WO2003104763A2

WO2003104763A2 - Apparatus and method for relative or quantitative comparison of multiple samples

Info

Publication number: WO2003104763A2
Application number: PCT/US2003/017034
Authority: WO
Inventors: David E. Clemmer; Amy E. Hilderbrand
Original assignee: Advanced Research And Technology Institute, Inc.
Priority date: 2002-06-05
Filing date: 2003-05-30
Publication date: 2003-12-18
Also published as: AU2003247442A8; WO2003104763A3; AU2003247442A1

Abstract

An apparatus and method are provided for conducting comparative relative or quantitative analysis of multiple samples. The apparatus and method may include post-ionization, gas-phase combination of one or more of the multiple samples with one or more reagents for quantitative or relative comparison of one or more of the multiple samples.

Description

APPARATUS AND METHOD FOR RELATIVE OR QUANTITATIVE COMPARISON OF MULTIPLE SAMPLES

FIELD OF THE INVENTION

The present invention relates generally to techniques for conducting comparative relative or quantitative analysis of multiple samples, wherein one or more such techniques may include post-ionization, gas-phase tagging of one or more of the multiple samples for quantitative or relative comparison thereof.

BACKGROUND OF THE INVENTION

The analysis of complex mixtures is emerging as a common theme across many areas of science, including areas relating to biological and medicinal chemistry, environmental science, the chemical sciences, and the like. Complex mixture analysis typically pursues two primary goals; namely component identification and determination of the abundances (concentration) of the different components. The first of these goals has been significantly impacted by recent advancements in mass spectrometry. Accurate and precise measurements of mass- to-charge ratios (m/z) has led to complete component identification in increasingly complex chemical and biological compositions.

The second of these goals has also been the subject of recent attention, particularly in emerging fields of extreme complexity such as proteomics and combinatorial chemistry. For example, while it is known that genes produce proteins, it is not enough to measure changes in gene concentration in order to understand variations of protein concentrations in the cell. Protein concentrations are affected by many other factors, especially factors relating to digestion and elimination of the protein from the cell. Additionally, proteins can be chemically modified after they are synthesized by a process known as post-translational modification. Although patterns are emerging for the sequences of DNAs and proteins and how they relate to one another, much less is understood about patterns that are associated with the post-translational modifications; although it is well known that some modifications are directly related to cellular function, including areas of disease such as cancer. It is accordingly desirable to record both the identities and abundances of large mixtures of proteins directly. It is further desirable to determine the abundances of large mixtures of metabolites, defined here as peptides, steroids and lipids, as well as other small molecules with biological function. A number of approaches for directly determining the relative and absolute abundances of proteins in complex mixtures are known. These approaches are all similar in the respect that, at some point, a complex molecular mixture is reacted in solution in such a way that it incorporates an isotopic label or tag. For example, proteolytic digestion can be carried out in the presence of heavy oxygen, ¹⁸O, such that every carboxylic acid end group of the peptide incorporates the heavy isotope. If a mixture was digested in the presence of ¹⁶O, then only the light isotope would be incorporated. If the heavy and light isotopic mixtures were combined, a series of doublets would result from a measurement of their mass spectrum. The intensities of the resulting pairs of peaks are a direct measure of the relative abundances of each peptide in the mixtures. An internal standard is thus created for every component of the mixture.

Several commercialized technologies currently exist that are based on the foregoing concepts. Examples of two such commercialized technologies include isotope-coded affinity tags (ICAT), described in "Quantitative Analysis of Complex Mixtures Using Isotope-Coded Affinity Tags", S. P. Gygi et al., Nature Biotechnology, Vol. 17, 1999, and global internal standard technology (GIST), described in "Comparative Proteomics Based on Stable Isotope Labeling and Affinity Selection", F. E. Regnier et al., J. Mass Spectrom. 2002, 37:133-145, both publications of which are incorporated herein by reference. All such technologies require reaction of complex mixtures in solution to incorporate an isotopic label or tag. These labeled or tagged mixtures are then ionized for subsequent spectrographic analysis.

SUMMARY OF THE INVENTION

The present invention is directed to techniques for conducting relative or quantitative comparison of multiple samples, and may comprise at least one or more of the following features or combinations thereof. An apparatus for comparative relative or quantitative analysis of a plurality of samples may comprising a plurality of ion sources each configured to generate ions from a different one of the plurality of samples. A capillary structure may have a plurality of ion inlets each coupled to a corresponding one of the plurality of ion sources and an ion outlet, wherein the capillary structure is configured to direct the ions generated by each of the plurality of ion sources to the ion outlet. A molecular analysis instrument may have an ion inlet coupled to the ion outlet of the capillary structure and configured to produce molecular intensity information as a function of at least one molecular characteristic for relative or quantitative comparison of the plurality of samples.

The apparatus may further include a computer configured to control introduction of ions from each of the plurality of ion sources into the capillary structure, wherein the computer includes a memory unit for storing therein the molecular intensity information produced by the molecular analysis instrument. The computer may be configured to control introduction of ions from each of the plurality of ion sources into the capillary structure such that ions generated by each of the plurality of ion sources are introduced into the capillary structure simultaneously. Alternatively, the computer may be configured to control introduction of ions from each of the plurality of ion sources into the capillary structure such that ions generated by each of the plurality of ion sources are introduced sequentially into the capillary structure, wherein the molecular intensity information produced by the molecular analysis instrument for each of the plurality of sets of sequentially introduced ions are stored by the computer in the memory unit for subsequent relative or quantitative comparison of the plurality of samples.

The apparatus may further include a first reagent source configured to introduce a first sample-labeling reagent into the capillary structure for combination with ions generated by one or more of the plurality of ion sources. In this embodiment, the apparatus may further include a computer configured to control introduction of ions from each of the plurality of ion sources and introduction of the first sample-labeling reagent from the first reagent source into the capillary structure, wherein the computer includes a memory unit for storing therein the molecular intensity information produced by the molecular analysis instrument. The computer may be configured to control introduction of ions from each of the plurality of ion sources and introduction of the first sample-labeling reagent into the capillary structure in pairs of ion sets such that a first set of ions generated by one of the plurality of ion sources is introduced into the capillary structure followed or preceded sequentially by a second set of ions generated by another one of the plurality of ion sources simultaneously with the first sample-labeling reagent so that the first sample-labeling reagent combines with the second set of ions, and wherein the molecular intensity information produced by the molecular analysis instrument for each of the pairs of sequentially introduced ions are stored by the computer in the memory unit for subsequent relative or quantitative comparison of the plurality of samples.

The capillary structure may include a reagent inlet coupled to the first reagent source, and the capillary structure may define a length between a junction of the reagent inlet and the ion inlet of the capillary structure coupled to the another one of the plurality of ion sources and the ion outlet of the capillary structure, wherein a reaction rate of the first sample-labeling reagent and the second set of ions is a function of the length.

The apparatus may further include means for modifying an operating temperature of at least a portion of the capillary structure carrying the first sample- labeling reagent, wherein a reaction rate of the first sample-labeling reagent and the second set of ions is a function of the operating temperature of the at least a portion of the capillary structure.

The apparatus may further include means for controlling the pressure of the first sample-labeling reagent supplied to the capillary structure by the first reagent source, wherein a reaction rate of the first sample-labeling reagent and the second set of ions is a function of the pressure of the first sample-labeling reagent.

The apparatus may further include a second reagent source configured to introduce a second sample-labeling reagent into the capillary structure for combination with ions generated by one or more of the plurality of ion sources. In this embodiment, the computer may be configured to control introduction of ions from each of the plurality of ion sources, introduction of the first sample-labeling reagent and introduction of the second sample-labeling reagent into the capillary structure in pairs of ion sets such that a first set of ions generated by one of the plurality of ion sources is introduced into the capillary structure simultaneously with the first sample- labeling reagent so that the first sample-labeling reagent combines with the first set of ions followed or preceded sequentially by a second set of ions generated by another one of the plurality of ion sources simultaneously with the second sample- labeling reagent so that the second sample-labeling reagent combines with the second set of ions, and wherein the molecular intensity information produced by the molecular analysis instrument for each of the pairs of sequentially introduced ions are stored by the computer in the memory unit for subsequent relative or quantitative comparison of the plurality of samples. The first and second regents may be selected to provide for a definable molecular characteristic shift in the molecular intensity information between the first set of ions and the second set of ions. In one embodiment, the first and second sample-labeling reagents may be deuterated and non-deuterated reagent pairs. The first set of ions may represent a distribution of ions generated from a sample of known composition and having known analyte concentration levels and the second set of ions represent a distribution of ions generated from a sample of same known composition but having unknown analyte concentration levels, wherein comparison of the molecular intensity information for the first set of ions and the molecular intensity information for the second set of ions provides for identification of abundances of the analyte concentration levels in the second set of ions.

In this embodiment, the capillary structure may include a first sample-labeling reagent inlet coupled to the first reagent source and a second reagent inlet coupled to the second reagent source, such that the capillary structure defines a first length between a junction of the first sample-labeling reagent inlet and the ion inlet of the capillary structure coupled to the one of the plurality of ion sources and the ion outlet of the capillary structure, and a second length between a junction of the second reagent inlet and the ion inlet of the capillary structure coupled to the another one of the plurality of ion sources and the ion outlet of the capillary structure. A reaction rate of the first sample-labeling reagent and the first set of ions is a function of the first length and a reaction rate of the second sample-labeling reagent and the second set of ions is a function of the second length.

The apparatus in this embodiment may further include means for modifying an operating temperature of at least a portion of the capillary structure carrying the first and second sample-labeling reagents, wherein a reaction rate of the first sample- labeling reagent and the first set of ions and a reaction rate of the second sample- labeling reagent and the second set of ions is a function of the operating temperature of the at least a portion of the capillary structure.

The apparatus in this embodiment may further include means for controlling the pressure of the first sample-labeling reagent supplied to the capillary structure by the first reagent source, and means for controlling the pressure of the second sample-labeling reagent supplied to the capillary structure by the second reagent source, wherein a reaction rate of the first sample-labeling reagent and the first set of ions is a function of the pressure of the first sample-labeling reagent and a reaction rate of the second sample-labeling reagent and the second set of ions is a function of the pressure of the second sample-labeling reagent.

One or more of the plurality of ion sources may be a matrix-assisted laser desorption ion (MALDI) source. Alternatively or additionally, one or more of the plurality of ion sources may be an electrospray ionization (ESI) source. Alternatively or additionally, one or more of the plurality of ion sources may be an electron impact ionization (El) source. Alternatively or additionally, one or more of the plurality of ion sources may be a chemical ionization (Cl) source. Alternatively or additionally, one or more of the plurality of ion sources may include an ion trap operable to selectively collect and release ions generated from a corresponding sample. Alternatively or additionally, one or more of the plurality of ion sources may include a gated ion collection chamber operable to selectively collect and release ions generated from a corresponding sample. Alternatively or additionally, one or more of the plurality of ion sources may include a charge normalization stage operable to selectively normalize the charge of ions generated from a corresponding sample. In one embodiment, the charge normalization stage may include a radiation source configured to emit radiation to normalize ions having various charge states to a predefined charge state. Alternatively or additionally, the charge normalization stage may include a reagent source providing a reagent selected to spread crowded ion mass peaks, resulting from multiply-charged ions generated from the corresponding sample, over a wider mass range. Alternatively or additionally, one or more of the ion sources may include a non-ionizing sample source producing particles from a corresponding sample, and a particle ionizing instrument configured to ionize the particles produced by the non-ionizing sample source.

One or more of the ion sources may include at least one ion separation instrument operable to separate ions in time as a function of a molecular characteristic. In this embodiment, the at least one ion separation instrument may be or include a mass spectrometer configured to separate ions in time as a function of ion mass-to-charge ratio. Alternatively or additionally, the at least one ion separation instrument may be or include an ion mobility spectrometer configured to separate ions in time as a function of ion mobility. Alternatively or additionally, the at least one ion separation instrument may be or include a liquid chromatograph configured to separate ions in time as a function of ion retention time. Alternatively or additionally, the at least one ion separation instrument may be or include a gas chromatograph configured to separate ions in time as a function of ion retention time. One or more of the ion sources may include an ion mass filter operable to selectively collect and release only ions within a predefined range of mass-to-charge ratios. Alternatively or additionally, one or more of the ion sources may include an ion fragmentation stage operable to selectively fragment ions generated from a corresponding sample into parent and daughter ions. The molecular analysis instrument may be or include at least one mass spectrometer. Alternatively or additionally, the molecular analysis instrument may be or includes at least one ion mobility spectrometer. Alternatively or additionally, the molecular analysis instrument may be or include at least one gas chromatograph. Alternatively or additionally, the molecular analysis instrument may be or include at least one liquid chromatograph. Additionally, the molecular analysis instrument may include at least one ion trap. Alternatively or additionally, the molecular analysis instrument may include at least one ion mass filter configured to selectively collect and release only ions within a predefined range of mass-to-charge ratios. Alternatively or additionally, the molecular analysis instrument may include at least one ion fragmentation stage operable to selectively fragment ions into parent and daughter ions. Alternatively or additionally, the molecular analysis instrument may include at least one charge normalization stage.

The apparatus may further include a reagent source configured to introduce a reagent into the capillary structure for combination with ions generated by one or more of the plurality of ion sources. The ions generated by at least one of the plurality of ion sources may be ions including a predetermined organic component contained in a larger organic structure, and the reagent may be selected to adduct to the predetermined organic component contained in the larger organic structure to shift molecular intensity information of the larger organic structure containing the predetermined organic component to different regions of one or more molecular characteristic spectra. The larger organic structure may be, for example, a peptide and the predetermined organic component may be, for example, one of a specific amino acid and a type of amino acid. In this embodiment, the molecular analysis instrument may include a number of cascaded molecular analysis units each configured to produce molecular intensity information as a function of a different molecular characteristic, and the molecular intensity information of the larger organic structure containing the predetermined organic component in this embodiment may thus be shifted to a different region of the molecular characteristic spectrum of at least one of the number of different molecular characteristics.

A method of conducting comparative relative or quantitative analysis of a plurality of samples may comprise one or more of the following acts of generating ions from each of a plurality of different samples, directing the ions generated from each of the plurality of different samples to an inlet of a molecular analysis instrument, analyzing the ions generated from each of the plurality of different samples via the molecular analysis instrument to produce molecular intensity information as a function of at least one molecular characteristic, and relatively or quantitatively comparing the molecular intensity information of the ions generated from each of the plurality of different samples.

The act of directing the ions generated from each of the plurality of different samples to an inlet of a molecular analysis instrument may include simultaneously directing the ions generated from each of the plurality of different samples into the inlet of the molecular analysis instrument. The method may further include combining ions generated from at least one of the plurality of different samples with a first sample-labeling reagent. In this embodiment, the act of directing the ions generated from each of the plurality of different samples to an inlet of a molecular analysis instrument may include sequentially directing ions generated from one of the plurality of different samples into the inlet of the molecular analysis instrument, and directing ions generated from another one of the plurality of different samples combined with the first sample- labeling reagent into the inlet of the molecular analysis instrument, wherein the act of relatively or quantitatively comparing the molecular intensity information of the ions generated from each of the plurality of different samples may include relatively or quantitatively comparing molecular intensity information for the ions generated from the one of the plurality of different samples with molecular intensity information for the ions generated from the another one of the plurality of different samples combined with the first sample-labeling reagent. The method may further including combining ions generated from at least another one of the plurality of different samples with a second sample-labeling reagent. In this embodiment, the act of directing the ions generated from each of the plurality of different samples to an inlet of a molecular analysis instrument may include sequentially directing ions generated from one of the plurality of different samples combined with the first sample-labeling reagent into the inlet of the molecular analysis instrument, and directing ions generated from another one of the plurality of different samples combined with the second sample-labeling reagent into the inlet of the molecular analysis instrument, wherein the act of relatively or quantitatively comparing the molecular intensity information of the ions generated from each of the plurality of different samples may include relatively or quantitatively comparing molecular intensity information for the ions generated from the one of the plurality of different samples combined with the first sample-labeling reagent with molecular intensity information for the ions generated from the another one of the plurality of different samples combined with the second sample-labeling reagent.

The first and second sample-labeling reagents may be selected to provide for a definable molecular characteristic shift in the molecular intensity information between the ions generated from the one of the plurality of different samples combined with the first sample-labeling reagent and the ions generated from the another one of the plurality of different samples combined with the second sample- labeling reagent. In one embodiment, the first and second sample-labeling reagents may be deuterated and non-deuterated reagent pairs.

The one of the plurality of different samples may be a sample of known composition and having known analyte concentration levels and the another one of the plurality of different samples may be a sample of the same known composition and having unknown analyte concentration levels, wherein the act of relatively or quantitatively comparing the molecular intensity information of the ions generated from each of the plurality of different samples may include comparing molecular intensity information for the ions generated from the one of the plurality of different samples combined with the first sample-labeling reagent with molecular intensity information for the ions generated from the another one of the plurality of different samples combined with the second sample-labeling reagent to determine the abundances of the analyte concentration levels in the ions generated from the another one of the plurality of different samples.

The relative or quantitative comparison apparatus may be operable in a temporal analysis mode to allow entrance of ions produced by a first one of the ion sources into the capillary structure for a first time period while inhibiting ions produced by the second one of the ion sources from entering the capillary structure, wherein the molecular analysis instrument is operable to analyze this first group of ions to produce a first molecular spectrum, and to thereafter allow entrance of ions produced by the second ion source into the capillary structure for a second time period while inhibiting ions produced by the first ion source from entering the capillary structure, wherein the molecular analysis instrument is operable to analyze this second group of ions to produce a second molecular spectrum, and to thereafter compare the first and second molecular spectra to determine quantitative or relative information relating to the first and second ion groups. The apparatus may include one or more isotopic labeling reagent sources coupled to the capillary structure such that ions produced by any one or more ion sources may be selectively reacted with a corresponding sample-labeling reagent prior to entrance into the inlet of the molecular analysis instrument to thereby tag or label such ionized samples. The one or more reagent sources may be or include a reagent gas source operable to supply reagent gas to the capillary structure. Alternatively or additionally, the one or more of the reagent sources may be or include a reagent solution source operable to supply reagent mist or droplets to the capillary structure. Alternatively or additionally, the one or more of the reagent sources may be or include a reagent sample; e.g., solid, liquid or gas, supplying reagent vapor to the capillary structure. Alternatively or additionally, the one or more of the reagent sources may be or include an ionization instrument configured to supply ionized reagent to the capillary structure.

The relative or quantitative comparison apparatus may be operable in a temporal labeling or tagging and analysis mode to allow entrance of ions produced by a first one of the ion sources and reagent produce by a reagent source into the capillary structure for a first time period while inhibiting ions produced by the second one of the ion sources from entering the capillary structure, wherein the molecular analysis instrument is operable to analyze the resulting reacted first group of ions to produce a first molecular spectrum, and to thereafter allow entrance of ions produced by the second ion source into the capillary structure for a second time period while inhibiting ions produced by the first ion source and reagent produced by the reagent source from entering the capillary structure, wherein the molecular analysis instrument is operable to analyze this second group of ions to produce a second molecular spectrum, and to thereafter compare the first and second molecular spectra to determine quantitative or relative information relating to the first and second ion groups. Such a comparison apparatus may include a number of such capillary structures each coupled to a separate inlet of an ion funneling stage having a single output coupled to the inlet of the molecular analysis instrument. Such a comparison apparatus may be operable to quantitatively or relatively compare molecular characteristic spectra of a number of different samples, one or more of which may have been reacted with a suitable reagent.

The relative or quantitative comparison apparatus may have a first capillary coupled to the inlet of the molecular analysis instrument, a first ion source coupled to the first capillary and producing ions from a first sample of known type and concentration, a first reagent source coupled to the first capillary and producing a first reagent, a second capillary coupled to the inlet of the molecular analysis instrument, a second ion source coupled to the second capillary and producing ions from a second sample of same type as the first sample but of unknown concentration, and a second reagent source coupled to the second capillary and producing a second reagent that is a deuterated form of the first reagent, wherein the first reagent reacts with ions produced by the first sample to form a first isotropic tag or label and the second reagent reacts with ions produced by the second sample to form a second isotropic tag or label, and a computer is operable to analyze ion peak information produced by the molecular analysis instrument to identify peaks of interest via the first and second isotropic tags or labels and determine therefrom quantitative or relative abundance information.

These and other objects will become more apparent from the following description of the illustrative embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a partial cross-sectional view of one illustrative embodiment of a post-ionization, multiple-sample tagging and relative or quantitative comparison instrument. FIG. 2A is a diagrammatic illustration of one embodiment an ion source suitable for use with the instrument illustrated in FIG. 1.

FIG. 2B is a partial cross-sectional view of an alternate embodiment of an ion source suitable for use with the instrument illustrated in FIG. 1.

FIG. 2C is a partial cross-sectional view of another alternate embodiment of an ion source suitable for use with the instrument illustrated in FIG. 1.

FIG. 3 is a block diagram illustrating a generalized multiple-stage ion source suitable for use with the instrument illustrated in FIG. 1.

FIG. 4A is a partial cross-sectional view of one embodiment of the generalized multiple-stage ion source shown in FIG. 3. FIG. 4B is a partial cross-sectional view of an alternate embodiment of the generalized multiple-stage ion source shown in FIG. 3.

FIG. 4C is a block diagram illustrating another alternate embodiment of the generalized multiple-stage ion source shown in FIG. 3.

FIG. 4D is a block diagram illustrating yet another alternate embodiment of the generalized multiple-stage ion source shown in FIG. 3.

FIG. 5A is a partial cross-sectional view of one embodiment of a charge neutralization device suitable for use with the multiple-stage ion source illustrated in FIG.4B.

FIG. 5B is a partial cross-sectional view of an alternate embodiment of a charge neutralization device suitable for use with the multiple-stage ion source illustrated in FIG. 4B.

FIG. 6A is a partial cross-sectional view of one embodiment of a labeling reagent source suitable for use with the instrument illustrated in FIG. 1.

FIG. 6B is a partial cross-sectional view of an alternate embodiment of a labeling reagent source suitable for use with the instrument illustrated in FIG. 1.

FIG. 6C is a diagrammatic illustration of another alternate embodiment of a labeling reagent source suitable for use with the instrument illustrated in FIG. 1. FIG. 7 is a partial cross-sectional view of a portion of the instrument of FIG. 1 incorporating a generalized multiple-stage molecular analysis instrument for analyzing the tagged or labeled analyte pairs.

FIG. 8 is a flowchart illustrating one embodiment of a process for post- ionization tagging and quantitative comparison of multiple samples.

FIG. 9 is a plot of ion intensity vs. mass-to-charge ratio illustrating ion intensity peaks for a sample of Bradykinin gas alone and a sample of Bradykinin gas labeled or tagged with 18-crown-6.

FIG. 10 is a diagrammatic illustration of another illustrative embodiment of a quantitative sample comparison instrument.

FIG. 11 A is a diagrammatic illustration of an alternate embodiment of the ion source arrangement illustrated in FIG. 10.

FIG. 11 B is a diagrammatic illustration of another alternate embodiment of the ion source arrangement illustrated in FIG. 10. FIG. 12 is a plot of valve-open time and resulting mass spectra vs. time illustrating various operational modes of the quantitative sample comparison instrument of FIGS. 10-11B.

FIG. 13 is a plot of drift time vs. mass-to-charge ratio of a sample of Tetralysine using a specific embodiment of the instrument of FIGS. 1 and/or 10. FIG. 14 is a similar plot of drift time vs. mass-to-charge ratio of another sample of Tetralysine combined with 18-crown-6 ether.

DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS

For the purposes of promoting an understanding of the principles of the invention, reference will now be made to a number of illustrative embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended.

Referring now to FIG. 1, one illustrative embodiment of a post-ionization, multiple-sample tagging and relative or quantitative comparison instrument 10 is shown. Instrument 10 includes a number, N, of ion sources 12ι - 12_N, wherein such ion sources will typically, but not necessarily, be provided in pairs. N may accordingly be any positive integer. The ion outlet of each ion source is fluidly coupled to a first inlet of an associated capillary having a second inlet fluidly coupled to the outlet of an associated isotopic labeling (or tagging) reagent source and an outlet fluidly coupled to a dedicated inlet of an N-inlet, single outlet sample tunneling stage 20. As illustrated in FIG. 1 , the ion outlet of ion source 12ι is thus fluidly coupled to a first inlet 13ι of a first capillary 14ι having an outlet fluidly coupled to a first inlet 22ι of a first passageway 24ι defined through the sample tunneling stage 20. A capillary branch 18ι defines an inlet 15ι fluidly coupled to an outlet of a first labeling (or tagging) reagent source 16ι and an outlet 17-ι fluidly coupled to capillary 14-ι as shown. Inlet 15ι defines the second inlet of capillary 14-|. Likewise, the ion outlet of ion source 12N is fluidly coupled to a first inlet 13N of an Nth capillary 14N having an outlet fluidly coupled to an Nth inlet 22_N of an Nth passageway 24N defined through the reaction cell 20. A capillary branch 18_N defines an inlet 15_N fluidly coupled to the outlet of an Nth labeling (or tagging) reagent source 16_N and an outlet 17_N fluidly coupled to capillary 14N as shown. Inlet 15N defines the second inlet of capillary 14N. It is to be understood that the junctions of the capillary branches 18ι - 18N with the corresponding capillaries 14ι - 14N may be positioned anywhere along the lengths of capillaries 14ι - 14N. In any case, the distances between these junctions and the corresponding inlets 22ι - 22N of the sample tunneling stage 20 define lengths L1 - LN, wherein these lengths may be substantially equal, or wherein one or more of these lengths may be different as illustrated in FIG. 1. The various capillaries 14ι - 14N and capillary branches 18ι - 18N are, in one embodiment, formed of aluminum, although these components may alternatively be formed of other suitable materials.

The sample tunneling stage 20 defines a single ion outlet 26, and the N passageways 24ι - 24N of the sample tunneling stage 20 converge at, and are fluidly coupled to, the ion outlet 26. Ion outlet 26 is positioned adjacent to, or coupled to, an ion inlet 28 of a molecular analysis instrument 30 configured to analyze ionized samples as a function of one or more molecular characteristics as will be described in greater detail hereinafter. Ions generated by each of the N ion sources 14ι - 14N, combined with the corresponding labeling reagents supplied by labeling reagent sources 16₁ - 16N, are directed to the ion outlet 26 of the sample tunneling stage 20, and accordingly to the inlet 28 of the molecular analysis instrument 30, by the corresponding passageways 24ι - 24_N. The lengths of each of the N passageways 24ι - 24N may be substantially equal as illustrated in FIG. 1 , although the lengths of one or more of these passageways may alternatively be different as will be described in greater detail hereinafter. In any case, the ion tunneling stage 20 is, in one embodiment, formed of aluminum, although stage 20 may alternatively be formed of other suitable materials.

Instrument 10 further includes a computer 40 having a memory 44 connected, or connectable, to a memory drive unit 46 via signal path 42, wherein unit 46 may be a floppy disk, CD ROM or other known drive unit operable to recall and/or store data to a corresponding data storage medium. Computer 40 is, in one embodiment, a commercially available, general-purpose, microprocessor-based computer, such as a personal computer (PC), laptop or notebook computer, or the like, that may be programmed to control the operation of instrument 10. It is to be understood, however, that computer 40 may alternatively be by any known computer or other signal processing circuit configured to control instrument 10 as described herein. As illustrated in FIG. 1 , instrument 10 may further include a display 50 electrically connected to computer 40 via signal path 52, a printer 54 electrically connected to computer 40 via signal path 56 and a keyboard 58 electrically connected to computer 40 via signal path 60. Display 50 and printer 54 provide a mechanism for displaying sample analysis results produced by instrument 10, and keyboard 58 provides a mechanism for allowing the user to input data into computer 40. The memory drive unit 46 may be used to provide computer 10 with additional data and/or executable programs, and/or additional data storage capacity.

The molecular analysis instrument 30 may be any known instrument operable to analyze samples introduced at inlet 28 as a function of one or more molecular characteristics. In the illustrated embodiment, for example, instrument 30 is a quadrupole mass spectrometer of known construction including ion focusing optics 32 positioned adjacent to the ion inlet 28, a quadrupole mass selection structure comprising quadrupoles 34ι - 34₄ (only three of the quadrupoles 34ι - 34₃ are shown in FIG. 1) having an ion inlet disposed adjacent to optics 32 and an ion outlet, and a detector 36 disposed adjacent to the ion outlet of the quadrupole structure. The quadrupole mass spectrometer 30 (or quadrupole mass filter, as it may otherwise be known) may be controlled in a scanning mode, as is known in the art, to selectively allow passage therethrough of ions having a desired range of mass-to-charge ratios. For example, the quadrupoles 34ι - 34₄ typically comprise four electrically conductive rods or plates that are disposed equidistant from a longitudinal axis therethrough. Two of the opposing rods 34₃ and 34₄ (not shown) are electrically connected to the positive terminal of a DC voltage source 62 via signal path 64, and to the output of a radio frequency (RF) voltage source 68 via signal path 70, wherein the DC source 62 has a control input connected to computer 40 via signal path 65ι and the RF source 68 has a control input connected to computer 40 via signal path 65₂. The negative terminal of DC source 62 is electrically connected to the remaining rods 34ι and 34₂ via signal path 66. Signal path 70 is further connected to a signal phase shifter 72 of known construction, wherein a signal output 74 of phase shifter 72 is electrically connected to rods 34ι and 34₂. Computer 40 is operable to control voltage supplies 62 and 68 to thereby control the DC potential applied between rod pairs 34-ι-34₂ and 34₃-34₄, and to control the RF voltage applied to rods 34₃-34₄. Phase shifter 72 is typically operable to shift the phase of the RF voltage on signal path 70 by 180°, and apply this phase shifted RF voltage to signal path 74. Those skilled in the art will recognize that phase shifter 366 may alternatively be replaced with a second RF voltage source that is controllable by computer 40 to produce an RF voltage identical to that produced by source 68 except shifted in phase by 180°.

In the operation of mass spectrometer 30, the RF voltages applied to rods 34_t - 34₄ alternately attract ions to rod pairs 34ι-34₂ and 34₃-34₄, wherein this attraction increases with decreasing ion mass-to-charge ratio (m/z). Below some threshold m/z value (i.e., lighter ions), the ions come into contact with one of the rods 34ι - 34 and are accordingly neutralized. The m/z value below which ions are neutralized is determined by the strength and frequency of the RF signal as is known in the art. The DC voltage applied to rods 34ι - 34₄ similarly attracts ions thereto wherein this attraction increases with increasing m/z values. Above some threshold m/z value (i.e., heavier ions), the ions come into contact with one of the rods 34ι - 34₄ and are accordingly neutralized. The m/z value above which ions are neutralized is determined by the strength of the DC signal as is known in the art. In a manner well known in the art, computer 40 is operable to control the DC voltage source 62 and the RF voltage source 68 in a scanning mode to sequentially allow passage therethrough of ions within a specified mass-to-charge range. In this manner, spectrometer 10 acts as a sequential mass selector, allowing detection by detector 36 of mass-to-charge ratio intensities within a specified mass range. In one embodiment, the detector 36 is an off-axis collision dynode/microchannel plate detector of known construction, although other known ion detectors may be used. Those skilled in the art will recognize that the molecular analysis instrument 30 may alternatively be any known instrument operable to analyze ions introduced thereto as a function of ion mass including, but not limited to, a linear time-of-flight mass spectrometer (TOFMS), a reflectron time-of-flight mass spectrometer, multi-pass time-of-flight mass spectrometer, Fourier Transform ion-cyclotron-resonance (FTICR-MS) mass spectrometer, ion trap mass spectrometer or other known mass spectrometer. Details of one linear TOFMS configuration that may be used as instrument 30, for example, are given in U.S. Patent Nos. 5,504,326, 5,510,613 and 5,712,479 to Reilly et al., the disclosures of which are all incorporated herein by reference.

The molecular analysis instrument 30 may alternatively be, or include, any known instrument operable to analyze ions as a function of one or more molecular characteristics other than mass-to-charge ratio. Examples of other such molecular characteristics include, but are not limited to, ion mobility, ion retention time, or the like, and instrument 30 may accordingly be, or include, an ion mobility spectrometer (IMS), liquid or gas chromatograph (LC or GC), or the like. Details of some configurations of such instruments that may be used as, and/or included within, instrument 30 of FIG. 1 are given in co-pending U.S. application Ser. No. 09/842,383, entitled INSTRUMENT FOR SEPARATING IONS IN TIME AS

FUNCTIONS OF PRESELECTED ION MOBILITY AND ION MASS, the disclosure of which is incorporated herein by reference.

The sample tunneling stage 20 may include a temperature adjustment structure for controlling the temperature within the various passageways 24ι - 24N. In one illustrative embodiment, for example, an electrically controllable heater 76 may be coupled to stage 20, as shown in FIG. 1, and electrically connected to computer 40 via signal path 77. Heater 76 may be implemented as a number of spaced-apart heater segments or as a continuous heater structure, that are mounted to the exterior surface of stage 20 as shown, or incorporated within stage 20. In any case, computer 40 is operable in this embodiment to control heater 76 to thereby control the temperature within the various passageways 24_! - 24_N. In an alternative embodiment, stage 20 may be surrounded by, or include therein, a variable temperature housing or passageway 78 which is connected to a variable temperature source 80 via path or conduit 82, all of which are shown in phantom in FIG. 1. In one embodiment, variable temperature source 80 may be a fluid holding tank and path 82 a conduit leading to housing or passageway 78. A return conduit (not shown) is also connected to the fluid holding tank so that fluid from within the tank may be circulated through housing or passageway 78. The fluid within the fluid holding tank may be a heated or cooled gas or liquid such as, for example, water, a suitable coolant fluid, liquid nitrogen or the like. In an alternate embodiment, variable temperature source 80 may be a known electrically actuatable temperature controller, and path 82 a pair of electrical conductors connected between the computer 40 and housing 78. In operation, temperature controller 80 is operable to controllably heat or cool housing 78 as desired. Regardless of the particular embodiment of housing or passageway 78, source 80 and path 82, source 80 may be controlled by computer 40 via signal path 84.

Computer 40 is electrically connected to each of the N ion sources 14ι - 14N by a number of signal paths, and computer 40 is operable to thereby control the production of ions produced by the various ion sources 14ι - 14N in a known manner. As illustrated in FIG. 1, for example, ion source 14ι is electrically connected to computer 40 via a number, J, of signal paths, wherein J may be any positive integer, and ion source 14N is electrically connected to computer 40 via a number, M, of signal paths, wherein M may be any positive integer, and wherein M may or may not be equal to J. Examples of a number of illustrative configurations of ion sources 14ι - 14N will be described hereinafter.

Computer 40 may also be electrically connected to one or more of the N labeling reagent sources 16ι - 16N by a number of signal paths, and computer 40 is operable in such cases thereby control the production of labeling reagent(s) produced thereby in a known manner. As shown in phantom in FIG. 1 , for example, labeling reagent source 16ι may be electrically connected to computer 40 via a number, P, of signal paths, wherein P may be any positive integer, and labeling reagent source 16N is electrically connected to computer 40 via a number, Q, of signal paths, wherein Q may be any positive integer, and wherein Q may or may not be equal to P. Examples of a number of illustrative configurations of labeling reagent sources 16-ι - 16N will be described hereinafter. Referring now to FIG. 2A, one illustrative embodiment 12_x of any one or more of the ion sources 12-ι - 12_N for use with instrument 10 of FIG. 1 is shown. Ion source 12_x includes a chamber 104 having a sample 106 mounted therein and an optical window 102 extending from the chamber 104. Signal path 90_x includes a first signal path 90χι electrically connecting a radiation source 100 to computer 40, wherein radiation source 100 is configured to direct radiation through optical window 102 to irradiate sample 106. Chamber 104 includes a conduit 110 extending therefrom to a pump 108 which may be electrically connected to computer 40 via signal path 90χ₂, as shown in phantom, wherein pump 108 may be controlled by computer 40, or independently of computer 40, to establish a desired vacuum or pressure within chamber 104. Ions produced by the irradiation of sample 106 are directed to an ion outlet 112 of ion source 12χ which is coupled, or disposed adjacent, to the ion inlet 13χ of capillary 14χ.

In one specific embodiment, ion source 12_x of FIG. 2A is a known MALDI arrangement wherein radiation source 100; e.g., a laser, is operable to desorb gaseous ions from a surface of the sample 106. Computer 40 is operable in this embodiment to control activation times of laser 100 to thereby control sample ionization events. The desorbed ions are directed by known internal structure of chamber 104 to the ion inlet 13χ of capillary 14χ. Pump 108 may be controlled, either by computer 40 or independently of computer 40, to pressurize chamber 104 to thereby conduct high pressure MALDI analysis in a manner well known in the art. Referring now to FIG. 2B, an alternate illustrative embodiment 12_x' of any one or more of the ion sources 12ι - 12N for use with the instrument 10 of FIG. 1 is shown. Ion source 12χ' includes a housing 120 defining an ion chamber 122 therein. A liquefied sample 124 has a spray hose or nozzle 126 extending toward an opening 130 defined in a desolvation region 128 coupled to chamber 122. Actuation of the spray nozzle 126 may be manually controlled, as is known in the art, or may be controlled by computer 40 via signal path 90χ₃. Desolvation region 128 is connected to computer 40 via signal path 90_X2, and is operable to convert charged sample droplets supplied thereto via nozzle 126 into gaseous ions and supply these ions to an ion optics member 132 contained within chamber 122. Ion optics member 132 is operable to focus the gaseous ions supplied by the desolvation region 128 and direct them to an ion outlet 112' of ion source 12χ' which is coupled, or disposed adjacent, to the ion inlet 13χ of capillary 14_x. Ion desolvation region 128 also includes a conduit 138 extending therefrom to a pump 136 which may be controlled by computer 40 via signal path 90χι, or independently of computer 40, to establish a desired vacuum or pressure within chamber 122. Ion source 12χ' is a known electrospray ionization (ESI) arrangement operable to convert a liquefied solution containing the sample to gaseous ions. Computer 40 is operable to control activation times of desolvation region 128 to thereby control sample ionization events. Pump 136 is operable to pressurize the ion source region 122 as is known in the art, and the desolvation region 128 is operable convert the liquefied solution to gaseous ions. The sample source 124 may include a solution containing a biomolecule of any size such as DNA, RNA, any of various proteins including blood, carbohydrates, glycoconjugates, and any other known biomolecules, although the solution within the sample source 124 may additionally or alternatively contain non-biomolecular structures. Referring now to FIG. 2C, another alternate illustrative embodiment 12_x" of any one or more of the ion sources 12ι - 12N for use with the instrument 10 of FIG. 1 is shown. Ion source 12χ" includes a housing 150 defining therein a ion source chamber 152 including an ion generation source 154 that may be either of the foregoing ion sources 12_x or 12χ' illustrated in FIGS. 2A and 2B, or may alternatively be any other known ion source arrangement including any one or more of the various ion source arrangements illustrated in the accompanying FIGS, and described hereinafter. In any case, ion generation source 154 is preferably controlled by computer 40, such as described hereinabove with respect to FIGS. 2A and/or 2B, via a number, R, of signal paths 90χι, wherein R may be any positive integer. Ion source chamber 152 also includes a first conduit 174 extending therefrom to a pump 172 which may be controlled by computer 40 via signal path 90χ₅, or independently of computer 40, to establish a desired vacuum or pressure within chamber 152. A second conduit 176 extends from chamber 152 to a source of buffer gas 178, wherein buffer gas source 178 may be controlled by computer 40 via signal path 90_X6, or independently of computer 40.

Ion source 12χ" further includes an ion trap 156 positioned between ion generation source 154 and an ion outlet 112" of ion source 12χ", wherein ion outlet 112" is coupled, or disposed adjacent, to the ion inlet 13χ of capillary 14_x. Ion trap 156 is preferably a known quadrupole ion trap, although other known ion trap arrangements may be used such as, for example, a hexapole or other multiple-pole ion trap. In the Illustrated embodiment, an endcap 158 of ion trap 156 is electrically connected to a first voltage source 160 via signal path 162ι, a center ring 164 is electrically connected to a second voltage source 166 via signal path 162₂ and an endcap 168 of ion trap 156 is connected to a third voltage source 170 via signal path 162₃. Voltage sources 160, 166 and 170 are electrically connected to computer 40 via signal paths 90χ₃, 90χ₄ and 90χ₅, respectively, and in the illustrated embodiment sources 160 and 170 are operable to produce DC voltages and source 166 is operable to produce AC voltages in the RF range.

The operation of ion trap 156 is known in the art, wherein computer 40 is configured to control voltage sources 160 and 170 to bias endcaps 158 and 168 such that ions generated by ion generation source 154 have enough energy to enter an ion inlet opening defined in the first endcap 158 but not enough to exit an ion outlet defined in the second endcap 168. Once inside the ion trap 156, the ions collide with the buffer gas provided by gas source 178 and lose energy. The RF voltage on center ring 164 is controlled so as to confine the reduced-energy ions within the trap 156. The confined ions undergo further collisions inside the trap 156 which causes the ions to correspondingly experience further energy loss, resulting in a concentration of the ions toward the center of ring 164. As long as the DC voltages on endcaps 158 and 168 and the RF voltage on center ring 164 are maintained, ions may enter the trap 156 and be collected therein as just described. Ions are ejected out of the trap 156 and into the ion inlet 13χ of the capillary14χ by turning off the RF voltage on center ring 164 and applying an appropriate DC pulse to one of the endcaps 158 or 168. For example, to eject a collection of positively charged ions from trap 156, either the voltage on endcap 158 may be pulsed to a DC level above that present on endcap 168, or the voltage on endcap 168 may be pulsed to a DC level below that present on endcap 158. In general, the magnitude of the RF and DC voltages supplied by sources 160, 166 and 170 may be varied to thereby collect ions of any desired mass-to-charge ratio within ion trap 156. Ions of all mass-to-charge ratios, or ions of any particular mass-to-charge ratio, may be selectively collected within ion trap 156 through proper choice of DC and RF peak magnitude. Referring now to FIG. 3, yet another illustrative embodiment 12_y of any one or more of the ion sources 12ι - 12_N for use with the instrument 10 of FIG. 1 is shown. Ion source 12γ is generally a multiple-stage ion source, and may include any number, S, of stages 190 - 190s, wherein S may be any positive integer. The first stage 190ι defines an outlet 192ι coupled to an inlet of a second stage 190₂ defining an outlet 192₂. Any subsequent stages 190₃ - 190_s similarly define outlets 192₃ - 192s, wherein outlet 192_s is coupled, or disposed adjacent, to the ion inlet 13_x of capillary 14χ. A number, T, of signal paths 90γι connect the first stage 190ι to computer 40, wherein T may be any positive integer. Likewise, a number, V, of signal paths 90_Y2 connect the second stage 190₂ to computer 40, wherein V may be any positive integer. Similarly, a number, V, of signal paths 90γ₃ and a number, W, of signal paths 90γs, connect any subsequent stages 190₃ - 190s to computer 40, wherein V and W may each be any positive integer.

Referring now to FIG. 4A, a cross-section of one illustrative embodiment 12γ of the multiple-stage ion source 12γ of FIG. 3 is shown. Multi-stage ion source 12γ includes a housing 200 defining an ion source chamber 204 separated from an ion collection chamber 206 by a wall or partition 202. Ion source chamber 204 includes a port having a conduit 208 connected thereto, wherein conduit 208 is connected to a pump or valve of known construction for changing gas pressure within region 204. An ion generation source 210 is disposed within region 204, wherein ion generation source 210 may be any of the ion sources 12χ, 12χ' or 12χ" described hereinabove with respect to FIGS. 2A-2C, and/or any combination thereof, and may additionally or alternatively be or include any one or more other known ion sources including, but not limited to, any ion source embodiment described herein. In any case, a number, Z, of signal paths 90γ₂ connect ion generation source 210 to computer 40, wherein Z may be any positive integer.

Wall or partition 202 defines an aperture 212 therethrough that is aligned with an ion outlet of ion generation source 190 (not shown), wherein aperture 212 defines an ion inlet to ion collection chamber 206. An electrically conductive grid, or series of vertically or horizontally parallel wires, 214 (hereinafter "grid") is positioned across an ion outlet aperture 112'" of ion collection chamber 206 coupled, or disposed adjacent, to ion inlet 13χ of capillary 14χ, wherein grid 214 is electrically connected to a voltage source 216 via signal path 218. Voltage source 216 is also electrically connected to computer 40 via signal path 90γ₂, and computer 40 is operable, in this embodiment, to control the voltage of grid 214 to selectively permit or inhibit entrance of ions into capillary 14χ. For example, computer 40 may be configured to inhibit entrance of ions into capillary 14χ by activating voltage source 216 to thereby cause ions in the vicinity of grid 214 to be attracted thereto and to be neutralized upon contact with grid 214. Likewise, computer 40 may be configured to permit entrance of ions into capillary 14χ by deactivating voltage source 216 to thereby permit passage of ions therethrough. Those skilled in the art will recognize that the ion collection chamber 206 is functionally similar to the ion trap 156 of FIG. 2C in that it provides for the collection of a large quantity of ions generated by ion generation source 210 prior to entrance into capillary 14χ. Through appropriate control of ion generation source 210 and grid 214 or equivalent structure or function, the quantity of ions entering capillary 14_x may thus be controlled.

Referring now to FIG. 4B, a block diagram of another illustrative embodiment 12_γ" of the multiple-stage ion source 12_γ of FIG. 3 is shown. Multi-stage ion source 12γ" includes an ion generation source 220 having an ion outlet 222 coupled to a charge normalization stage 224. Ion generation source 220 may be any of the ion sources 12χ, 12_x', 12_x", 12_γ or 12_γ' described hereinabove with respect to FIGS. 2A- 4A, and/or any combination thereof, and may additionally or alternatively be or include any one or more other known ion sources including, but not limited to, any ion source embodiment described herein. In any case, a number, A, of signal paths 90γι electrically connect ion generation source 220 to computer 40, wherein A may be any positive integer. The charge normalization stage 224 defines an ion inlet coupled to the ion outlet 222 of ion generation source 220 and an ion outlet 226 coupled, or disposed adjacent, to the ion inlet 13χ of capillary 14χ. A number, B, of signal paths 90γ₂ electrically connect the charge normalization stage 224 to computer 40, wherein B may be any positive integer.

Referring now to FIG. 5A, a cross-section of one illustrative embodiment 224' of the charge normalization stage 224 of the multi-stage ion source 12γ" of FIG. 4B is shown. In the embodiment illustrated in FIG. 5A, the charge normalization stage 224 is a charge normalization or reduction device 224' and includes a housing 230 defining a chamber 232 therein having an ion inlet 234 and an ion outlet 226. An axis of ion traversal 238 is defined between ion inlet 234 and ion outlet 226, and ion inlet 226 is coupled, or disposed adjacent, to the ion outlet 222 of ion generation source 220. A pump 240 may be controlled by computer 40 via signal path 90γ₂A, or may alternatively be controlled independent of computer 40, to set a desired pressure/vacuum within chamber 232. Device 224' further includes a radiation source 244 operable to emit radiation into the ion traversal path 238 as illustrated in FIG. 5A by arrows 246. In one embodiment, radiation source 244 is an alpha ionization source such as, for example, ²¹⁰Po, although other known radiation sources may be used. Device 224' further includes a source 242 of a suitable bath gas in fluid communication with chamber 232, wherein gas source 242 may be controlled by computer 40 via signal path 90_Y2B, or may alternatively be controlled independently of computer 40 as is known in the art.

For one or more of the techniques described hereinabove for generating ions, the resulting bulk of generated ions may yield a distribution of ions in various charge states with a correspondingly complex ion mass spectra. Analysis of such mixtures by molecular analysis instrument 30 may accordingly difficult since crowded mass-to- charge data typically exhibits excessive overlap in the mass peak information. In such cases, charge normalization or reduction device 224' may be disposed in-line between ion generating source 220 and capillary 14χ to normalize the charge states of all ions being passed to the molecular analysis instrument 30 to a predefined charge state (e.g., +1 charge state). This process serves to increase the mass separation of compact ions (e.g., to one mass peak per compound) to provide for more discernible molecular characteristic peaks. Charge normalization or reduction device 224' is thus operable to reduce peak congestion in the spectral data produced by instrument 30, wherein the result of this feature is more accurate and more highly resolved spectral information.

The operation of charge normalization or reduction device 224' is known wherein charge reduction or neutralization is achieved by exposure of ions passing therethrough to a bath gas which contains a high concentration of bipolar (i.e., both positively and negatively charged) ions. Collisions between the charged ions produced by ion source 220 and the bipolar ions within the bath gas supplied by gas source 242 to chamber 232 result in neutralization or normalization of the multiply charged ions produced by ion source 220. The rate of this process is controlled by the degree of exposure of the two sets of ions to radiation produced by radiation source 244. By controlling the degree of this exposure, the resulting charge state of ions produced by ion generation source 220 may, in turn, be normalized to any desired charge state. In one embodiment, for example, the charge distribution of ions produced by ion generation source 220 is reduced by device 224' such that ions exiting ion outlet 226 consist principally of singly charged ions.

Referring now to FIG. 5B, a cross-section of another illustrative embodiment 224" of the charge normalization stage 224 of the multi-stage ion source 12γ" of FIG. 4B is shown. In the embodiment illustrated in FIG. 5B, the charge normalization stage 224 is a reaction cell 224". Reaction cell 224" includes a housing 250 defining a chamber 252 therein having an ion inlet 254 coupled, or disposed adjacent, to the ion outlet 220 of ion generating source 220 and an ion outlet 226 coupled, or disposed adjacent, to the ion inlet 13_x of capillary 14_x. Reaction cell 224" defines an axis of ion traversal 258 between the ion inlet 254 and the ion outlet 226. Cell 224" includes a pump 260 that may be controlled by computer 40 via signal path 90γ_2A, or controlled independently of computer 40, to set a desired pressure/vacuum within chamber 252. In this embodiment, reaction cell 224" includes a source 262 of reagent gas in fluid communication with chamber 252 via passage 264. Reagent source 262 may be controlled by computer 40 via signal path 90γ₂s, or independently of computer 40. As an alternative to the charge normalization or reduction stage 224' illustrated and described with respect to FIG. 5A, reaction cell 224" may be used to separate crowded ion mass peaks resulting from multiply charged ions produced by ion generating source 220. In this embodiment, reagent source 262 may include any desired reagent gas such as, for example, D₂O. Ions passing through cell 224" in the presence of the reagent gas undergo a chemical reaction with the gas, as is known in the art, wherein isotopes of the ions separate in ion mass and to thereby provide for a spreading of mass peaks over a wider mass range. Albeit to a lesser extent than charge normalization or reduction stage 224', this serves to reduce peak crowding in the molecular analysis instrument 30 to accordingly provides for improved resolution with instrument 10. Alternatively, reagent source 262 may be a known charge neutralization or reduction gas that acts to neutralize or normalize the charge state of ions produced by ion generating source 220 in a manner similar to that described with respect to FIG. 5A. Referring now to FIG. 4C, a block diagram of yet another illustrative embodiment 12γ'" of the multiple-stage ion source 12γ of FIG. 3 is shown. Multistage ion source 12_γ'" includes an ion generation source 220 having an ion outlet 222 coupled, or disposed adjacent, to ion inlet of an ion separation instrument 270. Ion generation source 220 may be any of the ion sources 12_x, 12_x', 12_x", 12γ, 12_γ' or 12_γ" described hereinabove with respect to FIGS. 2A-4B, and/or any combination thereof, and may additionally or alternatively be or include any one or more other known ion sources including, but not limited to, any ion source embodiment described herein. In any case, a number, C, of signal paths 90_Yι electrically connect ion generation source 220 to computer 40, wherein C may be any positive integer. The ion separation instrument 270 defines an ion outlet 272 coupled, or disposed adjacent, to the ion inlet 13χ of capillary 14χ. A number, D, of signal paths 90_Y2 electrically connect the ion separation instrument to computer 40, wherein D may be any positive integer. In the embodiment illustrated in FIG. 4C, ions generated by ion generating source 220 are separated in time as a function of a molecular characteristic prior to entrance into capillary 14χ. The molecular characteristic may be for example, ion mass-to-charge ratio, ion mobility, ion retention or other molecular characteristic, and examples of known instruments that may be used as the ion separation instrument 270 include, but are not limited to, one or more mass spectrometers or mass analyzers, one or more ion mobility instruments, one or more liquid chromatographs, one or more gas chromatographs, and the like.

Referring now to FIG. 4D, a block diagram of a further illustrative embodiment 12γ"" of the multiple-stage ion source 12γ of FIG. 3 is shown. Multi-stage ion source 12γ"" includes a non-ionizing sample source 280 having a sample outlet 282 coupled, or disposed adjacent, to a sample inlet of an particle ionizing instrument 284. The non-ionizing sample source 280 may be any known device operable to convert a sample to mist or gas form. In one embodiment, for example, non-ionizing sample source 280 is an electrospray droplet source similar in many respects to the electrospray ionization source 12χ' illustrated and described hereinabove with respect to FIG. 2B, except that source 280 does not include a ionizing desolvation region or ion focusing optics. Rather, source 280 is configured in this embodiment to provide the sample to instrument 284 in the form of a non-ionized mist. In another embodiment, the sample may be in the form of a gas, and source 280 may in such a case be configured as a known gas interface to instrument 284. Those skilled in the art will recognize other non-ionizing sample sources, and any such sources are intended to fall within the scope of the claims appended hereto. In any case, a number, E, of signal paths 90γι electrically connect non-ionizing sample source 280 to computer 40, wherein E may be any positive integer.

The particle ionizing instrument 284 may be any known sample ionizing instrument or device operable to ionize a mist or gas sample provided by source 280. In one embodiment, for example, instrument 284 is a liquid or gas chromatograph operable to ionize samples provided by sample source 280. Those skilled in the art will recognize other particle ionizing instruments or devices, and any such instruments and devices are intended to fall within the scope of the claims appended hereto. In any case, the particle ionizing instrument 284 defines an ion outlet 286 coupled, or disposed adjacent, to the ion inlet 13χ of capillary 14χ. A number, F, of signal paths 90_Y2 electrically connect the particle ionizing instrument to computer 40, wherein F may be any positive integer.

It is to be understood that the various single and multiple-stage ion sources illustrated and described herein with reference to FIGS. 2A-5B are provided only by way of example, and that other single and/or multiple-stage ion source configurations, some of which may include additional ion processing stages as illustrated in FIG. 3, may be used. Examples of one or more additional ion processing stages that may form part of the multiple-stage ion source 12γ of FIG. 3 include, but are not limited to, a known ion mass filter configured to pass therethrough only ions having selectable mass-to-charge ratios, a known ion fragmentation device, such as a collision cell, configured to fragment parent ions into daughter ions, a known ion trap, such as that described hereinabove with respect to FIG. 2C, a known ion separating instrument configured to separate ions in time as a function of a specific molecular characteristic (e.g., ion mass-to-charge ratio, ion mobility, ion retention time), and the like. Those skilled in the art will recognize that such additional ion processing stages may be provided in various combinations to achieve desired results, and any such combinations will typically depend upon the application. Moreover, those skilled in the art will recognize that other known ion source arrangements may be used including, but not limited to, electron impact ionization sources, chemical ionization sources, and other known ion source arrangements.

Referring now to FIG. 6A, a diagrammatic illustration of one illustrative embodiment 16χ of any one or more of the sources of isotopic labeling reagent 16ι - 16N for use with instrument 10 of FIG. 1 is shown. Labeling reagent source 16χ includes a chamber 290 containing a reagent gas and defining an outlet 294 coupled, or disposed adjacent, to the reagent inlet 15χ of capillary branch 18χ. Reagent source 16χ includes a pump 292 that may be controlled by computer 40 via signal path 92_X3, or controlled independently of computer 40, to set a desired pressure/vacuum within chamber 290. A valve, needle or other known gas flow control mechanism 296 is disposed at the outlet 294, wherein valve 296 is electrically connected to computer 40 via signal path 92χ₂. Computer 40 is operable to control the position of valve 296 to thereby control the flow of reagent gas to capillary branch 18_x. Optionally, as shown in phantom in FIG. 6A, labeling reagent source 16χ may include a temperature adjustment structure 298 electrically connected to computer 40 via signal path 92_X2. The temperature adjustment structure 298 may take any of many known forms, two examples of which were described hereinabove with respect to the ion tunneling stage 20 of FIG. 1. The temperature adjustment structure 298 may be controlled by computer 40 or independently of computer 40 to thereby control the temperature of the labeling reagent gas supplied to capillary branch 18χ. Referring now to FIG. 6B, a diagrammatic illustration of another illustrative embodiment 16χ' of any one or more of the sources of isotopic labeling reagent 16ι - 16N for use with instrument 10 of FIG. 1 is shown. Labeling reagent source 16χ' includes a chamber 300 containing a liquid reagent source 304 having a spray nozzle 306 directed toward a chamber outlet 308 coupled, or disposed adjacent, to the reagent inlet 15_x of capillary branch 18_x. Reagent source 16_x' includes a pump 302 that may be controlled by computer 40 via signal path 92_X3, or controlled independently of computer 40, to set a desired pressure/vacuum within chamber 300. In one embodiment, a valve or other known flow control mechanism 310 is disposed at the outlet 308, wherein valve 310 is electrically connected to computer 40 via signal path 92χι. Computer 40 is operable, in this embodiment, to control the position of valve 310 to thereby control the flow of reagent to capillary branch 18_x. In an alternate embodiment, as shown in phantom in FIG. 6B, liquid reagent source 304 may be electrically connected to computer 40 via signal path 92χ₂, and source 304 may accordingly be controlled by computer 40, or independently of computer 40, to thereby control the rate and/or amount of reagent liquid sprayed toward inlet 15χ of capillary branch 18χ. In this embodiment, valve 310 may or may not be omitted. Optionally, as shown in phantom in FIG. 6B, labeling reagent source 16_x' may include a temperature adjustment structure 298 electrically connected to computer 40 via signal path 92χ₄ and operable as described hereinabove with respect to FIG. 6A.

Referring now to FIG. 6C, a diagrammatic illustration of yet another illustrative embodiment 16χ" of any one or more of the sources of isotopic labeling reagent 16ι - 16_N for use with instrument 10 of FIG. 1 is shown. Labeling reagent source 16χ" includes a chamber 320 containing a solid reagent 326, liquid reagent 328 or combination thereof. A chamber outlet 322 is coupled, or disposed adjacent, to the reagent inlet 15χ of capillary branch 18χ, and a valve or other known gas flow control mechanism 324 may be disposed at the outlet 32, wherein valve 324 is electrically connected to computer 40 via signal path 92χι. Computer 40 is operable, in this embodiment, to control the position of valve 324 to thereby control the flow of reagent vapor to capillary branch 18_x. Optionally, as shown in phantom in FIG. 6C, labeling reagent source 16χ" may include a temperature adjustment structure 330 that may be electrically connected to computer 40 via signal path 92χ₂ and operable as described hereinabove with respect to FIG. 6A to control the temperature of reagent 326 and/or 328. Generally, a reagent vapor is generated by reagent 326 and/or 328, and a pressure difference between chamber 320 and instrument 10 is operable to draw the reagent vapor into capillary branch 18χ. Generation of a reagent vapor from reagent 326 and/or 328 may be expedited by heating chamber 320 via temperature adjustment structure 330, wherein temperature adjustment structure 330 may or may not be computer controlled.

Those skilled in the art will recognize that the various isotopic labeling reagent sources 16χ, 16χ' and 16_x" illustrated and described with respect to FIGS. 6A-6C are provided only by way of example, and that other known reagent source structural arrangements may be used. It will be understood that any of the labeling reagent sources 16ι - 16 of FIG. 1 may include any known process operable to add an isotopic label or tag to ions generated by corresponding ion sources 12ι - 12N, wherein the labeling reagent may comprise charged (ionized) or uncharged molecules. Any one or more of the reagent sources 16ι - 16N may thus be, or include, an ion source or a molecule ionizing arrangement, some examples of which are illustrated and described hereinabove with respect to FIGS. 2A-5B. Referring now to FIG. 7, a partial cross-sectional view of a portion of the instrument 10 of FIG. 1 incorporating a generalized multiple-stage molecular analysis instrument 350 for analyzing the tagged or labeled analyte pairs. It is to be understood that the mass analysis instrument 30 illustrated and described hereinabove with reference to FIG. 1 was provided only as one example of a molecular analysis instrument that may be used to analyze ions produced at the outlet 26 of the ion tunneling stage 20, and in general, instrument 30 may be any known instrument configured to separate or select ions according to a molecular characteristic as described hereinabove. Instrument 30 may alternatively be a multiple-stage molecular analysis instrument 350 as illustrated in FIG. 7. In the embodiment illustrated in FIG. 7, instrument 350 may include any number, G, of stages 352ι - 352_G, wherein G may be any positive integer. The first stage 352ι defines an ion inlet coupled, or disposed adjacent, to the ion outlet 26 of ion tunneling stage 20, and an outlet 354 coupled to an inlet of a second stage 352₂ defining an outlet 354₂. Any subsequent stages 352₃ - 352G-I similarly define outlets 354₃ - 354_G-ι, and stage 352_G includes at its outlet end a detector 36 electrically connected to computer 40 via signal path 42. Detector 36 may be as described hereinabove with reference to FIG. 1. A number, "a", of signal paths 356 connect the first stage 352ι to computer 40, wherein "a" may be any positive integer. Likewise, a number, "b", of signal paths 358 connect the second stage 352₂ to computer 40, wherein "b" may be any positive integer. Similarly, a number, "c", of signal paths 360 and a number, "d", of signal paths 362 connect any subsequent stages 352₃ - 352_G-ι to computer 40, wherein "c" and "d" may each be any positive integer.

In the embodiment illustrated in FIG. 7, the multiple-stage molecular analysis instrument 350 may include any number and combination of known ion analysis instruments and ion processing devices. Examples of ion analysis instruments that may be included within instrument 350 include, but are not limited to, ion mass spectrometers, ion mobility spectrometers, liquid chromatographs, gas chromatographs, and the like. Examples of ion processing devices that may be included within instrument 350 include, but are not limited to, ion traps, ion mass filters, fragmentation devices including collision and non-collision disassociation devices, charge normalization devices, and the like.

One example configuration of the multiple-stage molecular analysis instrument configuration 350 may be a two-stage instrument comprising cascaded mass spectrometers. Another example configuration may be a two-stage instrument comprising an ion mobility instrument coupled between the ion tunneling stage 20 and a mass spectrometer. Either of the foregoing instrument configurations may further include, for example, an ion mass filter and/or ion fragmentation device disposed in front of the first instrument, between the two analysis instruments and/or between the second instrument and the detector 36. Those skilled in the art will recognize other ion analysis and/or ion processing device combinations, and any such combinations are intended to fall within the scope of the claims appended hereto. Further examples of various ion analysis and/or ion processing device combinations are detailed in co-pending U.S. application Ser. No. 09/842,383, entitled INSTRUMENT FOR SEPARATING IONS IN TIME AS FUNCTIONS OF PRESELECTED ION MOBILITY AND ION MASS, the disclosure of which was incorporated herein by reference.

In general, the post-ionization, multiple-sample tagging and quantitative comparison instrument 10 is operable in one illustrative embodiment to label or tag any number of pairs of gas-phase, post-ionized samples with suitable reagents for comparative relative or quantitative analysis. One or more like sample pairs may each be reacted with corresponding like reagent pairs, any number of like sample pairs may be reacted with a corresponding number of different reagent pairs, or any number of different sample pairs may be reacted with a corresponding number of different reagent pairs. In any case, the two samples in any one pair are generally different from each other, and the two corresponding reagent pairs may differ from each other generally in that one is deuterated while the other is non-deuterated.

For any one ionized sample, 12χ, and corresponding labeling reagent, 16χ, a reaction therebetween occurs in a reaction cell defined as including the portion of the capillary 14χ between the junction of the capillary branch 18χ therewith and the ion tunneling stage 20 and also including the corresponding passageway 24_x of the ion tunneling stage 20. For example, the reaction cell for ion source 12ι and labeling reagent 16ι is defined as the length L1 of capillary 14ι and the ion passageway 24ι. For any sample pair, it is desirable to select appropriate reagents that would react with the two different samples at known rates. Commonly used reagent pairs include, but are not limited to, H₂O and D₂O, deuterated and non-deuterated 18- crown-6, and the like, although any two different reagent pairs may be used (which may or may not be deuterated/non-deuterated reagent pairs) that will react at a known rate or to a known level with the two different samples making up the sample pair, and/or that result in a definable molecular characteristic shift (e.g., mass or mass-to-charge ratio) when the reagent-sample structures are analyzed by the molecular analysis instrument 30. In some cases, it is further desirable to select suitable reagents that provide for repeatable association with, or bonding to, one end or the other of the sample structures. It will be understood that the illustrative reagent pairs recited above are provided only by way of example, and that the choice of any specific reagent pair will typically depend on a number of factors including, but not limited to, ion sample composition, reagent-sample reaction rate, and the like. The reagent-sample pairs may include any known or created ion- molecule and/or ion-ion reaction that results in the formation of associative complexes or covalent conjugates suitable for subsequent comparative relative or quantitative comparison via molecular analysis instrument 30. The choice of any such suitable reagents will be within the knowledge of a skilled artisan and/or ascertainable via routine experimentation. Numerous examples of known reaction pairs and corresponding reaction rates may be found at http://webbook.nist.gov/chemistry/, the contents of which is incorporated herein by reference.

In general, the reaction of any sample/reagent pair within the corresponding reaction cell is governed by the equation:

I _ i *_ --nnσσLL ip - lo e (1 )>

where,

I_P is the peak intensity (concentration) of the sample, l₀ is the peak intensity (concentration) of the reactant ions, n is the number density of the reagent (proportional to reagent pressure), σ is the collision cross-section of the colliding pair (which may be a function of temperature), and L is the path length of the reaction cell.

As illustrated and described with reference to FIGS. 6A and 6B, the number density, n, and hence the pressure, of any of the labeling reagent sources may be adjusted via either of pumps 292 and 302, and/or via either of valves 296 and 310. The temperature, and hence, in some cases, the collision cross-section, σ, may be adjusted via the optional temperature control structures associated with either of the labeling reagent sources 16χ (see FIGS. 6A-6C) and the ion tunneling stage 20 (see FIG. 1). Finally, any of the path lengths, L, may be modified by adjusting either the capillary length, Lx, or the corresponding passageway defined through the ion tunneling stage 20. Accordingly, the reaction rates of any reagent/sample pair may be, at least to some extent, modified by modifying any one or more of n, σ or L. The reagent/sample pair reactions may thus be controlled so that they go to completion, or at least to some known level, by the time the ions reach the ion outlet 26 of the ion tunneling stage. It is to be understood that this ion labeling or tagging process may be carried out in any suitable reaction chamber, wherein the reaction chamber of FIG. 1 has been identified as including the length L_x of any capillary 14χ and a corresponding passageway 24_x defined through the ion tunneling stage 20. Those skilled in the art will recognize that the reaction chamber may alternatively be any definable structure such as a tube, capillary, chamber, drift tube portion of molecular analysis instrument 30 or the like, and/or may further include other known ion collection or storage instruments including for example, but not limited to, an ion trap, an ion mass filter, ion fragmentation cell, ion collection chamber, or the like. It should also be understood that any ion source 12χ may include an ion fragmentation cell as a final stage thereof, such that ion fragmentation occurs before the labeling or tagging process, and the ion labeling or tagging process is accordingly carried out on daughter ions of the parent ions generated from the sample source. In any case, analysis by the molecular analysis instrument 30 then follows in a known manner. Referring now to FIG. 8, a flowchart is shown illustrating a process 370 for operating and controlling instrument 10 to conduct comparative relative or quantitative analysis in the case that N=2; i.e., wherein instrument 10 includes two capillaries 14ι and 14₂, two ions sources 12ι and 12₂ and two reagent sources 16ι and 16₂. The process 370 may be implemented in the form of one or more software algorithms executable by computer 40, or may instead be controlled independently of computer 40. In any case, process 370 begins at step 372 where ionized sample or analyte pairs are introduced into corresponding capillaries 14χ. In this exemplary case, instrument 10 includes two such capillaries 14ι and 14₂, and step 372 in this case includes introducing the first ionized sample produced by ion source 12ι into capillary 14ι, and introducing the second ionized sample produced by ion source 12₂ (and different from the first sample) into capillary 14₂. Process 370 advances from step 372 to step 374 where labeling reagent pairs are introduced into corresponding capillary branches 18_x. Again, in the exemplary case, instrument 10 includes two such reagent sources 16-ι and 16₂ (one deuterated and one non-deuterated), and step 374 in this case includes introducing the first labeling reagent produced by reagent source 16ι into capillary branch 18-i and introducing the second labeling reagent produced by reagent source 16₂ into capillary branch 18₂.

Process 370 advances from step 374 to step 376 where the combined analyte-reagent pairs (or singlets) introduced into their respective reaction cells are directed via the tunneling stage 20 to the inlet 28 of the molecular analysis instrument 30. Referring to FIG. 9, an example of steps 372-376 is illustrated wherein ionized bradykinin is supplied by ion source 12χ to capillary 14χ, and 18- crown-6 is introduced as a reagent into capillary branch 18χ. As clearly illustrated in FIG. 9, the bradykinin ion peak occurs at approximately m/z = 550, whereas the bradykinin ions reacted with 18-crown-6 (18C6) in the reaction cell exhibit ion peaks at approximately m/z = 550, m/z = 675, m/z = 800 and m/z = 845.

Following step 376, ions emerging from the molecular analysis instrument 30 are detected at step 378 by ion detector 36, and such information is stored in the memory 44 of computer 40. Thereafter at step 380, the molecular peaks of interest are identified, and at step 382 the comparative relative or quantitative analysis is conducted. For example, in one embodiment of step 382, as illustrated in FIG. 8, the molecular peaks of interest from step 380 may be used to determine the concentration of unknown analytes. Those skilled in the art will recognize that step 382 represents only one example application, and that other comparative relative or quantitative analysis may be carried out at step 382.

As one example, assume that a peptide of known identity but of unknown concentration is contained in a solution, and it is desired to determine the concentration of this peptide. At step 372 of process 370, the peptide of unknown concentration in the solution is ionized by ion source 12-_t, using any one of the ionization sources described herein or using any other known sample ionization technique, and introduced into capillary 14-ι. The same peptide, but of known concentration in the same solution is prepared and ionized by ion source 12₂, again using any one of the ionization sources described herein or using any other known sample ionization technique, and introduced into capillary 14₂. In many cases, it is desirable to use a common ionization technique for any two sample pairs.

H₂0 and D₂0 are chosen as the labeling reagent pairs, and at step 374 of process 370, H₂0 of known concentration is introduced into capillary 18ι, using any one of the labeling reagent sources described herein or using any other known reagent introduction technique, for combination with the ionized sample in capillary 14ι. Likewise, D₂0 of known concentration is introduced into capillary 18₂ for combination with the ionized sample in capillary 14₂.

As the ions from each sample traverse their respective reaction cells, they undergo collisions with their respective light or heavy labeling reagents, and during these collisions they exchange their own heteroatom hydrogens for either H (source 12ι) or D (source 12₂), respectively. This exchange process occurs at known rates and can be driven to a desired level of completion within the respective reaction cells by adjusting any one or more of the parameters of equation (1) as described hereinabove. Upon exiting the ion tunneling stage 20, those analytes from ion source 12ι have mass-to-charge ratios that are the same as that which would be expected from any ion source. However, those analytes from ion source 12₂ have mass-to-charge ratios that are shifted to higher masses. The extent of the mass-to- charge ratio shift from ion source 12₂ will depend upon the number of heteroatom hydrogens that have exchanges for deuteriums. For example, a simple peptide sequence such as AVLGAVLG would be expected to exchange a maximum of seven backbone hydrogens, three end group hydrogens and possibly additional protons added by the ionization source - a maximum shift of approximately 11. This type of exchange is rapid, and it has been determined through experimentation that the exchange of even large systems (e.g., whole proteins) can be driven to completion in only a few milliseconds by simply heating the labeling reagent. Other approaches to drive the reaction to completion may include, for example, using more reactive deuterated reagents.

The concepts described herein have numerous applications including, but not limited to, determination of environmental pollutant content, determination of petroleum component content, determination of carbohydrate abundance, determination of peptide abundance, and the like. Those skilled in the art will recognize other applications of the concepts described herein, and any such applications are intended to fall within the scope of the claims appended hereto.

It is to be understood that while instrument 10 has been illustrated and described herein as including any number of ion sample source pairs and corresponding labeling reagent source pairs, other configurations of, and uses for, instrument 10 are contemplated. For example, one of the reagent sources in any one reagent source pair may be eliminated or deactivated such that ions from one of the ion sample sources are reacted with a reagent supplied by a corresponding reagent source while ions from the other ion sample source are supplied directly to the molecular analysis instrument without reacting these ions with a corresponding reagent. In general, the reagent in this case may be any reagent that will react at a known rate or to a known level with the corresponding ionized sample, and/or that result in a definable molecular characteristic shift (e.g., mass or mass-to-charge ratio) when the reagent-sample structure is analyzed by the molecular analysis instrument 30. In one embodiment, such an arrangement may take the general form of the instrument 10 illustrated in FIG. 1 with the exception that the reagent source 16χ and corresponding capillary branch 18χ associated with one of the ion sample sources of at least one sample pair is omitted.

In an alternative embodiment, such an arrangement may be accomplished with a single capillary having two ion sources coupled thereto. Referring to FIG. 10, one illustrative embodiment of such an instrument 10' is shown. Instrument 10' identical in some respects to instrument 10 illustrated in FIG. 1 , and like numbers are therefore used to identify like components. Instrument 10' includes a first ion source 12-i_A having an ion outlet coupled to an inlet 13-|_A of a first capillary branch 19IA, and a second ion source 12ι_B having an ion outlet coupled to an inlet 13-IB of a second capillary branch 19-|_B. Ion sources 12ι_A and 12ι_B may be, or include, any one or more of the various ion sources illustrated and/or described herein. The outlets of capillary branches 19-ι_A and 19-IB are coupled to an inlet of capillary 14-j. As shown in phantom, instrument 10' may include a reagent source 16ι having a reagent outlet coupled to the inlet 15^ of a third capillary branch 18ι having an outlet coupled to capillary 14-ι. Reagent source 16ι may be, or include, any one or more of the various reagent sources illustrated and/or described herein.

In one embodiment, the outlet of capillary 14ι may be coupled directly to the ion inlet of molecular analysis instrument 30 as illustrated in FIG. 11. Alternatively, capillary 14ι may be only one of a number of capillaries coupled to the molecular analysis instrument 30, in which case instrument 10' may include ion tunneling structure 20 disposed between capillary 14ι and molecular analysis instrument 30, as generally indicated in FIG. 10 by the capillary interruption lines 410. In the embodiment illustrated in FIG. 10, each of the ion sources 12ι_A and

12IB_> as well as the optional reagent source 16-i, include a valve member controllable via control computer 40, or independently of control computer 40, to selectively control supply of the corresponding source matter into capillary 14ι. For example, ion source 12-IA includes a valve 400IA, which may be a plate, needle or other known valve structure, disposed at the ion outlet of ion source 12 _A, and ion source 12-IB includes a valve 400ι_B, which may also be a plate, needle or other known valve structure, disposed at the ion outlet of ion source 12ι_B. Similarly, the optional reagent source 16ι includes a valve 400₂, which may also be a plate, needle or other known valve structure, disposed at the outlet of reagent source 16-ι. Valves 400ι_A, 400-iB and 400₂ may each be electrically coupled to control computer 40 via corresponding signal paths 90ι_A, 90-ι_B and 92ι, although valves 400-IA, 400-IB and 400₂ may alternatively be electrically or mechanically controlled independently of control computer 40.

It is to be understood that the angled capillary structures shown in FIG. 10 represent only one illustrative embodiment, and that other capillary structures may be used. For example, referring to FIG. 11 A, an alternate "parallel" structural arrangement of ion sources 12-ι_A and 12_1B, relative to capillary 14-ι, is illustrated. In this embodiment, a single capillary 19, disposed approximately 90 degrees relative to capillary 14_{1 ;} has a first inlet 13-ι_A coupled to the ion outlet of ion source 12IA, a second opposite inlet 13-ι_B coupled to the ion outlet of ion source 12ι_B and an outlet coupled to capillary 14ι. Referring to FIG. 11B, another alternate "series" structural arrangement of ion sources 12-|_A and 12-ι_B, relative to capillary 14ι, is illustrated. In this embodiment, a first capillary branch 19I , disposed approximately 90 degrees relative to capillary 14ι, has an inlet 13-IA coupled to the ion outlet of ion source 12IA and an outlet coupled to capillary 14-ι. A second capillary branch 19-IB, also disposed approximately 90 degrees relative to capillary 14ι, has an inlet 13ι_B coupled to the ion outlet of ion source 12ι_B and an outlet coupled to capillary 14ι downstream of the junction of capillary branch 19ι_A with capillary 14ι.

For any of the structural arrangements of instrument 10' illustrated in FIGS. 10-11 B, instrument 10' may be configured to operate in any one or more of a number of different operational modes. For example, one embodiment of instrument 10' includes reagent source 16ι, and in this embodiment, instrument 10' is operable in a temporal labeling (or tagging) and sample analysis mode to control reagent source 16ι to tag or label ions supplied by one of the ion sources 12IA or 12ι_B, while ions supplied by the other ion source 12IA or 12ι_B are passed, unreacted with a reagent, to instrument 30. In operation, the valves 400-ι_A and 400₂ of the first ion source 12-ι_A and labeling reagent source 16ι respectively are opened for some time period while the valve 400 ι_B of the second ion source 12_1B is maintained closed, such that ions from ion source 12-IA react with the reagent supplied by reagent source 16ι within capillary 14^ and the resulting reacted compound is then analyzed by the molecular analysis instrument 30. Thereafter, the valves 400-ι_A and 400₂ of the first ion source 12-|_A and the labeling reagent source 16ι respectively are closed, and the valve 400 _B of the second ion source 12ι_B is opened for another time period such that only the ions produced by ion source 12-IB pass through capillary 14ι and are analyzed by the molecular analysis instrument 30. Data from the analysis of the reacted compound (i.e., ions from ion source 12-|_A reacted with the reagent supplied by reagent source 16-t) and the subsequent analysis of ions from ion source 12_1B are then compared for quantitative and/or relative differences. This operation is shown graphically in FIG. 12 where, at time T1 , valves 400ι_A and 400₂ are opened while valve 400 ι_B is maintained in a closed position, and at time T2 valves 400ι_A and 400₂ are closed. Ions generated by ion source 12ι_A and reacted with the reagent produced by reagent source 16ι are then analyzed by the molecular analysis instrument 30; e.g., a mass spectrometer, which produces a first mass spectrum 440. Thereafter at time T3, valves 400_1A and 400₂ are maintained in closed positions while valve 400-IB is opened, and at time T4 valve 400IB is closed. Ions generated by ion source 12ι_B are then analyzed by the molecular analysis instrument 30; e.g., a mass spectrometer, which produces a second mass spectrum 450. Computer 40 is programmed in a known manner to compare spectrums 440 and 450, and determine relative or quantitative information therefrom. Alternatively, valves 400ι_A, 400₂ and 400-IB may be opened and closed simultaneously such that ions generated by source 12_1A, which react with the reagent produced by reagent source 16-i, and ions generated by source 12ι_B arrive at the inlet of the molecular instrument simultaneously. All such ions are then analyzed simultaneously by the molecular analysis instrument 30 to produce a molecular spectrum.

Other configurations of instrument 10' may be used wherein both of the reagent sources in any one reagent source pair may be eliminated such that ions from any one corresponding ion sample source pairs may be comparatively analyzed in the absence of reagents. With the instrument illustrated in FIGS. 10-11A, this may be accomplished by omitting optional reagent source 16ι. Instrument 10' is operable in a temporal comparative analysis mode in this embodiment to open valve 400ι_A of the first ion source 12ι_A for some time period while the valve 400-IB of the second ion source 12ι_B is maintained closed, such that only ions produced by ion source 12-IA pass through capillary 14ι and are analyzed by the molecular analysis instrument 30. Thereafter, valve 400-IA of the first ion source 12-IA is closed, and valve 400ι_B of the second ion source 12- is opened for another time period such that only the ions produced by ion source 12IB pass through capillary 14ι and are analyzed by the molecular analysis instrument 30. Data from the analysis of ions from the first ion source 12ι_A and the subsequent analysis of ions from ion source 12ι_B are then compared for quantitative and/or relative differences. In this manner, abundances of various molecules of a mixture relative to known abundances of these molecules within another mixture may be determined. This operation is shown graphically in FIG. 12 where the operation of valve 400₂ illustrated in FIG. 12 should be ignored as instrument 10', in this embodiment, does not include reagent source 16ι. At time T1 , valve 400-IA is opened while valve 400ι_B is maintained in a closed position, and at time T2 valve 400-IA is closed. Ions generated by ion source 12_1A are then analyzed by the molecular analysis instrument 30; e.g., a mass spectrometer, which produces a first mass spectrum 440. Thereafter at time T3, valve 400-|_A is maintained in a closed position while valve 400-ι_B is opened, and at time T4 valve 400-ι_B is closed. Ions generated by ion source 12-i_B are then analyzed by the molecular analysis instrument 30; e.g., a mass spectrometer, which produces a second mass spectrum 450. Computer 40 is programmed in a known manner to compare spectrums 440 and 450, and determine relative or quantitative information therefrom. Alternatively, valves 400-IA and 400-IB may be opened and closed simultaneously such that ions generated by source 12-IA and ions generated by source 12ι_B arrive at the inlet of the molecular instrument simultaneously. All such ions are then analyzed simultaneously by the molecular analysis instrument 30 to produce a molecular spectrum.

It is to be understood that the embodiment of instrument 10' illustrated in FIGS. 10-11 B is provided only by way of example, and that other structural alternatives may be implemented. For example, those skilled in the art will recognize that any number of ion sources and/or reagent sources may be coupled to a single capillary. In this manner, multiple samples of unknown analyte concentration may be quantitatively or relatively compared to a sample of known analyte concentration. Other structural modifications to instrument 10 of FIG. 1 and/or instrument 10' of FIGS. 10-11 B will occur to those skilled in the art, and such modifications that fall within the spirit of one or more of the concepts described herein are intended to fall within the scope of the claims appended hereto.

Another use for instrument 10 illustrated in FIG. 1 , and/or instrument 10' illustrated in FIG. 10, includes adducting, in the gas phase, reagent molecules, via one or more of the reagent sources 16-ι - 16N, to a certain organic component or components contained in larger organic ions, generated by one or more of the sample sources 13-i - 13N, for the purpose of shifting those larger organic ions containing the certain organic component or components to different regions of one or more molecular characteristic spectra. The reagent in this case is selected as a reagent that will generally combine with, or adduct to, only the certain organic component or components contained in the larger organic ions. This process allows for identification, for example, of ones of the larger organic ions that contain the certain organic component or components as compared with others of the larger organic ions that do not contain the certain organic component or components. Further, since this process provides for the shifting of larger organic molecules containing the certain organic component or components to different regions of more than one molecular characteristic spectrum, the process may accordingly be used to produce multi-dimensional spectral information. This allows the molecular characteristic data to be expanded over wider molecular characteristic ranges in multiple molecular characteristic dimensions, thereby providing for the expansion of crowded molecular information into identifiable molecules. The ability to so expand the molecular characteristic information in multiple molecular characteristic dimensions may be particularly useful when the molecular information is crowded in one or more dimensions due to fragmentation of the organic ions.

The process of adducting reagent molecules to a certain organic component or components contained in larger organic ions, as just described, may be carried out using any one or more of the instrument embodiments described hereinabove. For example, the instrument 10 of FIG. 1 or instrument 10' of FIG. 10 may be used to adduct any number of suitable reagent molecules to any number of correspondingly suitable organic ions. Alternatively, such instrumentation may be configured to adduct suitable reagent molecules from a single reagent source to one of a pair of sets of generated organic ions for comparative or quantitative analysis. Other examples will occur to those skilled in the art, and such other examples are intended to fall within the scope of the claims appended hereto. In any case, the molecular characteristic analysis instrument 30 may comprise any one or combination of the example molecular characteristic analysis instruments described hereinabove. It will be understood that the molecular characteristic analysis instrument 30 will necessarily comprise at least "G" molecular characteristic analysis instruments arranged in cascade fashion as illustrated in FIG. 7, wherein each of the "G" instruments is operable to analyze ions according to a different molecular characteristic, in order to produce "G"-dimensional spectral information, and wherein "G" may be any positive integer.

As one illustrative example of the foregoing process, specific amino acids or types of amino acids that may be present in certain peptides are known to combine with certain reagents. For example, 18-crown-6 ether is known to adduct specifically with basic amino acids; e.g., lysine, arginine, histidine, etc., and the N terminus of peptides. Using the gas-phase tagging or labeling concepts described herein, 18- crown-6 ether may thus be adducted to peptides that contain one or more basic amino acids, for the purpose of shifting molecular characteristic information of the peptides to different regions of one or more molecular characteristic spectra.

Referring to FIG. 13, for example, one embodiment of instrument 10 or 10' includes as a reagent source, 16 , 18-crown-6 ether, and the molecular analysis instrument 30 in this example embodiment includes a reflectron mass spectrometer positioned in cascade relationship with an ion mobility spectrometer. Initially, as illustrated in FIG. 13, one of the ion sources, 12χ, is operable to produce ions from the peptide Tetralysine in the absence of the 18-crown-6 ether reagent. The resulting drift time (ion mobility) vs. mass-to-charge ratio (m/z) spectral data reveals a [KKKK+2H]²⁺ peak 460 approximately at a drift time of 11 ms and a m/z of 266, and a [KKKK+H]⁺ peak 470 approximately at a drift time of 20 ms and a m/z of 531. As illustrated in FIG. 14, 18-crown-6 ether is introduced in the gas phase to ions produced from the peptide Tetralysine, and the 18-crown-6 ether adducts to the amino acid lysine contained in the peptide. The result of this adduction is to shift the location of both of the [KKKK+2H]²⁺ and [KKKK+H]⁺ peaks, 460' and 470' respectively, to higher mass- to-charge values and higher drift times. Specifically, the [KKKK+2H]²⁺ peak 470' in FIG. 14 is shifted to a drift time of approximately 18 ms and a m/z of approximately 530, and the [KKKK+H]⁺ peak 470' is shifted to a drift time of approximately 27 ms and a m/z of approximately 795. In this example, this technique may thus be used to identify quantitatively or comparatively peptides containing specific amino acids or types of amino acids, and to increase the peak capacity of one or more molecular characteristic dimensions.

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.

Claims

Claims:

1. An apparatus for comparative relative or quantitative analysis of a plurality of samples, comprising: a plurality of ion sources each configured to generate ions from a different one of the plurality of samples; a capillary structure having a plurality of ion inlets each coupled to a corresponding one of the plurality of ion sources and an ion outlet, the capillary structure configured to direct the ions generated by each of the plurality of ion sources to the ion outlet; and a molecular analysis instrument having an ion inlet coupled to the ion outlet of the capillary structure and configured to produce molecular intensity information as a function of at least one molecular characteristic for relative or quantitative comparison of the plurality of samples.

2. The apparatus of claim 1 further including a computer configured to control introduction of ions from each of the plurality of ion sources into the capillary structure, the computer including a memory unit for storing therein the molecular intensity information produced by the molecular analysis instrument.

3. The apparatus of claim 2 wherein the computer is configured to control introduction of ions from each of the plurality of ion sources into the capillary structure such that ions generated by each of the plurality of ion sources are introduced into the capillary structure simultaneously.

4. The apparatus of claim 2 wherein the computer is configured to control introduction of ions from each of the plurality of ion sources into the capillary structure such that ions generated by each of the plurality of ion sources are introduced sequentially into the capillary structure; and wherein the molecular intensity information produced by the molecular analysis instrument for each of the plurality of sets of sequentially introduced ions are stored by the computer in the memory unit for subsequent relative or quantitative comparison of the plurality of samples.

5. The apparatus of claim 1 further including a first reagent source configured to introduce a first sample-labeling reagent into the capillary structure for combination with ions generated by one or more of the plurality of ion sources.

6. The apparatus of claim 5 further including a computer configured to control introduction of ions from each of the plurality of ion sources and introduction of the first sample-labeling reagent from the first reagent source into the capillary structure, the computer including a memory unit for storing therein the molecular intensity information produced by the molecular analysis instrument.

7. The apparatus of claim 6 wherein the computer is configured to control introduction of ions from each of the plurality of ion sources and introduction of the first sample-labeling reagent into the capillary structure in pairs of ion sets such that a first set of ions generated by one of the plurality of ion sources is introduced into the capillary structure followed or preceded sequentially by a second set of ions generated by another one of the plurality of ion sources simultaneously with the first sample-labeling reagent so that the first sample-labeling reagent combines with the second set of ions; and wherein the molecular intensity information produced by the molecular analysis instrument for each of the pairs of sequentially introduced ions are stored by the computer in the memory unit for subsequent relative or quantitative comparison of the plurality of samples.

8. The apparatus of claim 7 wherein the capillary structure includes a reagent inlet coupled to the first reagent source, the capillary structure defining a length between a junction of the reagent inlet and the ion inlet of the capillary structure coupled to the another one of the plurality of ion sources and the ion outlet of the capillary structure; and wherein a reaction rate of the first sample-labeling reagent and the second set of ions is a function of the length.

9. The apparatus of claim 7 further including means for modifying an operating temperature of at least a portion of the capillary structure carrying the first sample-labeling reagent; wherein a reaction rate of the first sample-labeling reagent and the second set of ions is a function of the operating temperature of the at least a portion of the capillary structure.

10. The apparatus of claim 7 further including means for controlling the pressure of the first sample-labeling reagent supplied to the capillary structure by the first reagent source; wherein a reaction rate of the first sample-labeling reagent and the second set of ions is a function of the pressure of the first sample-labeling reagent.

11. The apparatus of claim 5 further including a second reagent source configured to introduce a second sample-labeling reagent into the capillary structure for combination with ions generated by one or more of the plurality of ion sources.

12. The apparatus of claim 11 further including a computer configured to control introduction of ions from each of the plurality of ion sources, introduction of the first sample-labeling reagent from the first reagent source and introduction of the second sample-labeling reagent from the second reagent source into the capillary structure, the computer including a memory unit for storing therein the molecular intensity information produced by the molecular analysis instrument.

13. The apparatus of claim 12 wherein the computer is configured to control introduction of ions from each of the plurality of ion sources, introduction of the first sample-labeling reagent and introduction of the second sample-labeling reagent into the capillary structure in pairs of ion sets such that a first set of ions generated by one of the plurality of ion sources is introduced into the capillary structure simultaneously with the first sample-labeling reagent so that the first sample-labeling reagent combines with the first set of ions followed or preceded sequentially by a second set of ions generated by another one of the plurality of ion sources simultaneously with the second sample-labeling reagent so that the second sample-labeling reagent combines with the second set of ions; and wherein the molecular intensity information produced by the molecular analysis instrument for each of the pairs of sequentially introduced ions are stored by the computer in the memory unit for subsequent relative or quantitative comparison of the plurality of samples.

14. The apparatus of claim 13 wherein the first and second regents are selected to provide for a definable molecular characteristic shift in the molecular intensity information between the first set of ions and the second set of ions.

15. The apparatus of claim 14 wherein the first and second sample-labeling reagents are deuterated and non-deuterated reagent pairs.

16. The apparatus of claim 14 wherein the first set of ions represent a distribution of ions generated from a sample of known composition and having known analyte concentration levels and the second set of ions represent a distribution of ions generated from a sample of same known composition but having unknown analyte concentration levels; and wherein comparison of the molecular intensity information for the first set of ions and the molecular intensity information for the second set of ions provides for identification of abundances of the analyte concentration levels in the second set of ions.

17. The apparatus of claim 16 wherein the first and second sample-labeling reagents are deuterated and non-deuterated reagent pairs.

18. The apparatus of claim 13 wherein the capillary structure includes a first reagent inlet coupled to the first reagent source and a second reagent inlet coupled to the second reagent source, the capillary structure defining a first length between a junction of the first reagent inlet and the ion inlet of the capillary structure coupled to the one of the plurality of ion sources and the ion outlet of the capillary structure, and a second length between a junction of the second reagent inlet and the ion inlet of the capillary structure coupled to the another one of the plurality of ion sources and the ion outlet of the capillary structure; and wherein a reaction rate of the first sample-labeling reagent and the first set of ions is a function of the first length and a reaction rate of the second sample- labeling reagent and the second set of ions is a function of the second length.

19. The apparatus of claim 13 further including means for modifying an operating temperature of at least a portion of the capillary structure carrying the first and second sample-labeling reagents; wherein a reaction rate of the first sample-labeling reagent and the first set of ions and a reaction rate of the second sample-labeling reagent and the second set of ions is a function of the operating temperature of the at least a portion of the capillary structure.

20. The apparatus of claim 13 further including: means for controlling the pressure of the first sample-labeling reagent supplied to the capillary structure by the first reagent source; and means for controlling the pressure of the second sample-labeling reagent supplied to the capillary structure by the second reagent source wherein a reaction rate of the first sample-labeling reagent and the first set of ions is a function of the pressure of the first sample-labeling reagent and a reaction rate of the second sample-labeling reagent and the second set of ions is a function of the pressure of the second sample-labeling reagent.

21. The apparatus of claim 1 wherein one or more of the plurality of ion sources is a matrix-assisted laser desorption ion (MALDI) source.

22. The apparatus of claim 1 wherein one or more of the plurality of ion sources is an electrospray ionization (ESI) source.

23. The apparatus of claim 1 wherein one or more of the plurality of ion sources is an electron impact ionization (El) source.

24. The apparatus of claim 1 wherein one or more of the plurality of ion sources is a chemical ionization (Cl) source.

25. The apparatus of claim 1 wherein one or more of the plurality of ion sources includes an ion trap operable to selectively collect and release ions generated from a corresponding sample.

26. The apparatus of claim 1 wherein one or more of the plurality of ion sources includes a gated ion collection chamber operable to selectively collect and release ions generated from a corresponding sample.

27. The apparatus of claim 1 wherein one or more of the plurality of ion sources includes a charge normalization stage operable to selectively normalize the charge of ions generated from a corresponding sample.

28. The apparatus of claim 27 wherein the charge normalization stage includes a radiation source configured to emit radiation to normalize ions having various charge states to a predefined charge state.

29. The apparatus of claim 27 wherein the charge normalization stage includes a reagent source providing a reagent selected to spread crowded ion mass peaks, resulting from multiply-charged ions generated from the corresponding sample, over a wider mass range.

30. The apparatus of claim 1 wherein one or more of the ion sources includes: a non-ionizing sample source producing particles from a corresponding sample; and a particle ionizing instrument configured to ionize the particles produced by the non-ionizing sample source.

31. The apparatus of claim 1 wherein one or more of the ion sources includes at least one ion separation instrument operable to separate ions in time as a function of a molecular characteristic.

32. The apparatus of claim 31 wherein the at least one ion separation instrument is a mass spectrometer configured to separate ions in time as a function of ion mass-to-charge ratio.

33. The apparatus of claim 31 wherein the at least one ion separation instrument is an ion mobility spectrometer configured to separate ions in time as a function of ion mobility.

34. The apparatus of claim 31 wherein the at least one ion separation instrument is a liquid chromatograph configured to separate ions in time as a function of ion retention time.

35. The apparatus of claim 31 wherein the at least one ion separation instrument is a gas chromatograph configured to separate ions in time as a function of ion retention time.

36. The apparatus of claim 1 wherein one or more of the ion sources includes an ion mass filter operable to selectively collect and release only ions within a predefined range of mass-to-charge ratios.

37. The apparatus of claim 1 wherein one or more of the ion sources includes an ion fragmentation stage operable to selectively fragment ions generated from a corresponding sample into parent and daughter ions.

38. The apparatus of claim 1 wherein the molecular analysis instrument is or includes at least one mass spectrometer.

39. The apparatus of claim 1 wherein the molecular analysis instrument is or includes at least one ion mobility spectrometer.

40. The apparatus of claim 1 wherein the molecular analysis instrument is or includes at least one gas chromatograph.

41. The apparatus of claim 1 wherein the molecular analysis instrument is or includes at least one liquid chromatograph.

42. The apparatus of claim 1 wherein the molecular analysis instrument includes at least one ion trap.

43. The apparatus of claim 1 wherein the molecular analysis instrument includes at least one ion mass filter configured to selectively collect and release only ions within a predefined range of mass-to-charge ratios.

44. The apparatus of claim 1 wherein the molecular analysis instrument includes at least one ion fragmentation stage operable to selectively fragment ions into parent and daughter ions.

45. The apparatus of claim 1 wherein the molecular analysis instrument includes at least one charge normalization stage.

46. The apparatus of claim 1 further including a reagent source configured to introduce a reagent into the capillary structure for combination with ions generated by one or more of the plurality of ion sources; and wherein ions generated by at least one of the plurality of ion sources are ions including a predetermined organic component contained in a larger organic structure, and the reagent is selected to adduct to the predetermined organic component contained in the larger organic structure to shift molecular intensity information of the larger organic structure containing the predetermined organic component to different regions of one or more molecular characteristic spectra.

47. The apparatus of claim 46 wherein the larger organic structure is a peptide and the predetermined organic component is one of a specific amino acid and a type of amino acid.

48. The apparatus of claim 46 wherein the molecular analysis instrument includes a number of cascaded molecular analysis units each configured to produce molecular intensity information as a function of a different molecular characteristic; and wherein the molecular intensity information of the larger organic structure containing the predetermined organic component is shifted to a different region of the molecular characteristic spectrum of at least one of the number of different molecular characteristics.

49. A method of conducting comparative relative or quantitative analysis of a plurality of samples, comprising: generating ions from each of a plurality of different samples; directing the ions generated from each of the plurality of different samples to an inlet of a molecular analysis instrument; analyzing the ions generated from each of the plurality of different samples via the molecular analysis instrument to produce molecular intensity information as a function of at least one molecular characteristic; and relatively or quantitatively comparing the molecular intensity information of the ions generated from each of the plurality of different samples.

50. The method of claim 49 wherein the act of directing the ions generated from each of the plurality of different samples to an inlet of a molecular analysis instrument includes simultaneously directing the ions generated from each of the plurality of different samples into the inlet of the molecular analysis instrument.

51. The method of claim 49 further including combining ions generated from at least one of the plurality of different samples with a first sample-labeling reagent.

52. The method of claim 51 wherein the act of directing the ions generated from each of the plurality of different samples to an inlet of a molecular analysis instrument includes sequentially: directing ions generated from one of the plurality of different samples into the inlet of the molecular analysis instrument; directing ions generated from another one of the plurality of different samples combined with the first sample-labeling reagent into the inlet of the molecular analysis instrument; and wherein the act of relatively or quantitatively comparing the molecular intensity information of the ions generated from each of the plurality of different samples includes relatively or quantitatively comparing molecular intensity information for the ions generated from the one of the plurality of different samples with molecular intensity information for the ions generated from the another one of the plurality of different samples combined with the first sample-labeling reagent.

53. The method of claim 51 further including combining ions generated from at least another one of the plurality of different samples with a second sample- labeling reagent.

54. The method of claim 53 wherein the act of directing the ions generated from each of the plurality of different samples to an inlet of a molecular analysis instrument includes sequentially: directing ions generated from one of the plurality of different samples combined with the first sample-labeling reagent into the inlet of the molecular analysis instrument; directing ions generated from another one of the plurality of different samples combined with the second sample-labeling reagent into the inlet of the molecular analysis instrument; and wherein the act of relatively or quantitatively comparing the molecular intensity information of the ions generated from each of the plurality of different samples includes relatively or quantitatively comparing molecular intensity information for the ions generated from the one of the plurality of different samples combined with the first sample-labeling reagent with molecular intensity information for the ions generated from the another one of the plurality of different samples combined with the second sample-labeling reagent.

55. The method of claim 54 wherein the first and second sample-labeling reagents are selected to provide for a definable molecular characteristic shift in the molecular intensity information between the ions generated from the one of the plurality of different samples combined with the first sample-labeling reagent and the ions generated from the another one of the plurality of different samples combined with the second sample-labeling reagent.

56. The method of claim 55 wherein the first and second sample-labeling reagents are deuterated and non-deuterated reagent pairs.

57. The method of claim 55 wherein the one of the plurality of different samples is a sample of known composition and having known analyte concentration levels and the another one of the plurality of different samples is a sample of the same known composition and having unknown analyte concentration levels; and wherein the act of relatively or quantitatively comparing the molecular intensity information of the ions generated from each of the plurality of different samples includes comparing molecular intensity information for the ions generated from the one of the plurality of different samples combined with the first sample- labeling reagent with molecular intensity information for the ions generated from the another one of the plurality of different samples combined with the second sample- labeling reagent to determine the abundances of the analyte concentration levels in the ions generated from the another one of the plurality of different samples.

58. The method of claim 57 wherein the first and second sample-labeling reagents are deuterated and non-deuterated reagent pairs.