission spray (e.g., electrospray, nanospray). The value of the threshold for the requisite electric field for generating emission spray between an emitter 20A, 20B and the inlet capillary 3 varies, for example, based on the internal diameter of the emitter 20A, 20B, the distance between the emitter 20A, 20B and the inlet of the inlet capillary 3, etc. The voltage differential that produces an electric field that is the same as or greater than the threshold electric field can be referred to as the onset voltage, or onset voltage differential.

[0081] However, in the example embodiment shown in FIGS. 1A and 1 B, the emitters 20A, 20B are positioned at a typical distance away from the inlet of the inlet capillary 3 (e.g., between about 5-10 mm, inclusive), such that the onset voltage required to generate an electric field of sufficient magnitude (e.g., at or above the threshold electric field) is between about -1.5 kV and about -2 kV, inclusive. As such, in FIG. 1 A, a voltage of about -1 .5 kV applied to the inlet capillary 3 by the third power supply 30C requires the first power supply 30A to apply a first voltage of about 0 V to the first emitter 20A to induce the emission spray 22A, while the application of a second voltage of about - 500 V to the second emitter 20B is sufficient to stop emissive activity (e.g., emission spray 22B, FIG. 1 B) from the second emitter 20B. Similarly, in FIG. 1 B, a voltage of about -1 .5 kV applied to the inlet capillary 3 by the third power supply 30C requires the second power supply 30B to apply a first voltage of about 0 V to the second emitter 20B to induce the emission spray 22A, while the application of a second voltage of about -500 V to the first emitter 20A is sufficient to stop emissive activity (e.g., emission spray 22A, FIG. 1A) from the first emitter 20A. This lower voltage requirement of each of the first and second power supplies 30A, 30B that are electrically connected to the first and second emitters 20A, 20B, respectively, is advantageous, in that this lower voltage requirement allows for faster voltage switching between the emitters 20A, 20B by the first and second power supplies 30A, 30B, since alternating between applying a higher voltage to the first and second emitters 20A, 20B would require longer durations of time between switching between the first and second voltages at each of the first and second power supplies 30A, 30B to allow sufficient time for any residual voltage to decay to a sufficient degree from the first or second emitter 20A, 20B from which the first or second voltage was previously (e.g., immediately before) applied so as to avoid inducing unintended emission spray from one of the first or second emitters 20A, 20B due to residual voltage.

[0082] Such a voltage-controlled ionization source 20 as is shown in the example embodiment of the system 10 in FIGS. 1A and 1 B advantageously allows for rapid, repeated sampling of a specimen from a sprayer, such as nanospray emitter(s) and/or electrospray emitter(s), thereby enabling a novel mode of selecting targets (e.g., molecules of interest, such as precursor ions) for tandem mass spectrometry (MS/MS) present within a specimen. As noted elsewhere herein, the traditional data-dependent acquisition (DDA) method for selecting targets for MS/MS uses the conventional “TopN” approach (e.g., N=10), where the N (e.g., 10) most abundant peaks in a spectrum are selected for MS/MS. Targeting the most abundant peaks using the TopN approach is an effective way to ensure that the MS/MS spectra that are collected are main constituents of the sample, although ionization efficiencies can obscure this, and also that the collected MS/MS spectra are of high quality in terms of signal-to-noise ratio. Flowever, lower-abundance species are often of greater clinical importance, or significance, in biological samples, yet these lower- abundance species and are not selected for MS/MS using the conventional TopN method. [0083] As such, a novel method (“DiffN”) for DDA using a dual nanospray design is disclosed herein, in which MS/MS targets are selected based on differential abundance of species between samples, independent of the proportion, concentration, or abundance of the species within the total specimen. Using differential abundance of targets, rather than absolute abundance of targets, within a specimen is more consistent with the goals of most lipidomics experiments. Furthermore, the ability to collect full scan (MS1 ) data from each sample type (one per emitter, or sprayer) to determine (e.g., calculate) differential abundance of species between a control specimen and a biological specimen of interest (e.g., from a human suspected of having any of a number of diseases) is a unique capability of the pulsing nanospray design disclosed herein.

[0084] An illustration of the results produced using the TopN and DiffN methods in selecting MS/MS target species in a pair of mass spectra is shown in FIGS. 2A and 2B. The mass spectra shown is the same between FIGS. 2A and 2B and the selected species within the mass spectra are designated using the stars, or asterisks. In the example mass spectra shown in FIGS. 2A and 2B, N=3 for both the TopN and the DiffN methods, respectively. As shown in FIGS. 2A and 2B, the differential abundance between the control and experimental specimens does not correlate with (e.g., is different from) the most abundant peaks in each spectrum. Rather, the overexpressed peaks for species 2, 3, and 5 in the experimental sample are not selected using the TopN method, as shown in FIG. 2A. The peaks associated with species 1 and 4 do not change between the control and experimental specimens, but species 1 and 4 are nevertheless selected in both the control and experimental specimens when the TopN method is used. As such, the example embodiment of the dual nanospray system disclosed herein is advantageous for use in validating the advantages provided in the DiffN method, especially since both the TopN and DiffN methods can be performed with the same ion source. [0085] A standard electrospray ionization source, or any other ion source, (e.g., a single ion source) can be used in performing compositional analysis of one or more specimens using the DiffN method described herein as well, in some embodiments. Thus, the DiffN method is not limited to only dual nanospray emission systems such as shown in FIGS. 1A and 1 B. In such embodiments, the single ion source is connected to two or more (e.g., a plurality of) specimen introduction lines (e.g., tubing) that can be alternated, such that the single ion source is supplied with sample from only one of the specimen introduction lines at a time. An example of a suitable specimen type that could be suitable for DiffN analysis using a single ion source is a gaseous specimen using a chemical ionization source. In such embodiments, the supply of the specimen from each of the specimen induction lines into the single ion source is controllable, for example, by using TTL (e.g., 0-5 V) signals to trigger a valve connected to one, both, or all of the specimen induction lines to allow the flow from only a first specimen induction line to the single ion source for a prescribed period of time, then moving the valve to a position to prevent the flow of the specimen from the first specimen induction line and the same or another valve could be commanded to allow the flow of a specimen from only a second specimen induction line to the single ion source for a same or different prescribed period of time. This process could be repeated for each specimen induction line until specimens from each of the specimen induction lines have been allowed to flow to the single ion source for a prescribed (e.g., same or different) period of time, at which point the process can be repeated to allow for the flow from the first specimen induction line or terminated. In some embodiments, each specimen induction line may be provided with a valve to control a flow of specimen therefrom to the single ion source. In some embodiments, multiple specimen induction lines may be connected to a single valve. The actuation of the valve(s) is analogous to triggering the emitter voltage disclosed elsewhere herein for multi-emitter ion sources.

[0086] As noted elsewhere herein, using external high voltage power supplies enables independent control of multiple nanospray emitters. The first and second power supplies 30A, 30B used in the example embodiment shown are controlled by TTL (0-5 V) signals, with a 5 V signal turning the power supply on (e.g., to apply a voltage selected by a dial to an associated sprayer) and 0 V turning the power supply off. A control system for the TTL input of the first and second power supplies 30A, 30B is provided, since the voltage applied to the inlet capillary 3 is held constant during operation of the system 10 and, as such, does not require active control. An external function generator (e.g., Standard Research Systems Model DS335) can be used to generate a square wave to trigger the TTL of one of the high voltage power supplies. Pulsing of a nanospray emitter containing a peptide with sequence of YAGFL at 2 mM is achieved using a 1.5 Hz 5 V0-P square wave. An extracted ion chronogram and mass spectrum of m/z 570.3, corresponding to [YAGFL + H] ⁺ , during the function generator-triggered pulsing using a 1.5 Hz square wave is shown in FIG. 3A. FIG. 3B is an enlarged view of a portion of the ion chronogram shown in FIG. 3A, showing signal increase and decrease during pulsing operation. FIG. 3C shows a mass spectra from scans 23 and 24, indicated in FIG. 3B, showing the presence and absence, respectively, of a signal for YAGFL in the scans 23 and 24.

[0087] As shown, pulsing nanospray using the external function generator can be achieved using the example embodiment of the system 10 disclosed herein, with the signal for YAGFL quickly increasing and decreasing over the duration of the experiment. However, closer inspection of the chronogram in FIG. 3B shows that the timing of the nanospray pulsing was not consistent with the timing of the sampling of the mass spectrometer, with the signal indicating the presence or absence of YAGFL remaining high or low for multiple sequential scans of the mass spectrometer. The mass spectra in FIG. 3C show that the signal for the YAGFL peptide can be pulsed on and off between scans using the example embodiment of the system 10 to provide a pulsing nanospray platform, demonstrating proof of concept for using a high voltage power supply to apply a stopping electrical potential (e.g., voltage) to the emitter to which it is electrically connected, the electrical potential having the same polarity as the electrical potential applied to the inlet capillary 3. Given the presence of the signal for YAGFL during multiple sequential scans, it was determined that the timing of the pulsed nanospray can be improved by syncing the activation of the first and second power supplies 30A, 30B to the sampling timing of the mass spectrometer to address the deficiencies shown in FIGS. 3A-3C.

[0088] In designing a suitable control system for syncing the timing of the pulsed emissive spray from the first and second emitters 20A, 20B to the sampling timing of the mass spectrometer 1 , the portion of the ion trap scan function used to trigger the first and second power supplies 30A, 30B, respectively, must be considered. Since each ion trap mass spectrometer scan begins with ion accumulation, the active emitter of the first and second emitters 20A, 20B is advantageously fully equilibrated prior to the start of the ion accumulation. If the active does not generate a stable spray during ion accumulation, the resulting mass spectrum will be of lower quality and/or fidelity. For this reason, the end of the ion accumulation step for the mass spectrometer 1 is advantageously selected as the trigger for switching the voltages generated by the first and second power supplies 30A, 30B. By enabling voltage switching at the end of the ion accumulation step, equilibration of a stable electrospray is produced during the mass analysis portion of the scan function of the mass spectrometer 1. Therefore, the subsequent ion accumulation is ensured to occur during an intense and reproducible emission spray (e.g., 22A, 22B, FIGS. 1A and 1 B, respectively), thereby minimizing variability in the resulting mass spectra. A similar sequence of pulsing of the first and second emitters 20A, 20B can be used with time-of-flight (TOF) mass spectrometers 1. After the ions from the active emitter (e.g., the first emitter 20A) of the first and second emitters 20A, 20B are pulsed into the TOF mass spectrometer 1 , the active sprayer is inactivated (e.g., turned off, such as by switching the voltage supplied thereto)) and the second emitter 20B is activated (e.g., by turned on, such as by switching the voltage supplied thereto) while the TOF mass spectrum is being acquired. This will allow the emission spray (e.g., 22B) of the newly activated emitter (e.g., the second emitter 20B) to stabilize prior to pulsing of the ions into the TOF. In cases in which a “beam type” mass spectrometer 1 is used (e.g., triple quadrupole), after the mass spectrum is acquired from an active emitter (e.g., the first emitter 20A), a delay may be incorporated before the next mass spectrum is obtained to allow the first emitter 20A to be turned off and for the second emitter 20B to be activated and allowed to stabilize.

[0089] In one example embodiment, synced timing can be achieved by setting the TTL output of the mass spectrometer to a high (5 V) value during the ion accumulation step and a low (0 V) value during the remainder of the scan function. A National Instruments DAQ and LabVIEW was used to generate an “HV Supply 1 Enable” signal and an “HV Supply 2 Enable” signal by triggering a 0-5 V square wave at the falling edge (e.g., the trailing edge) of the mass spectrometer output. The waveforms relevant to the timing of the first and second emitters 20A, 20B are shown in FIG. 4, which is a simplified illustration of the primary RF during an ion trap mass spectrometer scan and the waveforms used to time the pulsing (e.g., alternating) emission spray from the ionization source 20. “HV Supply 1 Enable” and “HV Supply 2 Enable” are triggered at the falling edge of the ion trap output, which is only high (e.g., 5 V) during the ion accumulation portion of the ion trap scan function.

[0090] Using the ion accumulation-synced timing shown in Figure 4 is also advantageous, in that the duration of the ion accumulation step and mass analysis is automatically accounted for in the pulsing operating mode disclosed herein. Ion accumulation times can vary from tenths of a millisecond to hundreds of milliseconds, and the duration of mass analysis depends on the scan rate (Da/s) and the mass range. An extracted ion chronogram of m/z 570.3 ([YAGFL + H] ⁺ ) collected while pulsing the high voltage power supply for a single emitter containing the same YAGFL peptide using the ion accumulation-synced “HV Supply 1 Enable” signal at different scan speeds is shown in FIGS. 5A-5D.

[0091] FIG. 5A is collected at a “Standard Enhanced” scan speed (e.g., 8,100 Da/s), while FIG. 5B is an enlarged portion of the graphical plot of FIG. 5A. FIG. 5C is collected at an “Ultra” Scan speed (e.g., 26,000 Da/s), while FIG. 5D is an enlarged portion of the graphical plot of FIG. 5C. Unlike the pulsed signal used in collecting the data shown in FIGS. 3A-3C, the extracted ion chronograms in FIGS. 5A-5D have a clear pattern of alternating between a high signal and zero signal after each ion trap scan. The 30 second pulsing using the Standard Enhanced (8,100 Da/s) scan speed, as shown in FIGS. 5A and 5B, has significantly fewer pulses than the same duration using the Ultra (26,000 Da/s) scan speed, as shown in FIGS. 5C and 5D, but the reproducibility of each pulse using the Standard Enhanced scan speed is improved from an RSD of 27%, using the Ultra scan speed, to 18%. While switching between (e.g., alternating) emitters at the end of each ion accumulation event is possible, the reproducibility and/or accuracy of each scan is lower than when scan averaging is used. In some embodiments, a typical operation of the mass spectrometer 1 can average between 6 and 20, inclusive, individual scans (e.g., one scan per ion accumulation) before outputting a mass spectrum to provide greater reproducibility between data points. This averaging functionality can be incorporated into the controller (e.g., in a LabVIEW program) by waiting to trigger the FIV Supply Enable signals until the occurrence of “N” falling edge events from the mass spectrometer output, where N is the desired number of scans to be averaged to produce the averaged value. An example of pulsing a single sprayer for 20 ion accumulations and the resulting averaged data is shown in FIG. 6.

[0092] The capability of specifying a number of ion accumulations (e.g., scans) before switching (e.g., activating or inactivating) an emitter is advantageous, in that it improves the integration of the pulsed nanospray ion source with the mass spectrometer 1. The raw data is processed to appear more similar to the data in FIGS. 5A-5D, as the desired output remains the average of the multiple ion accumulations. In some embodiments of scan averaging, each ion accumulation is saved (e.g., written to an output data file). Flowever, scan averaging using the built-in approach in the mass spectrometer software typically only saves the averaged value. Writing the data from each ion accumulation event increases the size of the data files proportionally, requiring additional storage space and processing power. However, data files are small for direct infusion experiments relative to long liquid chromatography-mass spectrometry experiments. Further integration of the timing of the voltage applied by each of the power supplies (e.g., 30A, 30B) with the mass spectrometer 1 is possible by accessing the timing that dictates the start and end of each set of averaged scans.

[0093] By using an effective pulsing mechanism and synchronizing the timing of emission spray using the ion trap scan function, operation of the pulsed ion source (e.g., 20, FIGS. 1A, 1 B) was evaluated using a lipid extract. Due to its pulsed operation, the duration of each electrospray pulse (e.g., emission spray) dictates the length of data collection. Faster pulses can be used but result in reduced signal reproducibility. The relative standard deviation (RSD) associated with pulsing after each ion accumulation (equivalent to a 1 spectral average, approximately 200 ms) is significantly higher for all measured lipids than the RSD associated with pulsing after 20 ion accumulations (equivalent to 20 spectral averages, approximately 4 s). These results are illustrated in the form of a box plot of extracted ion intensities and associated error measurements from five replicated pulses in FIG. 7.

[0094] Ultimately, the pulse time used in an experiment (e.g., a compositional analysis) performed using the system 10 is dictated by the needs of a given application. For example, a pulse time equivalent to 1 spectral average may be suitable for rapid qualitative, or screening, applications, while longer pulse times may be more suitable for quantitative applications. The inverse relationship between pulsing speed and signal reproducibility is caused by the relationship between measurement uncertainty and the number of measurements. Characterization of pulse times provides a fundamental understanding of both system performance and the length of time required to complete analysis of a specimen.

[0095] The presently disclosed subject matter was conceived on the basis of the data collected from a pulse-operated emitter, or sprayer, being equivalent to the data collected if that same emitter was operated in a continuous (e.g., conventional) operation mode. A comparison between pulsed and constant nanospray emissions of heart tissue lipid extract (25 pg/mL total lipid content) containing internal standards (50 ng/mL each) at 1 , 5, 10, and 20 ion accumulations per spectrum is shown in FIG. 8.

[0096] The overlap between total ion chronograms (TICs) of pulsed nanospray emission and constant nanospray emission in FIG. 8A suggests that the signal is equivalent between pulsed and constant operation. Further comparison between the two nanospray modes of operation in FIG. 8B shows that the signal for individual lipids is also consistent at variable numbers of scans per pulse (or number of averaged mass spectra). The solid traces, which show data obtained during pulsed operation, track closely with (e.g., are substantially similar to) the dotted traces, which show data obtained during constant operation, or emission. This is demonstrated for lipids spanning a large range of polarity, indicating minimal bias based on the chemical nature of the analytes. As demonstrated in FIG. 7, the standard deviation of each measurement in FIG. 8B decreases as the number of scans per pulse (or number of averaged mass spectra) increases between 1 and 20. These results verify that a pulsed nanospray that is equilibrated and stable prior to ion accumulation will not result in a signal that is significantly different from the signal generated from a continuously operated, or conventional, nanospray system.

[0097] Another advantageous feature induced by the operation of the pulsed nanospray ion source (e.g., 20, FIG. 1A, 1 B) is the absence of “crosstalk” (e.g., interference) between emitters (e.g., 22A, 22B) of the system 10. As described elsewhere herein, through the pulsed operation of the nanospray ion source, it is possible to measure differential abundances between specimen types. Interference between emitters would introduce measurement bias that would obscure differences between specimens, as the collected data would be the result of some mixture of ions generated from the first and second emitters 20A, 20B. Interference between the first and second emitters 20A, 20B was evaluated by loading a lipid specimen containing a tissue extract and lipid internal standards in the first emitter 20A and loading a lipid specimen containing only the tissue extract in the second emitter 20B. Pulsing between the first and second emitters 20A, 20B results in signal for detection of the lipid extract in every scan and signal for detection of the lipid internal standards only in scans corresponding to the first emitter 20A. The results of this experiment are shown in FIG. 9.

[0098] FIG. 9A shows a mass spectrum of bovine heart tissue extract containing lipid internal standards. FIG. 9B shows a mass spectrum of bovine heart tissue extract without lipid internal standards. FIG. 9C shows extracted ion chronograms of the summed intensity of m/z’s corresponding to lipid internal standards from pulsed dual nanospray (e.g., at 10 ion accumulations per mass spectrum) between a first emitter (e.g., 20A, FIGS. 1A, 1 B), which contains tissue extract and internal standards, and a second emitter (e.g., 20B, FIGS. 1A, 1 B), which contains only tissue extract. The shorter dashed trace corresponds to the first emitter 20A, which contains the tissue extract and internal standards, and the longer dashed trace near y=0 corresponds to the second emitter 20B, which only contains the tissue extract.

[0099] The data presented in FIGS. 9A-9C demonstrate that there is no crosstalk between the first and second emitters 20A, 20B when pulsing between lipid specimens at 10 ion accumulations per spectrum. Signal for the lipid internal standards, LPC (18:1 ), PE (28:2), PC (28:2), and SM (d36:2), is only observed for Emitter A (e.g., first emitter 20A, variable trace in FIG. 9C), with data from Emitter B (e.g., second emitter 20B, solid trace in FIG. 9C) only containing signal for lipids from the tissue extract.

[0100] The example embodiment for the pulsed dual nanospray source 20 of the system 10 disclosed herein addresses deficiencies in conventional lipidomics analytical methods using a combination of external high voltage power supplies and a voltage on the inlet capillary 3 of the mass spectrometer 1. The ability to control emission spray from each of the emitters (e.g., first and second emitters 20A, 20B) independently using these voltages provided by the first, second, and third power supplies 30A, 30B, 30C advantageously increases throughput of the system 1 , limits instrument variability between experiments by completing analyses relative to control conditions within seconds instead of minutes or hours, as was the case using conventional analytical techniques, and enables a new mode of DDA MS/MS acquisition where the most differentially abundant species (e.g., the species whose differential abundance between specimens is greatest) are targeted according to the DiffN method.

[0101] Example pulsing nanospray experiments were completed using a function generator in a control system to control the operation of each external high voltage power supply (e.g., of the first and second power supplies 30A, 30B). The timing of this control system is improved by syncing the external high voltage supplies to the scan function of the mass spectrometer 1 using, for example, LabVIEW. The analytical characteristics of pulsed nanospray operation of the system 10 are equivalent to constant nanospray operation under the instrument conditions that will be used for measurement of differential abundances. Additionally, the potential for crosstalk between the first and second emitters 20A, 20B of the pulsed dual nanospray source 20 is negligible, with the only examples of unexpected signal being attributed to electrical/chemical noise.

[0102] FIGS. 10A-10D are respective graphical plots for a DiffN method for analysis of analytes contained within two sample specimens in different concentrations. The respective mass spectra for “Specimen A” and “Specimen B” are shown in FIGS. 10A and 10B. Using a TopN approach where N = 4, MS/MS spectra is acquired for peaks 8, 9, 3, 5 in Mass Spectrum A. In Mass Spectrum B, MS/MS spectra would be acquired for peaks 7, 9, 3, 2. Only two peaks are common (e.g., having at least the fourth greatest concentration within the respective specimen) between the mass spectra for Specimens A and B, specifically, peaks designating concentrations for analytes 9 and 3. Flowever, in the example embodiment, analytes 9 and 3 are two species that have very little change from sample to sample; the difference between analytes 9 and 3 between Specimens A and B likely to be experimental uncertainty or biological variation rather than having any clinical significance. Furthermore, using the conventional TopN method, it is unknown how much difference there is between Specimens A and B for analytes in peaks 2, 5, and 7 because data was only acquired for one of the two specimens. Using the DiffN method (N = 4), MS/MS spectra would be acquired for peaks 1 , 6, 7, 5, since these are the species most likely to have statistically significant changes in abundance. When evaluating relative differences between analyte concentrations in samples, the conventional TopN method often results in acquiring data on species with very little difference between samples and will likely miss comparatively lower abundance species, even when there are relatively large relative changes in these species between Specimens A and B. However, the DiffN method will acquire data on species with the greatest relative difference between specimens and will detect such comparatively lower abundance species that nevertheless have relatively large relative changes between specimens that have previously gone undetected using the TopN method.

[0103] FIG. 11 shows an example flow chart for a method for the DiffN approach. In a first step, a mass spectrum for Sample A is acquired. In a next step, a mass spectrum for Sample B is acquired. In a next step, a ratio spectrum (see FIG. 10C) is generated, in which the mass spectrum acquired from Sample A is divided by the mass spectrum acquired from Sample B. In a next step, a DiffN spectrum (see FIG. 10D) is generated, in which any value in the plot of the ratio spectrum that is less than 1 is changed to the reciprocal value (e.g., the numerator and denominator are inverted). The N largest peaks (e.g., corresponding to the largest differences in abundance for particular analytes between Specimens A and B) are then determined in the DiffN spectrum, where N is a value between 1 and the maximum number of peaks in the DiffN spectrum. The MS/MS spectrum is then acquired for each of the N selected peaks in the DiffN spectrum. [0104] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the presently disclosed subject matter.

[0105] While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0106] All technical and scientific terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one skilled in the art. While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0107] In describing the presently disclosed subject matter, it will be understood that a number of techniques and steps are disclosed. Each of these has individual benefit and each can also be used in conjunction with one or more, or in some cases all, of the other disclosed techniques.

[0108] Accordingly, for the sake of clarity, this description will refrain from repeating every possible combination of the individual steps in an unnecessary fashion. Nevertheless, the specification and claims should be read with the understanding that such combinations are entirely within the scope of the invention and the claims.

[0109] Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to "a cell" includes a plurality of such cells, and so forth. [0110] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

[0111] As used herein, the term “about,” when referring to a value or to an amount of a composition, mass, weight, temperature, time, volume, concentration, percentage, etc., is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.

[0112] The term “comprising”, which is synonymous with “including” “containing” or “characterized by” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. “Comprising” is a term of art used in claim language which means that the named elements are essential, but other elements can be added and still form a construct within the scope of the claim.

[0113] As used herein, the phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. When the phrase “consists of” appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

[0114] As used herein, the phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps, plus those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter.

[0115] With respect to the terms “comprising”, “consisting of”, and “consisting essentially of”, where one of these three terms is used herein, the presently disclosed and claimed subject matter can include the use of either of the other two terms.

[0116] As used herein, the term “and/or” when used in the context of a listing of entities, refers to the entities being present singly or in combination. Thus, for example, the phrase “A, B, C, and/or D” includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D.

[0117] The subject matter disclosed herein can be implemented in software in combination with hardware and/or firmware. For example, the subject matter described herein can be implemented in software executed by a processor. In one exemplary implementation, the subject matter described herein can be implemented using a computer readable medium having stored thereon computer executable instructions that when executed by a processor of a computer control the computer to perform steps. Exemplary computer readable mediums suitable for implementing the subject matter described herein include non-transitory devices, such as disk memory devices, chip memory devices, programmable logic devices, and application specific integrated circuits. In addition, a computer readable medium that implements the subject matter described herein can be located on a single device or computing platform or can be distributed across multiple devices or computing platforms.

[0118] It will be understood that various details of the presently disclosed subject matter may be changed without departing from the scope of the presently disclosed subject matter. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.