WO2007140399A2

WO2007140399A2 - Method of correlating analyte separation information to a biomarker

Info

Publication number: WO2007140399A2
Application number: PCT/US2007/069959
Authority: WO
Inventors: David E. Clemmer; Stormy L. Koeniger; Samuel I. Merenbloom; Stephen J. Valentine; Stephen Naylor
Original assignee: Indiana University Research And Technology Corporation; Predictive Physiology And Medicine, Inc.
Priority date: 2006-05-30
Filing date: 2007-05-30
Publication date: 2007-12-06
Also published as: WO2007140401A3; WO2007140400A3; WO2007140399A3; WO2007140400A2; WO2007140401A2

Abstract

A method of correlating analyte separation information to a biomarker may comprise operating an analyte separation instrument (10) to produce a plurality of discrete analyte groups from a packet of a mixture of analytes, determining analyte intensities of each of the plurality of discrete analyte groups, creating a matrix (120) of the analyte intensities of each of the plurality of discrete analyte groups, identifying entries of the matrix that define the biomarker, and creating a map (140) correlating the biomarker to the identified entries of the matrix (120).

Description

METHOD OF CORRELATING ANALYTE SEPARATION INFORMATION TO A BIOMARKER

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S. C. 119(e) of U.S. Provisional Application Serial No. 60/803,454, filed May 30, 2006, which is expressly incorporated herein by reference.

FIELD OF THE INVENTION:

This disclosure relates generally to techniques for processing analyte separation information produced by analyte separation instruments, and more specifically to techniques for correlating analyte separation information to one or more biomarkers.

BACKGROUND OF THE INVENTION

Analytical instruments are known that are configured to separate analytes as a function of one or more analyte characteristics. It is desirable with such instruments to process analyte separation information in a manner that correlates the analyte separation information to one or more biomarkers.

SUMMARY OF THE INVENTION

The present invention may comprise one or more of the features recited in the attached claims and the following features and combinations thereof. A method of correlating analyte separation information to a biomarker may comprise operating an analyte separation instrument to produce a plurality of discrete analyte groups from a packet of a mixture of analytes, determining analyte intensities of each of the plurality of discrete analyte groups, creating a matrix of the analyte intensities of each of the plurality of discrete analyte groups, identifying entries of the matrix that define the biomarker, and creating a map correlating the biomarker to the identified entries of the matrix.

The analyte separation instrument may have an inlet with a first gate that is controllable to allow or inhibit entrance of analytes into the instrument from an analyte source, and a second gate that is separated by a distance from the first gate and that is controllable to allow or inhibit passage of analytes therethrough. Operating an analyte separation instrument may comprise activating the first gate to allow entrance of the packet of the mixture of analytes from the analyte source into the instrument, allowing the packet of analytes to separate between the first and second gates as a function of a first analyte characteristic, and activating the second gate at a plurality of successive discrete time periods after activating the first gate and before reactivating the first gate to allow passage therethrough of the plurality of discrete analyte groups separated from each of other according to the function of the first analyte characteristic. Creating a matrix of the analyte intensities may comprise entering an analyte intensity value of each of the plurality of discrete analyte groups into a separate row or column of a common column or row of the matrix. The method may further comprise repeatedly activating the first gate, allowing the packet of analytes to separate and activating the second gate a number of times, and for each of the number of times, consecutively adding an offset time, relative to activating the first gate, to each of the times at which the second gate is activated. Creating a matrix of analyte intensity values may comprise creating another common column or row each of the number of times that activating the first gate, allowing the packet of analytes to separate and activating the second gate are repeated.

The method may further comprise processing one of the plurality of discrete analyte groups to produce an additional analyte intensity value. Creating a matrix of the analyte intensities may comprise entering the additional analyte intensity value into a row or column of the matrix that is within the common column or row and that is adjacent to the row or column in which the analyte intensity value of the one of the plurality of discrete analyte groups was entered such that the analyte intensity value of the one of the plurality of discrete analyte groups and the additional analyte intensity value appear sequentially in the common column or row. The method may further comprise processing each of the plurality of discrete analyte groups to produce one or more additional analyte intensity values. Creating a matrix of the analyte intensities may comprise entering the one or more additional analyte intensity values for each of the plurality of discrete analyte groups into the matrix such that the analyte intensity value of each of the plurality of discrete analyte groups and the one or more corresponding additional analyte intensity values appear sequentially in the common column or row. The method may further comprise repeatedly activating the first gate, allowing the packet of analytes to separate and activating the second gate a number of times, and for each of the number of times, consecutively adding an offset time, relative to activating the first gate, to each of the times at which the second gate is activated. Creating a matrix of analyte intensity values may comprise creating another common column or row each of the number of times that activating the first gate, allowing the packet of analytes to separate and activating the second gate are repeated.

Processing one of the plurality of discrete analyte groups to produce an additional analyte intensity value may comprise processing the one of the plurality of discrete analyte groups within the analyte separation instrument in a manner that produces the additional analyte intensity value. Processing the one of the plurality of discrete analyte groups within the analyte separation instrument may comprise activating the one of the plurality discrete analyte groups within the analyte separation instrument. The analyte separation instrument may have a third gate that is separated by another distance from the second gate and that is controllable to allow or inhibit passage of analytes therethrough. Processing the one of the plurality of discrete analyte groups within the analyte separation instrument comprises allowing the one of the plurality of discrete analyte groups to separate between the second and third gates as a function of the first analyte characteristic.

The method may further comprise another analyte separation instrument having an analyte inlet coupled to an analyte outlet of the analyte separation instrument. Processing one of the plurality of discrete analyte groups to produce an additional analyte intensity value may comprise processing the one of the plurality of discrete analyte groups within the another analyte separation instrument in a manner that produces the additional analyte intensity value. Processing the one of the plurality of discrete analyte groups within the another analyte separation instrument may comprise allowing the one of the plurality of discrete analyte groups to separate within the another analyte separation instrument as a function of a second analyte characteristic.

The analyte separation instrument may be a liquid chromatograph, and the first analyte characteristic may be analyte retention time. Alternatively, the analyte separation instrument may be a gas chromatograph, and the first analyte characteristic may be analyte retention time. Alternatively still, the analyte separation instrument may be a capillary electrophoresis instrument, and the first analyte characteristic may be analyte charge-to-size ratio or electrophoretic mobility. Alternatively still, the analyte separation instrument may be a mobility spectrometer, and the first analyte characteristic may be analyte mobility.

The method may further comprise operating another analyte separation instrument to produce another plurality of discrete analyte groups from the plurality of discrete analyte groups, determining analyte intensities of each of the another plurality of discrete analyte groups, and creating another matrix of the analyte intensities of each of the another plurality of discrete analyte groups. Identifying entries of the matrix that define the biomarker may comprise identifying entries of either of the matrix and the another matrix that define the biomarker. Creating a map correlating the biomarker to the identified entries of the matrix may comprise creating a map correlating the biomarker to the identified entries of either of the matrix and the anther matrix.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram illustrating an example analyte separation instrument.

FIG. 2 is a timing diagram illustrating operation of the analyte separation instrument of FIG. 1.

FIG. 3 is a timing diagram further illustrating operation of the second gate of the instrument of FIG. 1.

FIG. 4 is a flowchart of one illustrative process for operating the instrument of FIG. 1. FIG. 5 is a plot of ion intensity vs. ion drift time for a human hemoglobin tryptic digest sample illustrating operation of the analyte separation instrument of FIG. 1 in the form of a two-stage ion mobility spectrometer.

FIG. 6 is a flowchart of one illustrative process for mapping one or more biomarkers to corresponding analyte intensity information. FIG. 7 is a chart illustrating one illustrative technique for creating a matrix of analyte intensity values according to the process of FIG. 6. FIG. 8 is a diagram illustrating identification of entries in a matrix of the type illustrated in FIG. 7 that define one or more biomarkers according to the process of FIG. 6.

FIG. 9 is a map illustrating one illustrative technique for creating a map correlating two biomarkers to corresponding matrix locations according to the process of FIG. 6.

DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS

For the purpose of promoting an understanding of the principles of this disclosure, reference will now be made to one or more embodiments illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended.

Referring now to FIG. 1, a generalized analyte separation instrument 10 is illustrated. As will become more apparent in the description that follows, the instrument 10 may take any of myriad forms. In any case, the instrument 10 includes an analyte source 12 coupled to an inlet of an analyte separation instrument 14 (ASI) having an analyte outlet. The analyte separation instrument 14 is generally configured to separate analytes in time, in space or in time and space, as a function of at least one analyte characteristic. The analyte source 12 may be conventional in construction, and may be configured to produce analytes in any known form including, but not limited to, neutrals, e.g., uncharged particles, and ions, e.g., charged particles. Examples of analyte sources that produce uncharged analytes include, but are not limited to, one or a combination of solutions, one or two-dimensional gels, and the like. Examples of analyte sources that produce charged particles include, but are not limited to, electrospray ion sources, laser desorption ionization structures and techniques, such as a matrix-assisted laser desorption ionization (MALDI), irradiation of samples via other radiations sources, and the like. Alternatively, the analyte source 12 may be integral with the analyte separation instrument 14. Alternatively or additionally, the analyte source 12 may be or include one or more analysis instruments configured to separate analytes in time, space or both, as a function of one or more corresponding analyte characteristics.

The analyte separation instrument 14 has an analyte inlet gate, G₁, which may be controlled to allow analytes from the analyte source 12 to enter the instrument 14 via its analyte inlet. In the case of analytes in the form of charged analytes, the analyte inlet gate, G₁, may be or include, for example, a grid, screen or other suitable structure that may be activated to allow charged analytes to pass therethrough, and that may be deactivated to inhibit passage therethrough of charged analytes. In the case of analytes in the form of uncharged analytes, the analyte inlet gate, Gi, may be or include, for example, a flow valve or other suitable structure that may be activated to allow uncharged analytes to pass therethrough, and that may be deactivated to inhibit passage therethrough of uncharged analytes. The analyte inlet gate, Gi, may be located at, or integral with, the analyte inlet of the instrument 14, downstream of the analyte inlet of the instrument 14 (i.e., within the instrument 14) or in the analyte source 12.

The instrument 14 may have any number, F, of gates, wherein F may be any positive integer greater than 2. In any case, the instrument 14 has an analyte outlet gate, Gp, which may be controlled to allow analytes to exit the active region of the instrument 14 via its analyte outlet. In the case of analytes in the form of charged analytes, the analyte outlet gate, Gp, may be or include, for example, a grid, screen or other suitable structure that may be activated to allow charged analytes to pass therethrough, and that may be deactivated to inhibit passage of charged analytes therethrough. In the case of analytes in the form of uncharged analytes, the analyte outlet gate, Gp, may be or include, for example, a flow valve or other suitable structure that may be activated to allow uncharged analytes to pass therethrough, and that may be deactivated to inhibit passage of uncharged analytes therethrough. The analyte outlet gate, GF, may be located at, or integral with, the analyte outlet of the instrument 14, upstream of the analyte outlet of the instrument 14, (i.e., within the instrument 14) or downstream of the analyte outlet of the instrument 14 (i.e., outside of the instrument 14). Between the analyte inlet gate, G₁, and the analyte outlet gate, G_F, the analyte separation instrument is configured to separate analytes in time as a function of an analyte characteristic.

As illustrated in FIG. 1, the analyte separation instrument 14 may be formed by cascading any number, F-I, of stages (Si - Sp-i) of equal or varying length each configured to separate analytes in time, space or both, as a function of an analyte characteristic, wherein F may be any positive integer greater than 1. In this embodiment, a total number of "F" analyte gates are included so that each stage of the instrument 14 has an analyte inlet gate and an analyte outlet gate. Any one or more of the "F" stages may further include one or more additional analyte processing structures. Examples of such one or more other additional analyte processing structures include, but are not limited to, analyte focusing structures such as one or more conventional analyte focusing funnels, one or more analyte activation regions, and the like. Examples of one such class of instruments, provided in the form of an ion mobility spectrometer, are illustrated and described in U.S. Patent Application Pub. No. US 2007/0114382 Al, entitled ION MOBILITY SPECTROMETER, the disclosure of which is incorporated by reference. As used herein, the term "analyte activation" refers to a process of inducing structural changes in analytes resulting from collisions of the analytes with a buffer gas or buffer gas mixture in the presence of a high AC and/or DC electric field. In the presence of sufficiently high electric fields, high energy collisions of analytes with the buffer gas or gas mixture result in fragmentation of at least some of the analytes. Analyte activation under sufficiently high electric fields thus corresponds to analyte fragmentation. In the presence of elevated electric fields that are not sufficiently high to result in analyte fragmentation, collisions of analytes with the buffer gas or gas mixture results in conformational changes, i.e., changes in the shape, of at least some of the analytes. Analyte activation, under electric field conditions that are not high enough to result in analyte fragmentation, thus corresponds to analyte conformational changes. In any case, examples of implementations of the analyte separation instrument 14 include, but are not limited to, a conventional reverse phase chromatograph, such as a liquid chromatograph that includes all partition, absorption, ion exchange and affinity selections, configured to separate analytes as a function of analyte retention time, a conventional gas chromatograph configured to separate analytes as a function of analyte retention time, a conventional capillary electrophoresis instrument capable of all capillary electrophoresis separations including capillary electrochromatography and configured to separate analytes as a function of analyte charge-to-size ratio or electrophoretic mobility, a conventional mobility spectrometer, configured to separate analytes as a function of analyte mobility and a conventional mass analyzer configured to separate analytes in time as a function of analyte mass-to-charge ratio. Examples of conventional mobility spectrometers include, but are not limited to, single and multiple-stage ion mobility spectrometers, high-field asymmetric waveform ion mobility spectrometers (FAIMS) employing a constant compensation voltage, high-field asymmetric waveform ion mobility spectrometers (FAIMS) employing a differential compensation voltage, and the like. Examples of such conventional mass analyzers include, but are not limited to, a quadrupole or ion trap mass analyzer, a linear quadrupole mass analyzer, a time- of-flight mass spectrometer (TOFMS), a Fourier Transform mass spectrometer (FTMS) and a cyclotron-based mass spectrometer.

The instrument 10 may, in some embodiments, include any number, G, of analyte processing instruments, APIi - APIQ, indicated by dashed-line representation generally in FIG. 1 at 16, that are located downstream of the analyte separation instrument 14 (i.e., after the analyte outlet of the instrument 14), where G may be any positive integer. In embodiments wherein the analyte separation instrument 14 is a gas chromatograph, mass analyzer or mass spectrometer, liquid chromatograph or capillary electrophoresis instrument, the number of analyte processing instruments 16 may be or include, for example, a conventional reverse phase chromatograph, such as a liquid chromatograph that includes all partition, absorption, ion exchange and affinity selections, configured to separate analytes as a function of analyte retention time, a conventional gas chromatograph configured to separate analytes as a function of analyte retention time, a conventional capillary electrophoresis instrument capable of all capillary electrophoresis separations including capillary electrochromatography and configured to separate analytes as a function of analyte charge-to-size ratio or electrophoretic mobility, a conventional mobility spectrometer configured to separate analytes as a function of analyte mobility and a conventional mass analyzer configured to separate analytes as a function of analyte mass-to-charge ratio. Examples of conventional mobility spectrometers include, but are not limited to, single and multiple-stage ion mobility spectrometers, high-field asymmetric waveform ion mobility spectrometers (FAIMS) employing a constant compensation voltage, high-field asymmetric waveform ion mobility spectrometers (FAIMS) employing a differential compensation voltage, and the like. Examples of such conventional mass analyzers include, but are not limited to, a quadrupole or ion trap mass analyzer, a linear quadrupole mass analyzer, a time- of-flight mass spectrometer (TOFMS), a Fourier Transform mass spectrometer (FTMS) and a cyclotron-based mass spectrometer. Alternatively or additionally, the number of analyte processing instruments 16 may be or include one or more conventional instruments configured to process analytes in a manner differently than separating analytes as a function of an analyte characteristic. Examples of such one or more analyte processing instruments include, but are not limited to, a conventional analyte filter configured to collect and/or allow passage therethrough only of analytes within a predefined range of analyte mass-to-charge ratios, a conventional analyte trap configured to collect and selectively eject analytes, a conventional collision cell configured to fragment analytes, a conventional charge neutralization device configured to normalize various analyte charge states to a target charge state (e.g., to a +1 charge state). Other conventional analyte processing instruments will occur to those skilled in the art, and any one or more such other conventional analyte processing instruments may be included in the analyte processing instrument 16.

In embodiments wherein the analyte separation instrument 14 is an ion mobility spectrometer, the number of analyte processing instruments 16 may be or include a conventional reverse phase chromatograph, such as a liquid chromatograph that includes all partition, absorption, ion exchange and affinity selections, configured to separate analytes as a function of analyte retention time, a conventional gas chromatograph configured to separate analytes as a function of analyte retention time, a conventional capillary electrophoresis instrument capable of all capillary electrophoresis separations including capillary electrochromatography and configured to separate analytes as a function of analyte charge-to-size ratio or electrophoretic mobility and a conventional mobility spectrometer configured to separate analytes as a function of analyte mobility.

The instrument 10 further includes an analyte detector 18 that is positioned to receive analytes exiting the analyte separation instrument 14 in embodiments that do not include any of the analyte processing instruments 16, and that is positioned to receive analytes exiting the last of the one or more analyte processing instruments 16 in embodiments that include one or more such analyte processing instruments 16. The analyte detector 18 may be a conventional analyte detector configured to produce analyte detection signals corresponding to arrival of analytes at the detector 18. Such signals are provided to a conventional processor 20 that is configured to process these signals and determine analyte separation information therefrom to determine single or multi-dimensional analyte separation information. In embodiments of the instrument 10 in which analytes are separated in only a single dimension, i.e., as a function of only a single analyte characteristic, the analyte separation information will be one-dimensional analyte separation information, and in all other embodiments the analyte separation information will be multi-dimensional analyte separation information.

The instrument 10 further includes a number of gating sources, indicated generally at 22. In some embodiments, the gating sources 22 include a number of voltage sources that are configured to provide AC and/or DC operating voltages and pulsed voltages to the various sections of the instrument 10 in a conventional manner. In other embodiments, the gating sources 22 may alternatively or additionally include a number of conventional flow control mechanisms configured to control the flow of liquid and/or gas into and/or out of the various sections of the instrument 10 in a conventional manner. The manner in which the gating sources are controlled to operate the instrument 10 in accordance with this disclosure will be described hereinafter. In any case, the gating source 22 provides a number, H, of gating signals to the analyte source 12, where H may be any positive integer. Likewise, the voltage source 22 provides a number, J, of gating signals to the analyte separation instrument 14, where J may be any positive integer, and provides a number, K, of gating signals to the analyte processing instrument 16, where K may be any positive integer. In the case of voltage sources, the gating signals may include

AC, DC and/or pulsed voltages, and in the case of flow control mechanisms the gating signals may include conventional control signals for controlling operation of the flow control mechanisms.

The instrument 10 may, in some embodiments, further includes a gas source 24 that includes a number, L, of different sources of buffer or other gas, Gl - GL, where L may be any positive integer. The gas source 24 is illustrated as being fluidly coupled to the analyte separation instrument 14 and to the analyte processing instrument 16, and the gas source 24 is configured to supply any one or combination of gases to each of the instruments 14 and 16. In one embodiment, the gating sources 22 may comprise one or more programmable gating sources such that the gating sources 22 are self-operating and self-controlled. Alternatively, as shown by dashed-line representation, the processor 20 may be configured in a conventional manner to control operation of one or more of the gating sources 22. The gas source 24, in one embodiment, includes one or more manually activated and/or programmable flow control mechanisms that provide for control over the supply of gas to the analyte separation instrument 14 and/or analyte processing instrument 16. Alternatively, as shown by dashed-line representation, the processor 20 may be configured in a conventional manner to control operation of the gas source 24.

Referring now to FIG. 2, a timing diagram illustrating operation of the analyte separation instrument 10 of FIG. 1 is shown. In one simplified implementation of the instrument 10, the analyte processing instrument 16 of FIG. 1 is omitted and the instrument 14 comprises two stages (e.g., F=3), with an analyte inlet gate, Gi, an analyte gate separated by a distance from Gi, e.g., G₂, and an outlet gate, e.g., G_F- This example case will be used herein to demonstrate operation of the analyte separation instrument 10, although it will be understood that such concepts are directly applicable to more complex embodiments of the analyte separation instrument 10. In describing either of the gates Gi and G₂ as being activated or deactivated, it will be understood that the actual activation and deactivation of these gates may be carried out by supplying one or more appropriate gating signals to the gates Gi and/or G₂ via the gating sources 22, either under manual and/or programmable control of one or more of the gating sources 22 or under at least partial control of the processor 24. In FIG. 2, the activation state of the gate Gi is represented by the gating signal 30, and the activation state of the gate G₂ is represented by the gating signal 32.

A packet of a mixture of analytes from the analyte source 12 are initially "gated" into the analyte separation instrument 14 by activating the gate Gi for a gate activation time period, TG_IA, 34. Thereafter, the gate Gi is deactivated for a time period PG_ID- During the time period T_GIA, analytes from the analyte source 12 enter the analyte separation instrument 14 through the gate Gi and the inlet of the analyte separation instrument 14, and into the first stage, Si, of the instrument 14. After entering the inlet of the analyte separation instrument 14, the analytes travel through Si toward the gate G₂ while separating as a function of an analyte characteristic. Upon the passage of a delay time, T_D, following activation of the first gate, Gi, and before the next activation of the first gate, Gi, the second gate, G₂, is activated at a plurality of successive discrete time periods to allow a corresponding plurality of discrete analyte groups to pass through the second gate, G₂, and into the second stage, S₂, of the analyte separation instrument 14. In the illustrated embodiment, the plurality of successive time periods, Pc, are illustrated as being periodic up to the next activation of the gate G₁. In this embodiment, corresponding periodic groups of analytes will thus be transmitted through the gate G₂ and into the second stage, S₂, of the analyte separation instrument 14 where they will further separate as a function of the analyte characteristic. It will be understood, however, that the plurality of successive time periods, Pc, need not be periodic from T_D until the next activation of the gate G₁. For example, one or more gaps in the G₂ activation times that are greater than Pc may be implemented to inhibit transmission of one or more corresponding groups of analytes through the second gate, G₂. In either case, the activation times, T_A, of the second gate, G₂, at each of the successive time periods Pc resemble the "teeth" of a comb, and the process of successively activating the gate G₂ may accordingly be referred to herein as a "comb", as "combing" or as a "combing technique."

As a result of this combing technique, there will be gaps in the analyte separation information corresponding to the portions of the time periods Pc where the gate G₂ is inactive. Analyte separation information corresponding to these gaps is captured by incrementally shifting the plurality of successive discrete G₂ activation times, i.e., the "teeth" of the comb, forward in time following each subsequent activation of the gate Gi until the time periods Pc where the gate G₂ is inactive have been spanned. Thus, for example, referring to FIGS. 2 and 3, at the next activation 36 of the gate G₁, an offset time period, Δ, is added to value of the most recent time delay value, TD, resulting in a delay between activation of the gate Gi and the first one of the plurality of successive discrete G₂ activation times of TD + Δ. This adds the offset time, Δ, to each of the G₂ activation times, T_A, relative to the activation of the first gate, Gi, which effectively shifts all of the "teeth" on the G₂ comb forward in time from their previous positions by an amount equal to the value of Δ, or equivalently a resolution time period, P_R, as illustrated in FIG. 3. Thereafter at the next activation 38 of the gate G₁, an offset time period, Δ, is again added to the value of the most recent time delay value, T_D, resulting in a delay between activation of the gate Gi and the first one of the plurality of successive discrete G₂ activation times of T_D + 2Δ. This again shifts all of the "teeth" on the G₂ comb forward in time from their previous positions by an amount equal to the value of Δ or PR. This process continues until the time period Pc is spanned by a sufficient number of successive additions to the delay time, T_D, of the offset value Δ.

Referring now to FIG. 4, a flowchart is shown of one illustrative process 50 for operating the analyte separation instrument 14 as just described with respect to FIGS. 2 and 3. The process 50 may be implemented in the form of one or more sets of programming instructions in embodiments of the instrument 10 wherein appropriate ones of the gating sources 22 are themselves programmable, or in the form of one or more software algorithms that are stored in a memory associated with the processor 20 and are executable by the processor 20 to control operation of one or more of the gating sources 22 in embodiments of the instrument 10 wherein operation of appropriate ones of the gating sources 22 are under the control of the processor 20.

The process 50 begins at step 52 where the delay time, T_D, is determined. In embodiments wherein it is desirable to "comb" the entire range of analytes traveling through the instrument 14, T_D is generally selected to correspond to the arrival at the gate G₂ of analytes having the shortest travel time between the gates G] and G₂. Alternatively, in embodiments where it is desirable to "comb" only a subset of the entire range of analytes traveling through the instrument 14, TD may be selected to correspond to the arrival at the gate G₂ of analytes in the subset of the entire range of analytes traveling through the instrument 14 that have the shortest travel time between the gates Gi and G₂. It will be understood that the delay time, TD, may have a positive or zero value.

Following step 52, the process 50 advances to step 54 where the period, Pc, between the comb "teeth" is determined. Generally, Pc will be selected based on a number of competing concerns. For example, in cases where there exists a large number of analytes traveling through the instrument 14 and/or the analytes are densely populated, e.g., tightly packed, in one or more analyte ranges or throughout the entire analyte range, it is desirable to select larger values of Pc so that manageable amounts of analyte separation information may be captured with each set of the plurality of successive discrete G₂ activation times. However, the total amount of data captures will increase with increasing values of Pc as large values of Pc will generally necessitate a large number of subsequent, time-shifted sets of the plurality of successive discrete G₂ activation times. The value of Pc will therefore generally be chosen based on a tradeoff between at least these concerns.

Following step 54, the process 50 advances to step 56 where the gate G₂ activation time, T_A, is determined. Selection of T_A will generally be limited at the lower end by the reaction time of the gate G₂ and on the amount of time required to allow a useful amount of ions to travel through the gate G₂. The upper limit of T_A will depend upon the desired peak resolution of the analyte separation information. Generally, TA will be selected to be a suitable value between these two limits.

In the example illustrated in FIGS. 2 and 3, the activation times, T_A, of the second gate, G₂, are illustrated as being a constant, predefined value. It will be understood, however, that the activation times, TA, may alternatively change, e.g., increase or decrease, linearly between adjacent activations of the first gate, G₁. Alternatively still, the activation times, T_A, may change, e.g., increase or decrease, non-linearly between adjacent activations of the first gate, Gi. The extent to which such activation times, T_A, change linearly or non-linearly will depend upon the type of analyte separation instrument(s) implemented in the instrument 10 and the underlying physics governing movement of analytes therethrough.

Following step 56, the process 50 advances to step 58 where the resolution period, P_R, or equivalently the offset time, Δ, is determined. P_R (or Δ) may be greater or less than T_A, or may be identical to T_A. In the embodiment illustrated in FIG. 3, for example, P_R (Δ) is selected to be equal to T_A so that data over the entire analyte separation range is captured with no overlap and no gaps between the information.

Following step 58, the process 50 advances to step 60 where a repetition value, REPS, is calculated as the ratio P_C/P_R. REPS corresponds to the number of time-shifted sets of the plurality of successive discrete G₂ activation times required to span Pc. Thereafter at step 62, the offset time value, Δ, is set to zero and a counter value, CNT, is set to 1. Thereafter at step 64, it is determined whether the gate Gi has been activated. If not, the process 50 loops back to step 64. If, however, it is determined at step 64 that the gate Gi has been activated, the process 50 advances to step 66 where the gate G₂ is activated for an activation time T_A every time period Pc beginning at a delay time TD + Δ from the most recent activation of the gate Gi. It will be noted that step 66 represents an embodiment wherein the time period, Pc, between activations of the gate G₂ is periodic between T_D + Δ and the next activation of the gate G₁, although it will be understood that this need not be the case as described hereinabove. Modifications to the process 50 to implement an embodiment wherein the time period, Pc, between activations of the gate G₂ is not periodic would be a mechanical step for a skilled artisan.

Following step 66, the process 50 advances to step 68 where the offset value Δ is incremented by the resolution period, P_R, and the count value, CNT, is incremented by 1. Thereafter at step 68, it is determined whether the count value, CNT, is equal to REPS. If so, the entire time period, Pc, between successive activations of the gate G₂ has been spanned and the process stops. If, however, it is determined at step 70 that the count value, CNT, is not equal to REPS, the process 50 loops back to step 64 to await the next activation of the gate Gl . When that occurs, the loop comprising step 64-70 is again executed.

Referring now to FIG. 5, an example of the process 50 is illustrated. In this example, the instrument 10 was essentially as described with respect to FIGS. 2 and 3. Specifically, the analyte processing instrument 16 was omitted, and the analyte separation instrument 14 was provided in the form of a two-stage ion mobility spectrometer. Ions in the form of a mixture of tryptic peptides were generated from a sample of human hemoglobin tryptic digest using an electrospray ion source as the analyte source 12. The first gate, Gj, was positioned at the ion inlet of the ion mobility spectrometer 14 as illustrated in FIG. 1, the second gate, G₂, was separated by a first distance from the first gate, G₁, and the final gate, G_F, was positioned at the ion outlet of the ion mobility spectrometer 14 and was separated by a second distance from the second gate, G₂. The first and second distances were selected such that the second distance was twice that of the first distance. The detector 18 was positioned to receive ions exiting the final gate, GF, of the ion mobility spectrometer 14. Ions from the electrospray ion source 12 were gated via Gi into the first stage of the ion mobility spectrometer 14 with a 100 microsecond gate pulse, and the ions then separated in time through the first stage, S₁. Ion groups were then gated out of the first stage, S₁, and into the second stage, S₂, via G₂ as illustrated in FIGS. 2 and 3 with a time delay, T_D, of zero, an activation time, TA, of 100 microseconds, and a period, Pc, between G₂ activations of approximately 1.0 millisecond, and with a total number of eight G₂ activations per comb. The G2 comb teeth were then advanced in time by an offset value, Δ, of 100 microseconds, and a total of 1 1 combs were used to span Pc.

FIG. 5 shows a plot 80 of ion intensity (detected by the detector 18 of FIG. 1) vs. ion drift time (through the ion mobility spectrometer 14) when operating the ion mobility spectrometer 14 in a conventional operating mode with the gate G₂ continuously activated or open to allow passage of ions therethrough. With all ions allowed to pass through G₂ as just described, the plot 80 illustrates a broad distribution of unresolved features that span drift times from approximately 22-55 milliseconds. This is effectively the time required for these ions to travel through all of the drift regions of the ion mobility spectrometer 14, and therefore represents a one-dimensional ion mobility spectrometer experiment.

FIG. 5 also shows a plot 82 of ion intensity vs. ion drift time with the second gate, G₂, operated as a comb having eight teeth following the delay period, TD, as described above. As ions from this comb are allowed to diffuse through the rest of the instrument 14, the illustrated pulses diffuse into peaks, the shapes of which are defined by the total diffusion of each packet of ions.

FIG. 5 further shows a plot 84 of ion intensity vs. ion drift time with the second gate, G₂, operated as second eight-tooth comb in which an offset time of approximately 100 microseconds is added to the delay time, TD- In this case, each of the observed peaks corresponds to a slightly different distribution of ions, differing slightly in mobilities, as compared with the first comb. As with the first comb, ions from the second comb diffuse through the rest of the instrument 14, and the illustrated pulses diffuse into peaks having shapes defined by the total diffusion of each packet of ions. By sequentially increasing the delay by multiples of the G₂ pulse width, the entire distribution of ions spanning each Pc is sampled. As shown in the last plot 86 of FIG. 5, the entire distribution of ions is sampled using 11 consecutive 8-tooth combs. It will be noted that the summation 86 of the 11 consecutive 8-tooth combs has a shape that is substantially similar to the original ion distribution 80. As the foregoing example illustrates, operating the ion separation instrument 14 according to the combing technique described herein sends fewer ions out of the ion mobility instrument 14 at any one time than by operating the instrument 14 using conventional techniques. This technique provides for the ability to enhance peak detection by providing more space in each of the successive analyte groups to further separate in one or more downstream separation stages and/or instruments.

It should be noted that while the method described herein of operating a analyte separation instrument is disclosed with reference to the analyte separation instrument 14 of FIG. 1 , it will be understood that this method may alternatively or additionally be implemented with any one or more analyte separation instrument included in the analyte source 12 and/or analyte processing instrument 16, and within one or multiple stages of the analyte separation instrument 14. The combing technique described herein in the context of analyte separation in one dimension may thus extend to a "brush" technique where analytes may be separated using the combing technique in two or more dimensions.

As an example of such a brush technique, another embodiment of the instrument 10 may comprise the analyte separation instrument 14 as just described, and also a single or multiple-stage analyte processing instrument 16 provided in the form of another analyte separation instrument. In this embodiment, the analyte separation instrument 14 may be operated using the combing technique just described, and the analyte separation instrument 16 may also be operated as a comb, i.e., using the combing technique described herein, to produce multi-dimensional ion separation information. In particular, the analyte separation instrument 16 will have an inlet with a first gate that is controllable to allow or inhibit analytes into the analyte separation instrument 16 from the analyte separation instrument 14. A second gate will be separated by a distance from the first gate of the analyte separation instrument 16, and this gate will be controllable as described above to allow or inhibit the passage of analytes therethrough. The detector 18 will be positioned to detect analytes exiting the analyte separation instrument 16.

In the operation of the analyte separation instrument 16 relative to that of the analyte separation instrument 14, the first gate of the analyte separation instrument 16 is activated to allow entrance therein of consecutive ones of the plurality of discrete analyte groups exiting the analyte separation instrument 14. The discrete analyte groups will then separate between the first and second gates of the analyte separation instrument 16 according to a second analyte characteristic. The second gate of the analyte separation instrument 16 is activated at a plurality of successive discrete time periods after activating the first gate of the analyte separation instrument 16 and before reactivating the first gate of the analyte separation instrument 16 to allow passage therethrough of a corresponding plurality of discrete groups of analytes separated from each of other according to the second analyte characteristic. The first gate of the analyte separation instrument 16 is repeatedly activated which allow the various discrete analyte groups to separate between the first and second gates of the analyte separation instrument 16, and between repeated activations of the first gate the second gate of the analyte separation instrument 16 is activated a plurality of times. For each of the plurality of times, an offset time, relative to activating the first gate of the analyte separation instrument 16, is consecutively added to each of the times at which the second gate of the analyte separation instrument 16 is activated. The analyte separation instrument 16 may take the form of a liquid chromatograph configured to separate analytes as another function of analyte retention time, a gas chromatograph configured to separate analytes as yet another function of analyte retention time, a capillary electrophoresis instrument configured to separate ions as a function of ion charge-to-size ratio or electrophoretic mobility, an ion separation instrument configured to separate ions as a function of ion mass-to-charge ratio and an ion mobility spectrometer configured to separate ions as a function of ion mobility, in embodiments in which the analyte separation instrument 14 is any of a gas chromatograph, a liquid chromatograph, a capillary electrophoresis instrument and a mass analyzer or spectrometer. In embodiments in which the analyte separation instrument 14 is an ion mobility spectrometer, the analyte separation instrument 16 may take the form of a liquid chromatograph configured to separate analytes as another function of analyte retention time, a gas chromatograph configured to separate analytes as yet another function of analyte retention time, a capillary electrophoresis instrument configured to separate ions as a function of ion charge-to-size ratio or electrophoretic mobility and another ion mobility spectrometer configured to separate ions as another function of ion mobility.

Referring now to FIG. 6, a flowchart is shown of one illustrative process 100 for mapping one or more biomarkers to corresponding analyte intensity information resulting from an analyte separation process that produces discrete groups of analytes. One example of such an analyte separation process is that illustrated in FIG. 4 and described herein, although it will be understood that the mapping process 100 is not limited to the analyte separation process illustrated in FIG. 4 and described herein. At least some of the process 100 may be provided in the form of one or more software algorithms that may be stored in a memory associated with the processor 20 and that may be executed by the processor 20. Alternatively, one or more such algorithms may be stored in a memory associated with a remote processor, such as a conventional personal computer, laptop computer or the like, and may be executed by such a remote processor. In any case, the process 100 begins at step 102 where the analyte intensity data that was generated according to the analyte separation process of FIG. 4 is used to create a matrix of analyte intensity values. Referring to FIG. 7, one illustrative technique for creating a matrix 120 of analyte intensity values according to step 102 of the process 10 is shown. The matrix 120 generally has a number, M, of rows, wherein M may be any positive integer. Each row represents a comb number, corresponding to a single set of analyte intensity values resulting from a corresponding set of the plurality of successive discrete activation times of the gate G₂. Thus, for example, row 1 represents the analyte intensity values resulting from comb number 1, corresponding to the analyte intensity values resulting from the plurality of successive discrete gate G₂ activation times that begin after the delay time T_D from the first activation of the gate Gi. Row 2 represents the analyte intensity values resulting from comb number 2, corresponding to the analyte intensity values resulting from the plurality of successive discrete gate G₂ activation times that begin after the delay time T_D + Δ from the second activation of the gate Gi and so forth. The value of M corresponds to the total number of sets of gate G₂ activation times required to span the complete range of analytes traveling through the instrument 14. Alternatively, the analyte intensity values resulting from the various different comb numbers could be entered in consecutive columns.

The matrix 120 generally has N "coarse" columns, wherein N may be any positive integer. Each of the N coarse columns corresponds to a tooth of the comb, i.e., to one of the plurality of successive activation times of the gate G₂ in each set of activation times. Thus, for example, the coarse column 1 holds analyte intensity values resulting from the first activation of the gate G₂ following each activation of the gate G₁, column 2 holds analyte intensity values resulting from the second activation of the gate G₂ following each activation of the gate Gi, and so forth. The value of N thus corresponds to the total number of gate G₂ activation times following each activation of the gate Gi. Alternatively, in embodiments in which the analyte intensity values resulting from the various different comb numbers are entered in consecutive columns, the analyte intensity values corresponding to the various teeth of any comb number could be entered in "coarse" rows. In the simplest form of the analyte separation instrument 14, e.g., a single-stage instrument having no additional analyte processing mechanism, the matrix 120 would be an M x N matrix populated with analyte intensity values as just described. However, other forms of the analyte separation instrument 14 may include two or more analyte separation stages, one or more analyte activation regions, and/or the like. The analyte separation, analyte activation and/or other analyte processing may occur before, during and after the combing process 50 described herein. During any such analyte separation, analyte activation and/or other analyte processing that occurs after the combing process, analytes may continue to resolve, i.e., further separate, change in conformation, fragment or undergo one or more additional analyte processing that results in additional analyte intensity information. Accordingly, the matrix 120 of FIG. 7 includes a number, P, of additional "fine" columns following each "coarse" column where the additional analyte intensity information resulting from such further analyte separation, analyte activation and/or other analyte processing of each "coarse" group of analytes is stored. P may be any positive integer, and the value of P corresponds to the total number of additional analyte intensity data values that result from further analyte separation, analyte activation and/or other analyte processing of each "coarse" group of analytes. In the general case, this then results in a total number of PP +N columns of the matrix 120. Alternatively, in embodiments in which the analyte intensity values resulting from the various different comb numbers are entered in consecutive columns, the additional analyte intensity values could be entered in "fine" rows next to the "coarse" rows. Step 102 of the process 100 presupposes that an analyte separation instrument has been operated in a manner that produces a plurality of discrete analyte groups from a packet of a mixture of analytes, as described herein, and that analyte intensities of each of the plurality of discrete analyte groups have been determined. In one embodiment of step 102, the matrix of the analyte intensities can be created by entering an analyte intensity value of each of the plurality of discrete analyte groups into a separate row or column of a common, i.e., the same, column or row of the matrix. Thus, if the analyte intensity values are entered in separate columns, they must appear in the same, or common, row of the matrix, as illustrated in FIG. 7. Conversely, if the analyte intensity values are entered in separate rows of the matrix, they must then appear in the same, or common, column of the matrix. For each new comb, another common column or row is created in the matrix to accommodate the analyte intensity values associated with the new comb. If an additional analyte intensity value is generated from one of the discrete analyte groups, the additional analyte intensity value is entered into a row (or column) of the matrix that is within the common column (or row), and that is adjacent to the row (or column) in which the analyte intensity value of the discrete analyte group was entered such that the analyte intensity value of the discrete analyte group and the additional analyte intensity value appear sequentially in the common column (or row). Multiple analyte intensity values generated by any discrete analyte group likewise appear sequentially in the matrix as illustrated in FIG. 7. It will be appreciated that in embodiments wherein the combing technique described herein is additionally implemented two or more times in the analyte separation instrument 14, in the analyte source 12 and/or in the analyte processing instrument 16, multiple matrices 120 will result.

Referring again to FIG. 6, the process 100 advances from step 102 to step 104 where the matrix entries that define one or more biomarkers are identified. This is illustrated graphically in FIG. 8 which shows identification of three locations in a 12 x 19 matrix 130 that define a first biomarker and four locations in the matrix 130 that define a second biomarker. Generally, biomarker may be any substance that is used as an indicator of a biological state. For example, one type of biomarker may be any kind of analyte indicating the existence (past or present) of living organisms. Another type of biomarker may be any substance that is introduced in an organism for the purpose of examining organ function or other aspects of health. Yet another type of biomarker may be any substance whose detection indicates a particular disease state or exposure to any environmental substance such as a toxin. Another type of biomarker may be a fragment of DNA sequence that is associated with a disease, that changes susceptibility to disease or that causes disease. Still another type of biomarker may be one of a number of components of a biological sample. For example, biomarkers of a sample of human tissue or fluid may include, but are not limited to, all proteins, proteins remaining after abundant protein removal, low analyte weight proteins, glycans, lipids, peptides without glycans, phosphorylated peptides and metabolites. Other types and/or examples of biomarkers will occur to those skilled in the art, and any such other types and/or examples are contemplated by this disclosure.

Generally, any substance that defines a biomarker of interest relating to the sample being analyzed may be identified as being defined by a combination of analyte intensity values from the matrix generated at step 102 of the process 100. Thus, in FIG. 8 for example, the biomarker Bl is identified as being defined by the analyte intensity values stored in the matrix at row 5, column 4, at row 8, column 8 and at row 3, column 17. Similarly, the biomarker B2 is identified as being defined by the analyte intensity values stored in the matrix at row 2, column 6, at row 4, column 9, at row 3, column 11 and at row 10, column 16. This identification process may be done manually, or may instead be automated. The identification process may be assisted by consulting one or more databases of biomarkers and/or substances. In embodiments in which multiple matrices have been generated, step 104 may be carried out by identifying entries in any one or more of the matrices that define the biomarker.

Referring again to FIG. 6, the process 100 advances from step 104 to step 106 where a map is created that correlates one or more biomarkers to corresponding locations in the matrix created at step 102 of the process 100. Referring to FIG. 9, an example map 140 is shown illustrating one illustrative technique for creating a map correlating the two biomarkers Bl and B2 of FIG. 8 to corresponding locations in the matrix 130. In the illustrated embodiment, the first two digits of the biomarker map values contained in the map 140 indicate the number of matrix locations that define the biomarker. Thus, the biomarker Bl is defined by three locations in the matrix 130 and the biomarker B2 is defined by four locations in the matrix 130. The next two digits of each biomarker map value identify the number of digits used to identify the row and column of the first matrix location. In the example illustrated in FIG. 9, the second two digits in each of the biomarker map values indicate that the row and column values of the first matrix location are each single digits. The next two digits of each biomarker map value are the actual row and column of the first location of the matrix 130 that defines the corresponding biomarker. In the example illustrated in FIG. 9, the first matrix location that defines the biomarker Bl is row 5, column 4, and the first matrix location that defines the biomarker B2 is row 2, column 6. The remaining digits of each of the biomarker map values are processed in like manner to determine all of the matrix locations that define each of the biomarkers of interest. It will be understood that simpler or more complicated techniques may be implemented to create the map correlating one or more biomarkers to corresponding matrix locations, and any such simpler or more complicated techniques are contemplated by this disclosure. In embodiments in which multiple matrices have been generated, step 106 may be carried out by creating a map that correlates the biomarker to the identified entries in any one or more of the matrices.

Referring again to FIG. 6, the process 100 may advance to step 108 where the map 140 is used to identify specific analyte separation data to analyze when investigating one or more biomarkers of other samples. In one embodiment, for example, the analyte intensity values identified by the map created at step 106 may represent baseline values to which corresponding analyte intensity values of other samples may be compared. In another embodiment, the analyte intensity values identified by the map created at step 106 may be averaged with corresponding analyte intensity values of multiple maps to create baseline values to which corresponding analyte intensity values of other samples may be compared.

Alternatively or additionally, the process 100 may advance from step 106 to step 110 where the map 140 is used to identify specific comb and teeth numbers to monitor when investigating biomarkers of future samples of the same type used to generate the matrix created at step 102. For example, the matrix locations of the analyte intensity information that define a biomarker correspond, and may be mapped back to, specific comb and tooth numbers from which the matrix was generated. In subsequent analyses of samples of the same type used to generate the matrix, the comb and tooth numbers may then be used to identify specific analyte intensity values to monitor for such analyses. While the invention has been illustrated and described in detail in the foregoing drawings and description, the same is to be considered as illustrative and not restrictive in character, it being understood that only illustrative embodiments thereof have been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected. For example, while one or more implementations of the analyte separation instrument 14 and analyte processing instruments 16 have been described herein as including at least first and second gates, it will be understood that such gates may in some embodiments be implanted in the form of actual gates as described above, or may instead be implemented in the form of two or more cascaded analysis instruments. As one specific example, which is not intended to be limiting in any was, the instrument 10 may comprise a two-stage ion mobility spectrometer 14, followed by two cascaded capillary electrophoresis instruments. In this embodiment, the "gates" of the two- stage ion mobility spectrometer 14 may be provided in the form of actual gates, as described herein. The first gate of the capillary electrophoresis instrument may correspond to an inlet gate of a first one of the cascaded capillary electrophoresis instruments, and the second gate of the capillary electrophoresis instrument may correspond to the outlet gate of the first one of the cascaded capillary electrophoresis instruments, and/or the inlet gate of the second one of the cascaded capillary electrophoresis instruments. Those skilled in the art will recognize that the term "gate," as used herein, thus covers implementations using physical gates and also implementations using two or more cascaded instruments.

Claims

CLAIMS:

1. A method of correlating analyte separation information to a biomarker comprising: operating an analyte separation instrument to produce a plurality of discrete analyte groups from a packet of a mixture of analytes, determining analyte intensities of each of the plurality of discrete analyte groups, creating a matrix of the analyte intensities of each of the plurality of discrete analyte groups, identifying entries of the matrix that define the biomarker, and creating a map correlating the biomarker to the identified entries of the matrix.

2. The method of claim 1 wherein the analyte separation instrument has an inlet with a first gate that is controllable to allow or inhibit entrance of analytes into the instrument from an analyte source, and a second gate that is separated by a distance from the first gate and that is controllable to allow or inhibit passage of analytes therethrough, and wherein operating an analyte separation instrument comprises: activating the first gate to allow entrance of the packet of the mixture of analytes from the analyte source into the instrument, allowing the packet of analytes to separate between the first and second gates as a function of a first analyte characteristic, and activating the second gate at a plurality of successive discrete time periods after activating the first gate and before reactivating the first gate to allow passage therethrough of the plurality of discrete analyte groups separated from each of other according to the function of the first analyte characteristic.

3. The method of claim 2 wherein creating a matrix of the analyte intensities comprises entering an analyte intensity value of each of the plurality of discrete analyte groups into a separate row or column of a common column or row of the matrix.

4. The method of claim 3 further comprising: repeatedly activating the first gate, allowing the packet of analytes to separate and activating the second gate a number of times, and for each of the number of times, consecutively adding an offset time, relative to activating the first gate, to each of the times at which the second gate is activated.

5. The method of claim 4 wherein creating a matrix of analyte intensity values comprises creating another common column or row each of the number of times that activating the first gate, allowing the packet of analytes to separate and activating the second gate are repeated.

6. The method of claim 3 further comprising processing one of the plurality of discrete analyte groups to produce an additional analyte intensity value, wherein creating a matrix of the analyte intensities comprises entering the additional analyte intensity value into a row or column of the matrix that is within the common column or row and that is adjacent to the row or column in which the analyte intensity value of the one of the plurality of discrete analyte groups was entered such that the analyte intensity value of the one of the plurality of discrete analyte groups and the additional analyte intensity value appear sequentially in the common column or row.

7. The method of claim 6 further comprising processing each of the plurality of discrete analyte groups to produce one or more additional analyte intensity values, wherein creating a matrix of the analyte intensities comprises entering the one or more additional analyte intensity values for each of the plurality of discrete analyte groups into the matrix such that the analyte intensity value of each of the plurality of discrete analyte groups and the one or more corresponding additional analyte intensity values appear sequentially in the common column or row.

8. The method of claim 6 further comprising: repeatedly activating the first gate, allowing the packet of analytes to separate and activating the second gate a number of times, and for each of the number of times, consecutively adding an offset time, relative to activating the first gate, to each of the times at which the second gate is activated.

9. The method of claim 8 wherein creating a matrix of analyte intensity values comprises creating another common column or row each of the number of times that activating the first gate, allowing the packet of analytes to separate and activating the second gate are repeated.

10. The method of claim 6 wherein processing one of the plurality of discrete analyte groups to produce an additional analyte intensity value comprises processing the one of the plurality of discrete analyte groups within the analyte separation instrument in a manner that produces the additional analyte intensity value.

1 1. The method of claim 10 wherein processing the one of the plurality of discrete analyte groups within the analyte separation instrument comprises activating the one of the plurality discrete analyte groups within the analyte separation instrument.

12. The method of claim 10 wherein the analyte separation instrument has a third gate that is separated by another distance from the second gate and that is controllable to allow or inhibit passage of analytes therethrough, and wherein processing the one of the plurality of discrete analyte groups within the analyte separation instrument comprises allowing the one of the plurality of discrete analyte groups to separate between the second and third gates as a function of the first analyte characteristic.

13. The method of claim 6 further comprising another analyte separation instrument having an analyte inlet coupled to an analyte outlet of the analyte separation instrument, and wherein processing one of the plurality of discrete analyte groups to produce an additional analyte intensity value comprises processing the one of the plurality of discrete analyte groups within the another analyte separation instrument in a manner that produces the additional analyte intensity value.

14. The method of claim 13 wherein processing the one of the plurality of discrete analyte groups within the another analyte separation instrument comprises allowing the one of the plurality of discrete analyte groups to separate within the another analyte separation instrument as a function of a second analyte characteristic.

15. The method of claim 3 wherein the analyte separation instrument is a liquid chromatograph, and wherein the first analyte characteristic is analyte retention time.

16. The method of claim 1 wherein the analyte separation instrument is a gas chromatograph, and wherein the first analyte characteristic is analyte retention time.

17. The method of claim 1 wherein the analyte separation instrument is a capillary electrophoresis instrument, and wherein the first analyte characteristic is analyte charge-to-size ratio or electrophoretic mobility.

18. The method of claim 1 wherein the analyte separation instrument is a mobility spectrometer, and wherein the first analyte characteristic is analyte mobility.

19. The method of claim 1 further comprising: operating another analyte separation instrument to produce another plurality of discrete analyte groups from the plurality of discrete analyte groups, determining analyte intensities of each of the another plurality of discrete analyte groups, and creating another matrix of the analyte intensities of each of the another plurality of discrete analyte groups.

20. The method of claim 19 wherein identifying entries of the matrix that define the biomarker comprises identifying entries of either of the matrix and the another matrix that define the biomarker.

21. The method of claim 20 wherein creating a map correlating the biomarker to the identified entries of the matrix comprises creating a map correlating the biomarker to the identified entries of either of the matrix and the anther matrix.