A method for resolving charge state ambiguities in high- and very-high-mass mass spectra.

Abstract

The mass spectrometry method consists of the following steps: identifying charge state distribution (CSD) groups within the deconvoluted mass spectrometry data, each CSD consisting of at least one common mass spectral peak value assigned to each distinct charge state within each CSD in each group; assigning a weighting factor to each CSD within each group; calculating a score-weighted average molecular weight for each compound CSD within each group using the weighting factor; identifying a single target CSD within each group that corresponds to the molecular weight closest to the calculated average; summing the intensities of each common assigned mass spectral peak across the entire group for each identified common peak in each group; assigning the summed intensity to a single target CSD for that group; discarding all CSDs other than the target CSD; and using the summed intensity to calculate the abundance of each component compound.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims priority to U.S. Provisional Patent Application No. 18 / 151,321, filed January 6, 2023.

[0002] The present invention relates to mass spectrometry and mass spectrometers, and more particularly to mass spectrometry of macromolecular compounds having molecular weights of 450 kilodaltons (450 kDa) or greater. [Background technology]

[0003] Mass spectrometry has advanced over the past few decades to become one of the most widely applicable analytical tools for detecting and characterizing a wide class of molecules. Mass spectrometry is applicable to virtually any molecular species that can be ionized to form ions in the gas phase. Thus, mass spectrometry offers perhaps the most widely applicable quantitative analytical method. Furthermore, mass spectrometry is a highly selective technique, particularly suited to the analysis of complex mixtures of different compounds at various concentrations. Mass spectrometry offers extremely high detection sensitivity, approaching detection limits of 10 parts per trillion for some molecular species. As a result of these beneficial properties, there has been considerable interest over the past few decades in developing mass spectrometry methods for the analysis of complex mixtures of biomolecules, such as peptides, proteins, carbohydrates, oligonucleotides, and complexes of these molecules.

[0004] One common type of application of mass spectrometry to the analysis of natural samples involves characterizing and / or quantifying components of complex mixtures of biomolecules. Many of these notable biological molecules are biopolymers, such as polynucleotides (RNA and DNA), polypeptides, and polysaccharides. Generally, the chemical composition (related to the specific set of monomers that make up the polymer) and the sequence of the monomers are characteristic analytical properties of a given class of biopolymer molecule. Nevertheless, because biopolymer molecules of a given class typically have high molecular weights and can generate ions with a wide range of charge states, the process of distinguishing between various molecules in such mixtures of molecules by mass spectrometry can be challenging.

[0005] Biomolecules are often introduced into the ionization source of a mass spectrometer dissolved in water or a mixture of aqueous buffer and organic solvent. Compounds from the soluble analyte can be separated from insoluble compounds using, for example, a solid-phase extraction device. The soluble fraction typically consists of multiple compounds, many of which are macromolecules such as peptides and proteins. These fractions may then be further fractionated using reversed-phase chromatography. The various soluble biomolecules, whether separated by chromatography or simply injected, can then be readily ionized by electrospray ionization. Ion species thus generated are referred to herein as "primary" ion species. The mass analyst then detects and quantifies the primary ion species or intentionally generated fragment ion species (or other product ion species) by their respective mass-to-charge (m / z) ratios.

[0006] An important feature of electrospray ionization is that it proceeds by adding charged units from the solution to each molecular framework, and therefore tends to maintain molecular structure without excessive fragmentation. Thus, each organic molecular species of interest within the unionized analyte

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[0007] More specifically, organic molecular species

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[0008] In fact, mass analysis of a single analyte can result in many overlapping charge state distributions such as those described above. Accordingly, many computer software programs and algorithms are known or commercially available that can separate ("deconvolute") and identify the various overlapping distributions. Generally, the output of such a deconvolution program consists of the following steps: (i) a list of the median values ​​of the recognized peaks; (ii) grouping the medians into charge state distributions and assigning the likely charge state to each peak in each group; and (iii) for each identified charge state distribution, the calculated molecular weight of the molecular species.

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[0009] For example, techniques used in one such computer program are disclosed in U.S. Patent No. 10,217,619. Figure 1 shows the results of deconvolution of a five-component protein mixture consisting of cytochrome c, lysozyme, myoglobin, trypsin inhibitor, and carbonic anhydrase, calculated by the method described in U.S. Patent No. 10,217,619. The top display panel 103 shows the data obtained from mass spectrometry displayed as centroids. The centrally located main display panel 101 shows each peak value with its respective symbol. A horizontally located mass-to-charge (m / z) scale 107 is displayed below the central panel 103 for both the top panel 103 and the center panel 101. Each horizontal line 101 in the main panel connects the centroid symbols of the peak values ​​assigned to a single charge state distribution. The numerical values ​​101 attached to the diagonal dotted lines in the panel are the assigned charge states. The left panel 105 of the display displays the calculated molecular weights (in Daltons) of the protein molecules. The molecular weight (MW) scale 105 on the side panel is oriented vertically on the display and is orthogonal to the horizontal m / z scale 107 associated with the detected ions.

[0010] The conventional mass spectrometry methods described above are effective for proteins with low to medium molecular weights. However, the inventors recognize that continued improvements in mass spectrometer performance may result in a larger mass spectrometry range of m / z measurements. Specifically, as molecular weights approach and exceed approximately 450 kDa, ambiguity in charge state measurements may arise, potentially resulting in false positives or mismeasurements of the abundance of compounds actually present in the sample. This ambiguity is due to the uncertainty in the assigned charge states (e.g., the standard deviation of the assigned charge states).

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[0011] A method for resolving charge state measurement ambiguities that arise when resolving ultra-high mass (>450 kDa) mass spectrometry data is described. The method identifies component compounds that share one or more assigned m / z peaks identified by a deconvolution routine, but the shared m / z peaks are assigned different charge states within the different component compounds. The method further determines which components are genuine and which are false positives. The method then discards the false positives and combines their signals with those of components actually present to correct for the abundances of various components and, optionally, generate a final spectrum of the molecular weights of those components.

[0012] According to aspects of the current teachings, there is provided a method for eliminating false positive identifications and correcting for the abundance of constituent compounds of a sample having a molecular weight of about 450 kDa or greater, as determined by deconvolution of mass spectrometry data, the method comprising: identifying groups of deconvoluted charge state distributions within the resolved mass spectrometry data, wherein all charge state distributions of the identified groups contain at least one common assigned mass spectral peak, and each common assigned mass spectral peak is assigned a different charge state within each charge state distribution of the group; Recognizing a charge state distribution within the identified charge state distribution group that corresponds to a false positive compound identification; summing the peak intensities of commonly assigned mass spectral peaks of the identified groups of charge state distributions identified as corresponding to false positive compound identifications with the peak intensities of the target charge state distributions of groups not identified as corresponding to false positive compound identifications; discarding from the group of identified charge state distributions all charge state distributions corresponding to false positive compound identifications; and Using the summed peak intensities to calculate the abundances of the component compounds corresponding to the target charge state distribution.

[0013] In some cases, the procedure for identifying charge state distributions within a recognized charge state distribution group that correspond to false positive compound identifications is: assigning a respective weighting factor to each charge state distribution in the identified charge state distribution group; calculating a score-weighted average molecular weight of each real or hypothetical component molecular species corresponding to each charge state distribution of the identified charge state distribution group within the identified charge state distribution group, using assigned weighting coefficients; and locating a target charge state distribution within the identified charge state distribution group as the charge state distribution having a value closest to the calculated average molecular weight; Includes: In some cases, weighting factors can be assigned based, at least in part, on the assigned or calculated mean squared error in the calculated molecular weights of the real or hypothetical component molecular species corresponding to the charge state distribution. In some cases, weighting factors can be assigned based, at least in part, on the intensity of the mass spectral peaks of each charge state distribution.

[0014] According to another aspect of the present teachings, there is provided a mass spectrometry system comprising: an electrospray ion source configured to receive one or more component compounds having a molecular weight of 450 kilodaltons (kDa) or greater; a mass analyzer configured to receive ions produced by ionization of the constituent compounds of the analyte; a detector configured to detect ions output from the mass analyzer and generate mass spectral data therefrom; a data storage device configured to receive the mass spectrometry data from the detector; and a programmable processor configured to receive the mass spectral data from either the detector or the data storage device, the programmable processor including computer readable instructions operable to: performing conventional deconvolution of said mass spectral data; and Automatically detect and remove false-positive compound identifications produced by conventional deconvolution, where the erroneous compound identification is caused by the standard deviation of the charge state assignments being equal to or greater than the conventional maximum. [Brief explanation of the drawings]

[0015] The above and various other aspects of the present invention will become apparent from the following description, which is given by way of example only, and with reference to the accompanying drawings, which are not necessarily drawn to scale: [Figure 1]This figure is a graph showing the mass spectrometry deconvolution results obtained from a five-component protein mixture consisting of cytochrome c, lysozyme, myoglobin, trypsin inhibitor, and carbonic anhydrase. [Figure 2A] This figure is a chromatogram showing the elution profiles of various multimers of the globular protein apoferritin. [Figure 2B] This figure is a low-resolution mass spectrometry analysis of the eluate corresponding to the elution profile in Figure 2A. [Figure 3A] This figure shows the calculated molecular weight spectrum of the eluted components, which is generated by applying a deconvolution algorithm to the data in Figure 2B. [Figure 3B] This figure is a graphical representation of how charge state uncertainty results in the assignment of individual peaks from the mass spectral data in Figure 2B to a distribution of multiple charge states. The graph includes a centrally located main display panel showing each peak's assignment with its respective symbol, a top display panel showing the raw mass spectral data, a left panel showing calculated molecular weights along a vertical molecular weight axis, and a mass-to-charge (m / z) scale located horizontally on both the top and center panels. [Figure 3C] This figure is a graph similar to FIG. 3B, showing the results of correcting mass spectrometry data to a single molecular weight value using the current teaching method. [Figure 3D] This figure is a spectrum of the corrected molecular weights of the eluted components of Figure 3A, as corrected according to current teachings. [Figure 4] This figure is a flow diagram of a method for resolving mass spectrometry charge state ambiguity according to current teachings. [Figure 5] This figure is a schematic diagram of a system for generating and automatically analyzing chromatographic / mass spectrometry spectra that may be employed in connection with the methods of the present teachings. DETAILED DESCRIPTION OF THE INVENTION

[0016] The following description is presented to enable any person skilled in the art to make and use the invention, and is provided in the context of a particular application and its requirements. Various modifications to the described embodiments will be readily apparent to those skilled in the art, and the general principles herein may be applied to other embodiments. Thus, the present invention is not intended to be limited to the embodiments and examples shown, but is to be accorded the widest possible scope in accordance with the features and principles shown and described. For a more detailed and complete understanding of the features of the present invention, please see Figures 1, 2A, 2B, 3A, 3B, 3C, 3D, 4, and 5 in conjunction with the following description.

[0017] In describing the invention herein, words denoted in the singular are understood to include plural equivalents, and words denoted in the plural are understood to include the singular equivalents, unless this is implicitly or explicitly understood or described. Furthermore, for any given component or embodiment described herein, it is understood that any of the possible candidates or alternatives listed for that component may generally be used individually or in combination with each other, unless otherwise understood or described, either implicitly or explicitly. Furthermore, as illustrated herein, the figures are not necessarily drawn to scale, and some elements may be shown for clarity of the matter only. Also, reference numerals may be repeated among the various figures to indicate corresponding or similar elements.

[0018] Figures 2A-2B and 3A-3D show examples of this unrecognizable problem. Figure 2A is a chromatogram showing the elution profile 61 of various multimers of the globular protein apoferritin, consisting of a broad elution peak that maximizes during retention time 62. Figure 2B is a low-resolution mass spectrometry spectrum within the m / z range of 4000-15000 Th of the elution corresponding to the elution profile in Figure 2A. Figure 3A shows the molecular weight spectrum of the eluting chemical components of the eluate, determined by applying a conventional deconvolution algorithm to the data in Figure 2B. The deconvolution routine calculates the molecular weights of the chemical components: 471,679.1 Da (molecular component peak value 71), 493,192.1 Da (molecular component peak value 72), 507,043.7 Da (molecular component peak value 73), and 517,193.1 Da (molecular component peak value 74). The calculated molecular weight of the molecular component peak value, 73, is close to the known molecular weight of the apoferritin 24-mer. However, as is evident from the charge state assignments listed in Table 1 below, in this example, a portion of the mass spectrometry signal intensity of the apoferritin 24-mer peak value (i.e., the data shown in Figure 2B) is falsely assigned as a molecular component peak value, 74. This conclusion is illustrated by the fact that, in this case, the deconvolution routine assigns each of the several observed mass spectrometry peaks (listed in the table as mass spectrometry peaks 2-5) to both the true molecular component peak value, 73, and the false molecular component peak, 74, resulting in the assignment of two different charge states to each mass spectrometry peak value. Notably, each charge state assignment, 74, used to falsely identify a molecular component peak is consistently one unit larger than the true molecular component peak value assignment, 73. Ambiguity in charge state assignment is due to the loss of accuracy in the charge state assignment, as described in equation (2). [Table 1]

[0019] Figure 3B, similar to the graph in Figure 1, illustrates how charge state uncertainty can cause individual peaks in the mass spectral data of Figure 2B to be assigned to multiple charge state distributions. The top display panel 103 in Figure 3B is a magnified version of the mass spectral data of Figure 2B, with the horizontal mass-to-charge ratio (m / z) scale 107 at the bottom of the figure. The left panel 105 shows a rotated view of the molecular weights calculated in a standard deconvolution software package, with the mass scale (i.e., molecular weight scale) oriented vertically. Finally, the center panel 101 correlates the mass spectral peak values ​​in Table 1 with their assigned charge states, as well as the horizontally oriented m / z scale and the vertically oriented molecular weight scale, showing the median peak values ​​in Table 1 as individual points in the center panel 101. Similar to the graph in Figure 1, the diagonal dotted lines 101 in the center panel are of constant charge state, labeled according to the charge state they represent. The diagonal dotted lines have different slopes.

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[0020] The stars above the mass spectrum in the top panel 103 of Figure 3B indicate the locations of the seven different peaks used for the assignment, as summarized in Table 1. (For reference, the diamond-shaped peak 103 in the top panel of Figure 3B represents the charge state distribution of another apoferritin multimer with a molecular weight of 493 kDa, represented by peak 72 of the molecular component in Figures 3A-3D.) Although only seven peaks are listed in Table 1, these seven peaks are plotted using 11 different points in the molecular weight and mass-to-charge plot in the middle panel (Figure 3B 101), and four of these peaks (peaks 2-5 in Table 1) are assigned by deconvolution to two hypothetical peaks 73 and 74, which correspond to different molecular weights as described above. Horizontal line 173 connects peak 73 to the corresponding charge state assignment of the tabulated mass spectral line, and horizontal line 174 connects peak 74 to the corresponding charge state assignment. Both sets of assignments are calculated by deconvolution.

[0021] above As noted in (e.g., Equation 2), when mass spectrometry is used to examine ions of compounds with molecular weights approaching or exceeding 500 kDa, the uncertainty in charge assignment can approach or exceed one charge unit. The charge assignment error bars can be, for example, as defined by Equation 2 below or multiples thereof:

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[0022] Figure 3C is a graph similar to Figure 3B, showing the results of correcting mass spectral data to a single molecular weight value using the methods of the present invention, as further described below. Using these methods, molecular component peak value 74 was identified as a false positive, and the corrected actual peak value 75 has an adjusted molecular weight of 507,353.41 Da (compared to the 507,043.7 Da originally calculated for peak value 73 for false positive peak value 74).

[0023] 4 is a flow diagram of a method 200 for resolving mass spectrometry charge state ambiguities in accordance with the current teachings. Method 200 applies to the analysis of macromolecules having molecular weights of 450 kDa or greater, which are ionized by an ionization technique in which ions of the macromolecule are generated by attaching multiple charged particles to the analyte molecule. Typically, but not necessarily, the method is directed to the analysis of organic macromolecules ionized by either electrospray or thermospray ionization.

[0024] In preparation for performing method 200, a mass spectrum of a sample containing macromolecular component compounds is measured by a mass spectrometer and / or obtained from a data storage device prior to performing method 100. Also, after measuring or obtaining the mass spectrum, a conventional "deconvolution" procedure is performed to convert the various mass-to-charge ratios P ​​of the peaks in the observed mass spectrum into tentatively identified component molecular species.

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[0025] In a first step 203 of method 200, the list of results obtained by the deconvolution procedure is searched to identify all instances in which one or more observed mass spectral peak values ​​are assigned by the deconvolution routine to multiple charge state distributions (i.e., "groups" of charge state distributions), each assigned to a different charge state within each charge state distribution in the group. Based on this search, one or more groups of charge state distributions in the deconvoluted mass spectrometry data are identified, where the criteria for identifying a group are that all charge state distributions in the group contain at least one common assigned mass spectral peak (i.e., a peak shared by all charge state distributions in the group), and further, that each common assigned mass spectral peak is assigned a different charge state within each charge state distribution.

[0026] In step 205, a weighting factor is assigned to each charge state distribution in each identified charge state distribution group. (Step 203), each weighting factor relates to the quality of the mass spectral data for the mass spectral peak corresponding to the species according to a selected metric. Any weighting factor may be based on mass spectral peak intensity, mass mean square error, or other quality indicator appropriate to the analytical instrument.

[0027] In step 207, these weighting factors are used to calculate a score-weighted average molecular weight for each group of charge state distributions identified in step 203. However, before performing step 207, in step 206, all of the P and z values ​​of the mass spectral peaks in each charge state distribution of each identified group are first used to calculate the molecular weights of the constituent compounds corresponding to that charge state distribution, regardless of whether the constituent compounds are real or virtual compounds, and regardless of whether the constituent compounds are actually present in the analyte. These individual molecular weights are then averaged in step 207 using the weighting factors assigned in step 205.

[0028] While each score-weighted average molecular weight calculated in step 207 generally does not correspond to an actual component species, it generally approximates the true molecular weight of a particular species actually present in the sample. Thus, in step 209, the component molecular species for which the initial calculated molecular weight (step 206) is the one most closely related to the intensity-weighted average molecular weight (step 207) of each group in the charge state distribution, and the component molecular species is positioned within the group. The individual molecular component species so positioned within each group is the most likely true positive species for that group, and is referred to herein as the "target" component species.

[0029] In the subsequent step 211, all component species other than the identified target component are discarded from each identified group. The discarded component molecular species are considered false positives. Therefore, for each commonly assigned peak in each group of each charge state distribution, the signal intensities initially assigned to the discarded species are summed with the signal intensities of the target component species located in the previous step. Finally, in step 213, the abundances of the various identified target components are recalculated from their respective summed peak signal intensities.

[0030] Figure 3B shows the results of applying this method to the deconvolution results shown in Figure 3A. Specifically, a false positive at 517 kDa (peak value 74 in Figure 3A) is identified and discarded, and its signal is combined with the true component at 507 kDa to obtain the corrected mass and intensity (shown in Figure 3B as molecular component peak value 75).

[0031] 5 is a schematic diagram of a system for generating and automatically analyzing chromatographic / mass spectrometry spectra in accordance with the methods of the present teachings. A chromatograph 33, such as a liquid chromatograph, high performance liquid chromatograph, or ultra-high performance liquid chromatograph, analyzes the analytes of a mixture to be analyzed. The analyte mixture is received by the mass analyzer 32 and at least partially separated into its individual chemical components according to well-known chromatographic principles. The at least partially separated chemical components are then transferred to a mass spectrometry system 34 for mass analysis at respective times. Upon receipt by the mass spectrometer, each chemical component is ionized by the mass spectrometry system ionization source 34a. The ionization source may generate multiple ion species (i.e., multiple precursor ion species) of different charge or mass from each chemical component. Thus, multiple ion species with different mass-to-charge ratios (e.g., charge state distributions) may be generated for each chemical component, with each component eluting from the chromatograph at its own characteristic time. These various ion species are analyzed by the mass analyzer 34b of the mass spectrometry system 34 and detected by the ion detector 35. As is well known, the combined effects of mass analysis and ion detection result in the mass spectrometer generating mass spectrometry data, which represent a record of the detected ion intensities as a function of the mass-to-charge ratios of ions generated from the analyte. Using the mass spectral data, various ion species can be appropriately identified according to their various mass-to-charge ratios. The mass spectrometer may include a mass filtering device (not shown) for isolating ion species within a particular selected m / z range, and a fragmentation cell (not shown) for fragmenting the selected ion species to produce product ions.

[0032] Still referring to Figure 5, the programmable processor 37 of the system 10 is electronically coupled to the detector of the mass analyzer and receives data generated by the detector during chromatography / mass analysis of the analyte. It may consist of a computer, or simply a circuit board or other programmable logic device operated by firmware or software. Optionally, the programmable processor may be electronically coupled to the chromatography and / or mass spectrometer to send electronic control signals to either of these instruments to control their operation. The nature of such control signals may be determined in response to data sent from the detector to the programmable processor or to analysis of that data. The programmable processor may also be electronically coupled to a display or other output 38 to output data and data analysis results to a user or directly to an electronic data storage device 36.

[0033] The programmable processor 37 of the system 10 is shown in FIG. 5 and generally includes computer-readable instructions operable to: control the individual operations and sequences of operations of the chromatograph 33; control the operations and sequences of operations of the mass spectrometer 34; receive mass spectra from the detector 35; perform conventional deconvolution procedures on the mass spectral data; and execute the logical steps of the method 100 (FIG. 4) to eliminate false positive identifications provided by the conventional deconvolution procedures and correct for compound abundances.

[0034] The discussion contained in this application is intended to serve as a basic description. The present invention is not intended to be limited in scope by the specific embodiments described herein, which are intended to outline individual aspects of the present invention. Functionally equivalent methods and components are within the scope of the present invention. Various other modifications of the present invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and accompanying drawings.

Claims

1. 1. A method for eliminating false positive identifications and correcting for abundances of constituent compounds of a sample having a molecular weight of 450 kilodaltons (kDa) or greater as determined from deconvoluted mass spectrometry data, the method comprising: (a) identifying groups of deconvoluted charge state distributions within the deconvoluted mass spectrometry data, wherein all charge state distributions of the identified groups contain at least one common assigned mass spectral peak, and each common assigned mass spectral peak is assigned a different charge state within each charge state distribution of the group; (b) recognizing, within the group of identified charge state distributions, charge state distributions that correspond to false positive compound identifications; (c) summing the peak intensities of the commonly assigned mass spectral peaks of the identified group of charge state distributions identified as corresponding to false positive compound identifications with the peak intensities of the target charge state distributions of the group not identified as corresponding to false positive compound identifications; (d) discarding from said group of identified charge state distributions all charge state distributions that correspond to said false positive compound identifications; and (e) using the summed peak intensities to calculate the abundances of component compounds corresponding to the target charge state distribution; A method comprising:

2. Step (b) of identifying charge state distributions within said identified charge state distribution groups that correspond to false positive compound identifications comprises: (b1) assigning a respective weighting factor to each charge state distribution of the identified charge state distribution group; (b2) calculating a score-weighted average molecular weight of each real or hypothetical component molecular species corresponding to each charge state distribution of the identified charge state distribution group within the identified charge state distribution group, using assigned weighting coefficients; and (b3) locating the target charge state distribution within the identified charge state distribution group as the charge state distribution having a value closest to the calculated average molecular weight.

3. 3. The method of claim 2, wherein each weighting factor is assigned based at least in part on the intensity of the mass spectral peak value of the respective charge state distribution.

4. 3. The method of claim 2, wherein each weighting factor is assigned based, at least in part, on an assigned or calculated mean squared error in the molecular weights of the actual or hypothetical component molecular species corresponding to the respective charge state distribution.

5. 1. A mass spectrometer system, comprising: an electrospray ion source configured to receive an analyte comprising one or more component compounds having a molecular weight of 450 kilodaltons (kDa) or greater; a mass analyzer configured to receive ions produced by ionization of the constituent compounds of the analyte; a detector configured to detect ions output from the mass analyzer and generate mass spectral data therefrom; a data storage device configured to receive mass spectrometry data from the detector; and a programmable processor configured to receive the mass spectral data from either the detector or the data storage device; The processor performing conventional deconvolution of said mass spectral data; and and automatically detecting and removing false positive compound identifications produced by said conventional deconvolution, wherein a false compound identification is caused by a standard deviation of charge state assignments being equal to or greater than a conventional maximum. Mass spectrometer system.

6. The reusable computer readable instructions for automatically detecting and removing false positive compound identifications generated by conventional deconvolution include: (a) identifying groups of deconvoluted charge state distributions within the deconvoluted mass spectrometry data, wherein all charge state distributions of the identified groups contain at least one common assigned mass spectral peak, and each common assigned mass spectral peak is assigned a different charge state within each charge state distribution of the group; assigning a respective weighting factor to each charge state distribution in each group within said identified groups of charge state distributions; using weighting factors within said group of identified charge state distributions to calculate a score-weighted average molecular weight of the actual or hypothetical component molecular species corresponding to each identified charge state distribution of said group; finding a single target charge state distribution within the group of identified charge state distributions that corresponds to a molecular weight closest to the calculated average molecular weight; and 6. The mass spectrometry system of claim 5, further comprising removing all charge state distributions other than the single target charge state distribution from the identified group of charge state distributions.

7. 6. The mass spectrometer system of claim 5, wherein the detected ions of one or more constituent compounds have a charge state of 50 or more.

8. 6. The mass spectrometer system of claim 5, wherein the detected ions of one or more constituent compounds have a charge state of 100 or more.

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