Patent Description:
The ability to acquire both accurate and precise qualitative and quantitative mass spectral data in the analysis of complex systems, such as proteins or other complex compounds, is predicated on the ability to measure the physico-chemical attributes of all ions independent of any surrounding matrix. Successful qualitative analysis requires ensuring that fragment and product ions are correctly aligned or matched to the parent or precursor ions from which they were derived.

MS coupled with chromatography, such as liquid chromatography (LC) methods, is a common approach to quantifying compounds in a sample. For example, MS/MS (or tandem MS) relates to a product ion spectrum of fragment ion from one or more precursor ions generated by a mechanism of disassociation, such as in-source fragmentation, surface-induced fragmentation, collision-induced dissociation, electron capture, electron-detachment, charge transfer, and photodissociation. The quantitation is typically performed by first separating compounds by LC to generate a sequence of chromatograms, then ionizing and detecting the separated compounds by MS/MS to produce a plurality of mass spectra having ion peaks. Using a peak area or a sum area of all peaks within a mass window that are associated with a target compound, quantitative information may be inferred about the compound of interest, assuming there is a correlation between the peak area and the compound concentration.

Structural elucidation of molecules in complex samples is determining which fragment ions in a product ion spectrum belong to which precursor. Conventional approaches experience challenges in quantifying a target compound in a complex sample, particularly under the time and resource constraints expected by operators and researchers. For example, as the complexity or dynamic range of a sample increases, the chance of multiple compounds co-eluting from LC in a same retention time (RT) window is also magnified, resulting in mass spectra where ions from different compounds occupy a same mass-to-charge-ratio (m/z) space and interfere with each other. In another example, as the complexity of a sample increases, the number of high-abundance compounds in the sample proportionally increases as well, which can saturate a detector and corrupt linearity of detector response. Accordingly, conventional MS techniques are frequently not able to provide a signal profile that is adequately correlated with the quantity of the compounds that constitute complex samples. The following document is known: <NPL> [<NUM>] <NUM>-<NUM> * abstract * * page <NUM>, paragraph <NUM> - page <NUM>, paragraph <NUM>. <NUM> * * page <NUM> * * page <NUM>, paragraph <NUM> *. Brad J Williams et al. disclose a multi-modal acquisition method, which the author asserts increases the peak capacity of complex proteomics sample dramatically, when compared with alternative methods.

The following presents a simplified summary in order to provide a basic understanding of some novel embodiments described herein. This summary is not an extensive overview, and it is not intended to identify key/critical elements or to delineate the scope thereof. Some concepts are presented in a simplified form as a prelude to the more detailed description that is presented later.

Various embodiments are generally directed to MS techniques for analyzing a sample. For example, some embodiments may include techniques and apparatus operative to segregate product ions in a composite product ion spectrum to their parent precursor ions based on, among other things, mass information of the constituent elements of the molecules under analysis. In exemplary embodiments, product ions may be aligned to their parent precursor via comparison of the rate of change of nominal (or integer) mass and mass defect (or fractional mass), for example, in descending order of nominal mass relative to that of the possible parent precursors.

The invention provides a computer-implemented method, apparatus and computer-readable storage medium as claimed. Other embodiments are described and claimed.

To the accomplishment of the foregoing and related ends, certain illustrative aspects are described herein in connection with the following description and the annexed drawings. These aspects are indicative of the various ways in which the principles disclosed herein can be practiced and all aspects and equivalents thereof are intended to be within the scope of the claimed subject matter. Other advantages and novel features will become apparent from the following detailed description when considered in conjunction with the drawings.

Various embodiments may be generally directed to methods and apparatus for generating MS spectral data and aligning product ions to precursor molecules. Techniques and embodiments will now be described with reference to illustrative embodiments for analyzing samples, for instance, in a system analyzing samples using an MS process, such as LC/MS/MS. It will be appreciated that the techniques described herein may be used in connection with other systems, methods, and/or embodiments and may have a broader application than provided for purposes of illustration in this detailed description.

In this description, numerous specific details, such as component and system configurations, may be set forth in order to provide a more thorough understanding of various embodiments. It will be appreciated, however, by one skilled in the art, that the embodiments may be practiced without such specific details. Additionally, some well-known structures and other features have not been shown in detail, to avoid unnecessarily obscuring the present invention.

In the following description, references to "one embodiment," "an embodiment," "example embodiment," "various embodiments," etc., indicate that the embodiment(s) of the invention so described may include particular features, structures, or characteristics, but more than one embodiment may and not every embodiment necessarily does include the particular features, structures, or characteristics. Further, some embodiments may have some, all, or none of the features described for other embodiments.

As used in this description and the claims and unless otherwise specified, the use of the ordinal adjectives "first," "second," "third," etc. to describe an element merely indicate that a particular instance of an element or different instances of like elements are being referred to, and is not intended to imply that the elements so described must be in a particular sequence, either temporally, spatially, in ranking, or in any other manner.

<FIG> illustrates an example of an operating environment <NUM> that may be representative of various embodiments. The operating environment <NUM> depicted in <FIG> may include a chromatography device <NUM>, including, without limitation, an LC device. A sample may be injected into chromatography device <NUM> through an injector. A pump may direct the sample through a column of chromatography device <NUM> to separate the sample into component parts according to retention time through the column. The output of chromatography device <NUM> may be directed to a mass spectrometer <NUM> for analysis. The system of chromatography device <NUM> and mass spectrometer <NUM> may be configured, for example, as an LC-MS or LC-MS/MS device. Although LC and MS/MS are used as examples, embodiments are not so limited, as any type of MS system capable of operating according to some embodiments is contemplated herein.

LC-MS and LC-MS/MS acquisition techniques typically involve acquiring one or more parent (precursor) ion data sets (spectra) and one or more corresponding product ion data sets (spectra) for a sample eluting from chromatography device <NUM>. In these techniques, eluent from a liquid chromatography column of chromatography device <NUM> is passed into an ion source, where it is ionized. A fragmentation, collision or reaction device (or cell) is typically arranged downstream of the ion source to selectively fragment or react parent ions to produce product ions. In MS <NUM>, the output of collision cell is directed to a mass analyzer. The mass analyzer can be any mass analyzer, including, for example, quadrupole, time-of-flight (TOF), ion trap, magnetic sector mass analyzers as well as combinations thereof. A detector of the mass spectrometer detects ions emanating from the mass analyzer. The detector can be integral with mass analyzer. For example, in the case of a TOF mass analyzer, the detector can be a microchannel plate detector that counts intensity of ions, i.e., counts numbers of ions impinging it. For example, MS <NUM> may include a TOF MS arranged downstream of the fragmentation, collision or reaction device, that may be used used to acquire the parent and product ion data sets.

After acquiring the data sets, product and parent ions may be detected in the data sets, and product ions are assigned or "binned" to parent ions. Binning may be accomplished, for example, by chromatographic retention time alignment. A product ion may be assigned (binned) to one or more particular parent ions if the product ion's retention time is the same as that of the one or more parent ions, within measurement error. Measurement error is typically taken to be a fraction of the peak width of the one or more parent ions. For example, a product ion may be assigned to one or more particular parent ions if the product ion's retention time is within +/- the half-width at half maximum ("HWHM") of the chromatographic peak width of the one or more parent ions.

Another known acquisition technique additionally makes use of ion mobility. In such arrangements, an ion mobility separation ("IMS") device is arranged downstream of the ion source and upstream of the fragmentation, collision or reaction device. Binning is accomplished by retention time alignment as well as ion mobility alignment. Thus, different components of the sample can more readily be distinguished, and product ions can be more accurately assigned to the appropriate parent ions. However, there are still a significant number of cases where components have retention times and drift times that cannot be distinguished.

MS <NUM> may be operated according to various acquisition modes of operation. Non-limiting examples of modes of operation may include Data Dependent Acquisition (DDA), Data Independent Acquisition (DIA), and/or variations thereof. In DDA, fragment or product ions are formed from parent or precursor ions that are mass resolved (for example, using a quadrupole mass filter isolation window). In DIA, fragment or product ions are formed from parent or precursor ions that are either time resolved (MSE) or time and ion mobility drift time resolved (HD-MSE).

Precursor and product ion alignment in DDA may include use of the product ions from the isolated (for instance, 1st mass filter precursor) m/z precursor ion being assigned to the parent precursor with no additional filtering other than the width of the m/z isolation window. The assumption is the narrower the mass isolation window the more selective the MS/MS product ion spectra will be. Such a relationship may occur when dealing with simple mixtures; however, a small mass isolation window, even as small as <NUM> Th, may not contain only one precursor molecule when sampling complex mixtures. Biomolecules, for example, primarily constructed of (carbon (C), nitrogen (N), hydrogen (H), oxygen (O), sulfur (S), and phosphorus (P), may create compounds of very different structures albeit nearly identical m/z. Moreover, certain elemental compositions can produce precursor ion m/z values that are nearly identical but of different charge states. Although biomolecules, such as proteins/peptides, are used as examples herein, embodiments are not so limited. Indeed, any type or class of compound capable of being processed and analyzed according to some embodiments is contemplated herein, such as pesticides, hydrocarbons, and/or the like.

Conventional systems generally use one of two commercially available types of DIA, SWATH (Sequential Window Acquisition of all THeoretical Mass Spectra) and MSE. In general, SWATH operates substantially similar to DDA acquisition, however the m/z isolation window width is larger (> <NUM> Thomson (Th)). In MSE, all precursor ions are fragmented all the time within, either, a constant or variable m/z isolation window. Although these types of experiments have higher product ion sampling rates, they are limited in that every MS/MS spectra contains product ions from more than one precursor. SWATH uses spectral libraries for matching product ions between known identifications and the composite product ion spectra. In addition, product ion spectra can also be searched using very wide precursor mass tolerances (for example, the width of the isolation window at all available charge states). For MSE, precursor and product ions are aligned by center mass retention and, if ion mobility separation is applied, centered drift times. Although DIA samples more of the ions complement of a complex sample, the ability to correctly identify and quantify across a wide dynamic range of the added complement of compounds sampled is limited by complexity.

MS <NUM> may be communicatively coupled to an apparatus <NUM>, such as a computing or logic device, having a processor circuitry <NUM>, a memory unit <NUM>, and a transceiver <NUM>. Processing circuitry <NUM> may be communicatively coupled to memory unit <NUM> and/or transceiver <NUM>. In some embodiments, apparatus <NUM> and/or components thereof may be components of MS <NUM>. In various embodiments, apparatus <NUM> and/or components thereof may be operative to control MS <NUM> and/or certain functions thereof.

Processing circuitry <NUM> may include and/or may access various logic for performing processes according to some embodiments. For instance, processing circuitry <NUM> may include and/or may access MS data analysis logic <NUM>, for example, NM-MD logic <NUM>, fragmentation efficiency logic <NUM>, and/or precursor-product alignment logic <NUM>. MS data analysis logic <NUM>, for example, NM-MD logic <NUM>, fragmentation efficiency logic <NUM>, and/or precursor-product alignment logic <NUM> may be implemented in hardware, software, or a combination thereof. As used in this application, the terms "logic, "component," "layer," "system," "circuitry," "decoder," "encoder," and/or "module" are intended to refer to a computer-related entity, either hardware, a combination of hardware and software, software, or software in execution, examples of which are provided by the exemplary computing architecture <NUM> of <FIG>. For example, a logic, circuitry, or a layer may be and/or may include, but are not limited to, a process running on a processor, a processor, a hard disk drive, multiple storage drives (of optical and/or magnetic storage medium), an object, an executable, a thread of execution, a program, a computer, hardware circuitry, integrated circuits, application specific integrated circuits (ASIC), programmable logic devices (PLD), digital signal processors (DSP), field programmable gate array (FPGA), a system-on-a-chip (SoC), memory units, logic gates, registers, semiconductor device, chips, microchips, chip sets, software components, programs, applications, firmware, software modules, computer code, combinations of any of the foregoing, and/or the like. In some embodiments, at least a portion of MS data analysis logic <NUM> (and logic thereof) may be arranged within processor circuitry <NUM>. In other embodiments, for example, MS data analysis logic <NUM> may be located within an accelerator, a processor core, an interface, an individual processor die, and/or the like.

Transceiver <NUM> may be operative to provide communication processes for apparatus <NUM>. For example, apparatus <NUM> may be in communication with nodes 182a-n accessible via network <NUM> via transceiver <NUM>. Memory unit <NUM> may include various types of computer-readable storage media and/or systems in the form of one or more higher speed memory units, such as read-only memory (ROM), random-access memory (RAM), dynamic RAM (DRAM), Double-Data-Rate DRAM (DDRAM), synchronous DRAM (SDRAM), static RAM (SRAM), programmable ROM (PROM), erasable programmable ROM (EPROM), electrically erasable programmable ROM (EEPROM), flash memory, polymer memory such as ferroelectric polymer memory, ovonic memory, phase change or ferroelectric memory, silicon-oxide-nitride-oxide-silicon (SONOS) memory, magnetic or optical cards, an array of devices such as Redundant Array of Independent Disks (RAID) drives, solid state memory devices (e.g., USB memory, solid state drives (SSD) and any other type of storage media suitable for storing information. In addition, memory unit <NUM> may include various types of computer-readable storage media in the form of one or more lower speed memory units, including an internal (or external) hard disk drive (HDD), a magnetic floppy disk drive (FDD), and an optical disk drive to read from or write to a removable optical disk (e.g., a CD-ROM or DVD), a solid state drive (SSD), and/or the like.

In some embodiments, various data may be stored in memory unit <NUM>, such as ion data sets <NUM>, mass information <NUM>, and/or nominal mass (NM)-mass defect (MD) relationship information <NUM>. In various embodiments, sample analysis results of MS <NUM> may be provided to apparatus <NUM> and stored, for example, as ion data sets <NUM>. Non-limiting examples of ion data sets <NUM> may include m/z information, retention time information, mobility drift information, intensity information, spectra, and/or the like. In some embodiments, ion data sets <NUM>, mass information <NUM>, and/or NM-MD relationship information <NUM> may include information accessed and/or stored in one or more remote locations, such as in a database, cloud computing environment, and/or the like.

MS data analysis logic <NUM> may be operative to provide various analysis processes for data from MS <NUM>. For example, MS data analysis logic <NUM>, alone or via one or more of NM-MD logic <NUM>, fragmentation efficiency logic <NUM>, and/or precursor-product alignment logic <NUM>, may be operative to provide a MS data analysis process to analyze MS data (for instance, ion data sets), generate MS data spectra, align product ions and precursors, and/or the like.

NM-MD logic <NUM> may be operative to provide various processes relating to mass information associated with one or more molecules being analyzed by MS <NUM>. In some embodiments, one or more molecules may be selected or otherwise designated as a target precursor. In general, a target precursor is the precursor of interest in a MS analysis, such as a particular protein or peptide that is of interest in a complex sample.

NM-MD logic <NUM> may access, generate, and/or provide mass information <NUM> associated with the target precursor and/or constituent elements or potential fragments thereof. In various embodiments, mass information <NUM> may include, but is not limited to, nominal mass and mass defect information of constituent elements (or building blocks) and/or potential fragments of the target precursor. For example, if the target precursor is a peptide, the constituent elements would include amino acids as well as any other potential elements, moieties, and/or the like that may be included in the target precursor. In general, the nominal mass of an element includes the integer mass of the most abundant naturally occurring stable isotope of an element or the sum of the integer masses of the constituent elements of a molecular ion or molecule. The mass defect is the difference between the actual mass of a specific nucleus and the nominal mass. The mass defect may represent the equivalent of the different binding energies required for nuclear stabilization of the different elements. By convention, the mass excess of 12C is defined as zero. The mass defect of the elements may be positive (for example, <NUM>H: <NUM> and <NUM>N: <NUM>) or negative (e.g. <NUM>O: <NUM>, <NUM>S: <NUM>, and <NUM>P: <NUM>). The mass defect of a compound may include the fractional mass, which is the mass to the right of the decimal point. For example, the amino acid alanine has a nominal mass of <NUM> and a mass defect or fractional mass of <NUM>.

<FIG> depicts Table <NUM><NUM> providing mass information <NUM> for the constituent elements of a peptide precursor molecule. More specifically, <FIG> provides NM-MD mass information for the amino acids <NUM> (with cysteine modified by carboxyaminomethylation), which includes, without limitation, the elemental makeup <NUM> of each amino acid, the molecular mass (Mr) at the charge states of z=<NUM><NUM>, z=<NUM><NUM>, z=<NUM><NUM>, and z=<NUM><NUM>, and the mass defect <NUM>, <NUM>, <NUM>, and <NUM> at each charge state. As depicted in Table <NUM><NUM>, minimum <NUM>, maximum <NUM>, median <NUM>, average <NUM>, standard deviation <NUM>, and CV <NUM> values exist for both Mr <NUM>, <NUM>, <NUM>, and <NUM> and mass defect <NUM>, <NUM>, <NUM>, and <NUM>. Table <NUM><NUM> represents non-weighted values for the constituent elements of a peptide precursor molecule.

MS data analysis logic <NUM>, for example, via NM-MD logic <NUM>, may use the integer and mass defect of the constituent building blocks of the target compounds relative to that of their parent molecules' intact molecular mass. In some embodiments, given the m/z and z of the precursor ion(s) are known, the maximum charge reduced (<NUM> charge at a time) m/z value(s) of the highest m/z product ion are known. In some instances, a single eluting component when ionized can exist at multiple charge states. For those components of multiple charge states each charge state is treated separately in both DDA and high-definition mass spectrometry (HDMS or HDMSE)DIA acquisitions. Though the eluting component is singular in the liquid phase, when ionized it may exist at multiply charge states. In DDA acquisitions the charge states are separated by the mass isolation window. In HDMSE DIA acquisitions, the ion mobility separation (IMS), which is internal to the ion path, separates the charge states by their collisional cross-sectional area (CCSA<NUM>). For MSE and SWATH type DIA acquisitions, both charge states will fragment simultaneously producing a product ion spectra containing both <NUM>+ and <NUM>+ fragment ions. Current precursor selection schemes based on DDA and/or DIA involve processes where fragmentation mass candidates are selected by intensity and are included in an exclusion directory to avoid constant refragmentation of highly abundant species. Conventional DDA and/or DIA methods do not fully utilize valuable information available based on the fractional mass of high-accuracy precursor mass measurements delivered by current instrumentation. Accordingly, the MS data analysis process according to some embodiments may, among other things, separate the product ions by charge state using one set of product ions to validate the other.

When a peptide bond is dissociated, two fragment ions are generated, Y-ions and B-ions (the compliment to the Y-ion). More particularly, if a peptide fragments at the peptide bond, then if a charge is retained on the N terminal fragment, that fragment ion is termed a B-ion. If the charge is retained on the C terminal fragment, the fragment ion is termed a Y-ion. A more comprehensive list of possible fragments and their nomenclature is provided in <NPL>, and <NPL>,. The fragment containing only the amino terminal amino acid is termed b<NUM>, the fragment containing the first two amino terminal amino acids is termed b<NUM>, and so forth.

The highest m/z product ion in an MS/MS spectrum from a precursor of charge z is referred to as Ymax, in which Ymax = [Mr + (z-<NUM>)*H]/z, where Mr is the molecular mass of the precursor, z is the charge number of the precursor, and H is the mass of hydrogen. The maximum fragment ion mass for a B-ion is Bmax = [(Mr + (z-<NUM>)*H)-H<NUM><NUM>]/z, where Mr is the molecular mass of the precursor, z is the charge number of the precursor, H is the mass of hydrogen, and H<NUM>O is the molecular mass of water.

In some embodiments, NM-MD logic <NUM> may be operative to generate, access, and/or provide NM-MD relationship information <NUM>. In various embodiments, NM-MD relationship information <NUM> may include associations between the nominal m/z and the fractional m/z of elements associated with a target precursor, such as product ion fragments. In exemplary embodiments, NM-MD relationship information <NUM> may include a determination of nominal m/z and the fractional m/z for product ion fragments, such as represented in <FIG>. For example, <FIG> represent a density plot or graph of nominal m/z and the fractional m/z for product ion fragments of certain z values. In some embodiments, the product ion fragments depicted in <FIG> may be a result of empirical mass analysis via a MS. In other embodiments, the product ion fragments depicted in <FIG> may be the result of in silico digests or graphical representations of other computer-generated data (for instance, UniProt and/or the like).

<FIG> depicts NM-MD relationship information <NUM> in the form of a graph <NUM> or plot representing nominal m/z <NUM> versus fractional m/z <NUM> for ions, such as product ions, of z=<NUM><NUM>. As shown in <FIG>, plot <NUM> may include a single charge vector <NUM> of product ions. <FIG> depicts NM-MD relationship information <NUM> in the form of a graph <NUM> representing nominal m/z <NUM> versus fractional m/z <NUM> for ions of z=<NUM><NUM>. As shown in <FIG>, plot <NUM> may include a plurality of charge vectors <NUM>, <NUM>, and <NUM> of product ions. <FIG> depicts NM-MD relationship information <NUM> in the form of a graph <NUM> representing nominal m/z <NUM> versus fractional m/z <NUM> for ions of z=<NUM><NUM>. As shown in <FIG>, plot <NUM> may include a plurality of charge vectors <NUM>, <NUM>, <NUM>, and <NUM> of product ions. <FIG> depicts NM-MD relationship information <NUM> in the form of a combined density graph <NUM> for ions of z=<NUM>-<NUM>.

In various embodiments, NM-MD logic <NUM> may be operative to select candidate product ion fragments from the population of potential product ion products associated with a sample, target precursor(s), or class of compound (for instance, peptides). In exemplary embodiments, the MS data analysis process may provide precursor and product ion alignment using generated relationships associated with the nominal mass and mass defect of a precursor ions (for example, [Mr + (z-<NUM>)*H]/z) and that of its principal product ions. For instance, as the target precursor molecule fragments, each fragment may decrease in both nominal mass and mass defect by the nominal mass and mass defect of the constituent component (building block) of the parent. For example, an MS data analysis process according to some embodiments may calculate a weight averaged nominal mass and mass defect as well as standard deviations of each from the set of building blocks of the molecules understudy.

<FIG> depicts Table <NUM><NUM> providing mass information <NUM> in the form of constituent element distribution information. For peptides, mass information <NUM> may be in the form of an average frequency distribution <NUM> of amino acids <NUM> in the proteomes of yeast, human and mouse. In some embodiments, average frequency distribution <NUM> may be used to determine the weight averaged median nominal and fractional mass values for a sample containing peptides.

An MS data analysis process configured according to some embodiments may use the nominal and mass defect of both Ymax and Bmax as the maximum values, and the weight average nominal and mass defect of Y<NUM> and B<NUM>, respectively, as the minimums to create two separate linear regression line fits for NM-MD relationship information (see, for example, <FIG>). In some embodiments, the Y<NUM> and B<NUM> values may be held constant for all regression line fits. In acquisitions employing IMS, product ions may be pre-filtered, for example, by comparison of their nominal drift time to that of each parent precursor. Elimination by drift time is not absolute as the method employs an isolation window the width which is dictated by an adaptable function reflecting the precursor ion m/z and z values. Accordingly, the width of the isolation window increases as the m/z and z increase. In various embodiments, upper and lower boundary lines may be provided for each of the regression line fits for product ion extraction by adding either a user- or algorithmically- derived multiple of the respective standard deviations for both the nominal mass and mass defect of the constituent building blocks resident in the mass information <NUM> (for instance, <FIG>, Table <NUM><NUM>). In exemplary embodiments, all or substantially all product ions outside the upper and lower boundary lines are removed for consideration in the next stage of processing.

<FIG> depicts regression line fits according to some embodiments. As shown in <FIG>, product ions may be plotted on a graph <NUM> of nominal m/z <NUM> versus fractional m/z <NUM>, including Bmax ([(Mr + (z-<NUM>)*H)-H<NUM><NUM>]/z) <NUM> and Ymax (Mr + (z-<NUM>)*H]/z) <NUM>. A first regression line fit <NUM> and a second regression line fit <NUM> are provided according to some embodiments. First regression line fit <NUM> may be associated with upper and lower boundaries <NUM> and <NUM>, which are a threshold distance from first regression line fit <NUM>. Second regression line fit <NUM> may be associated with upper and lower boundaries <NUM> and <NUM>, which are a threshold distance from second regression line fit <NUM>. Values of regression line <NUM> are also depicted in <FIG>. According to some embodiments, the MS data analysis process may locate Ymax and Y<NUM> on the graph <NUM> and generate a line connecting Ymax and Y<NUM> to generate one regression line fit <NUM>. In some embodiments, the MS data analysis process may locate Bmax and B<NUM> on the graph <NUM> and generate a line connecting Bmax and B<NUM> to generate another regression line fit <NUM>. In some embodiments, the threshold distance may include a standard deviation used to determine the upper and lower boundaries <NUM>, <NUM>, <NUM>, <NUM>, which may be based on the integer mass and mass defect of a constituent element, such as tryptophan. In some embodiments, the threshold distance may be a manual- or algorithmic-based value. In some embodiments, regression line fit <NUM> may represent a maximum regression line fit and regression line fit <NUM> may represent a minimum regression line fit. Referring to <FIG>, therein are depicted regression line fits for the ions depicted therein, including regression line fits <NUM> and <NUM> for <FIG>, regression line fits <NUM> and <NUM> for <FIG>, and regression line fits <NUM> and <NUM> for <FIG>.

As shown in <FIG>, Y-ions are predominately below the regression line fit(s) whereas B-ions are predominately (><NUM>%) above. Accordingly, MS data analysis processes according to some embodiments may provide increased selectivity in the identification process by allowing Y- ions to be segregated from the B-companions. Product ions between the regression line fit upper and lower boundaries <NUM> and <NUM> and/or regression line fit upper and lower boundaries <NUM> and <NUM> may be selected and designated as candidate ion fragments.

The process of selecting and designating candidate ion fragments may be replicated for charge values of interest. For example, the process of determining regression line fits (and their respective upper and lower boundaries) for charge vectors may be repeated for precursor ions of z ><NUM>. For certain fragmentation processes, such as collision-induced dissociation (CID), precursor ions of z > <NUM> can produce fragment ions from <NUM>+ to a maximum of z-<NUM>. Extraction of the <NUM>+ complement of fragment ions may be performed as depicted for <FIG>. Eue to the elemental composition of amino acids and the ability of peptide ions to exist at multiple charge states in a MS, some of the higher z fragment ions will be captured in the <NUM>+ processing. For <NUM>+ product ions, those that do not fall within the <NUM>+ upper and lower boundary lines, their mass defects will toggle between an upper and lower charge vector. <FIG> illustrates in <NUM>-dimensional space the nominal mass <NUM> versus mass defect <NUM> of the <NUM>+ product ions where the center charge vector <NUM> is within <NUM>+ upper and lower boundary lines the other isotopes are distributed within the upper charge vector <NUM> and lower charge vector <NUM>. Referring to Table <NUM><NUM>, therein is depicted the toggling in fraction mass between the three charge vector lanes.

In some embodiments, the regression line fits for the upper and lower charge vector lanes are calculated using [Mr + (z-<NUM>)*H]/z as the maximum and the median weight averaged B<NUM> and Y<NUM> [Mr + (z-<NUM>)*H]/z for the minimum for precursor ions of z > <NUM>. The upper charge vector may be calculated using the [Mr + (z-<NUM>)*H]/z of the precursor as the maximum and the median weight averaged B<NUM> and Y<NUM> [Mr + (z-<NUM>)*H]/z for the minimum. The lower charge vector may be calculated similarly; however, the maximum value may be set to the precursor ion's [Mr + (z-<NUM>)*H]/z minus 1x the median nominal and mass defect from the mass information of the constituent building blocks (for instance, Table <NUM><NUM>). In various embodiments, the minimal nominal mass and mass defect may be identical to the upper charge vector. The number of unique charge vector lanes is proportional to (z-<NUM>) for precursor ions of z > <NUM>. Higher z precursor ions may be processed similarly. For example, <FIG> depicts the <NUM>+ precursor ion charge vector lanes.

Once all regression line fits have been constructed, the MS analysis process according to some embodiments may create a stripe (in <NUM>- or <NUM>-dimensional space), depending on whether IMS was employed, selecting only the candidate product ions that were captured between the upper and lower boundary lines of their respective regression line fits. The stripe, such as the candidate ions of the stripe <NUM> of <FIG>, is defined by upper and lower boundary lines. These boundary lines reflect the theoretical values plus/minus standard deviations for each of the two values, nominal mass and mass defect. These widths (or number of standard deviations) in the <NUM> dimensions of nominal mass and mass defect project a stripe of candidate ion fragments above and below the regression line fit. Ions that are not within the stripe of candidate ions may be removed from further consideration and/or analysis of the target precursor or potential fragment ions.

Once the candidate ion fragments on the stripe are extracted, the fragmentation efficiency logic <NUM> may be operative to perform a fragmentation filtering process of the MS data analysis process. In some embodiments, the fragmentation filtering process may be operative to further filter the candidate ion fragments based on the fragmentation efficiency to determine target ion fragments. In some embodiments, fragmentation efficiency may be determined as <NUM> minus the ratio of the residual ion intensity/precursor ion intensity. In various embodiments, sensitivity as a function of qualitative analysis directly relates to fragmentation efficiency. For example, too much residual precursor ion intensity in the MS/MS spectrum indicates poor fragmentation and, therefore, lower intensity product ions. Similarly, the absence of any residual precursor ion intensity in the MS/MS spectrum, provided the precursor ion cluster intensity is high enough to produce a statistically significant number of product, may indicate over-fragmentation. In exemplary embodiments, the identification of constituent components across the widest dynamic range is a function of product ion intensity.

The fragmentation filtering process according to some embodiments determines the residual precursor ion cluster in the product ion spectrum and calculates the difference in their respective intensities to calculate the fragmentation efficiency. <FIG> depicts a graph <NUM> of fragmentation efficiency <NUM> versus percent maximum and minimum product ion intensity <NUM>. In some embodiments, the calculated fragmentation efficiency may be compared to <FIG>, for example, and the maximum product ion intensities tolerance <NUM> and minimum product ion intensities tolerances <NUM> relative to the precursor ions are set. In some embodiments, <FIG> may be generated by statistical analysis of a set of previously acquired data sets, such as DDA data sets and/or simulated data sets. DDA data sets may be used because the instrument uses a lookup table of optimized collision energy values for each switched on precursor ion's nominal mass and z. The collision energy may be varied from the optimal settings to characterize the fragmentation efficiency. From these datasets, the regression line fits for the upper (most intense product) and lower (least intense product) boundary lines may be calculated based on the precursor ion's fragmentation efficiency.

<FIG> and <FIG> depict nominal mass versus mass defect for potential product ions of interest for a target precursor. <FIG> depicts potential product ions that have not been filtered according to some embodiments; rather, they are unfiltered or filtered according to conventional methods. <FIG> depicts (a stripe of) target fragment ions that were located on a charge vector strip between the upper and lower bounds of nominal mass and mass defect and within the intensity windows of the upper and lower intensity bounds. <FIG> and <FIG> depict spectra generated from the ion data sets based on the target fragment ions of <FIG> and <FIG>, respectively, labeled by z value and fragment ion type (Y-ion or B-ion). The spectrum of <FIG> includes significantly more peaks than the spectrum of <FIG>. Accordingly, precursor-product analysis and/or alignment may be more efficient and accurate using the filtered spectrum of <FIG>. For example, the peptide match or "ions score" for <FIG> is <NUM>, while the ions score for <FIG> is <NUM>, which is a significant improvement over the ions score for the conventional methods of <FIG>. In some embodiments, precursor-product alignment logic <NUM> may be operative to align product ion fragments with a target precursor, for example, using the ion data sets <NUM> and/or associated spectra, such as the spectra depicted in <FIG> and <FIG>.

In some embodiments, the MS data analysis process may perform a de-novo sequencing process and/or a sequence tag formation process based on, among other things, fractional mass, nominal mass, z, and fragment ion type. A de-novo sequencing process may include determining the delta nominal mass between the precursor ions' [Mr + (z-<NUM>)*H]/z and its near Y-ion neighbors (below the regression line) within the maximum integer mass of all the constituent elements, for example, as provided in Table <NUM><NUM>. Given a nominal mass match, the delta of the mass defects is compared to validate the match, if the match is made within a match tolerance or threshold, the attributes of the [Mr + (z-<NUM>)*H]/z are replaced with those of the matched product ion and the process may be repeated. In instances where there is no initial match (for example, no Ymax - <NUM> amino acid), the algorithm looks for the delta nominal mass and mass defect of two amino acids in an attempt to connect to Ymax - <NUM> amino acids. If no connection is made for Ymax - <NUM> amino acid or Ymax - <NUM> amino acids, the process takes the most intense product ion, knowing its fragment ion type (above b or below y), and subtracts its nominal mass from the precursor ion's [Mr + (z-<NUM>)*H]/z nominal mass and looks for the companion (upper/lower stripe) for its complementary pair. The process repeats this process until all the extracted product ions are queried for their complementary pair. If a complementary pair is found the [Mr + (z-<NUM>)*H]/z value from the first pass is replaced with that of the complementary pair and the same process is repeated lane-by-lane.

Included herein are one or more logic flows representative of exemplary methodologies for performing novel aspects of the disclosed architecture. While, for purposes of simplicity of explanation, the one or more methodologies shown herein are shown and described as a series of acts, those skilled in the art will understand and appreciate that the methodologies are not limited by the order of acts. Some acts may, in accordance therewith, occur in a different order and/or concurrently with other acts from that shown and described herein. For example, those skilled in the art will understand and appreciate that a methodology could alternatively be represented as a series of interrelated states or events, such as in a state diagram. Moreover, not all acts illustrated in a methodology may be required for a novel implementation.

A logic flow may be implemented in software, firmware, hardware, or any combination thereof. In software and firmware embodiments, a logic flow may be implemented by computer executable instructions stored on a non-transitory computer readable medium or machine readable medium, such as an optical, magnetic or semiconductor storage.

<FIG> illustrates an embodiment of a logic flow <NUM>. Logic flow <NUM> may be representative of some or all of the operations executed by one or more embodiments described herein, such as apparatus <NUM> and/or MS <NUM>. In some embodiments, logic flow <NUM> may be representative of some or all of the operations of a MS data analysis process.

Logic flow <NUM> may determine a target precursor at block <NUM>. For example, an operator of MS <NUM> may enter at least one target precursor of interest (for instance, a protein or peptide) during analysis of a sample. At block <NUM>, logic flow <NUM> may access precursor composition information for elements of the target precursor. For example, for a peptide target precursor, MS data analysis logic <NUM> may access mass information <NUM> that includes nominal mass and mass defect for multiple charge values for amino acids, such as provided in Table <NUM><NUM>. Logic flow <NUM> may determine NM-MD relationship information for ion fragments associated with the target precursor at block <NUM>. For example, NM-MD <NUM> may determine, generate, access, and/or provide plots of nominal m/z versus mass defect m/z for certain charge values, such as depicted in <FIG>.

At block <NUM>, logic flow <NUM> may determine an ion fragment upper boundary and an ion fragment lower boundary for the ion fragments. For example, the MS data analysis process configured according to some embodiments may use the nominal and mass defect of both Ymax and Bmax as the maximum values, and the weight average nominal and mass defect of Y<NUM> and B<NUM>, respectively, as the minimums to create two separate linear regression line fits for NM-MD relationship information. In addition, upper and lower boundary lines may be provided for each of the regression line fits for product ion extraction by adding either a user- or algorithmically- derived multiple of the respective standard deviations for both the nominal mass and mass defect of the constituent building blocks resident in the mass information <NUM>. At block <NUM>, logic flow <NUM> may extract candidate ion fragments based on applying the ion fragment upper boundary and the ion fragment lower boundary to the NM-MD relationship information. For example, referring to <FIG>, product ions between the regression line fit upper and lower boundaries <NUM> and <NUM> and regression line fit upper and lower boundaries <NUM> and <NUM> may be selected and designated as candidate ion fragments.

Logic flow <NUM> may determine fragment efficiency information associated with ion fragments of a target precursor at block <NUM>. For example, the fragmentation filtering process may be operative to further filter the candidate ion fragments based on the fragmentation efficiency to determine target ion fragments. In some embodiments, fragmentation efficiency may be determined as <NUM> minus the ratio of the residual ion intensity/precursor ion intensity. At block <NUM>, logic flow <NUM> may determine minimum and maximum product ion intensity tolerances. For example, the calculated fragmentation efficiency may be compared to <FIG> for example, and the maximum product ion intensities tolerance <NUM> and minimum product ion intensities tolerances <NUM> relative to the precursor ions are set. Logic flow <NUM> may determine target product ions by filtering candidate product ions based on the minimum and maximum product ion intensities at block <NUM>. For example, <FIG> depicts target fragment ions that were located on a charge vector strip between the upper and lower bounds of nominal mass and mass defect and within the intensity windows of the upper and lower intensity bounds. At block <NUM>, logic flow <NUM> may generate spectral data for the target ion fragments of the product ion data set. For example, <FIG> depicts spectra generated from the ion data sets based on the target fragment ions of <FIG> labeled by z value and fragment ion type (Y-ion or B-ion).

<FIG> illustrates an example of a storage medium <NUM>. Storage medium 1400may comprise an article of manufacture. In some examples, storage medium <NUM> may include any non-transitory computer readable medium or machine readable medium, such as an optical, magnetic or semiconductor storage. Storage medium <NUM> may store various types of computer executable instructions, such as instructions to implement logic flow <NUM> and/or <NUM>. Examples of a computer readable or machine readable storage medium may include any tangible media capable of storing electronic data, including volatile memory or non- volatile memory, removable or non-removable memory, erasable or non-erasable memory, writeable or re-writeable memory, and so forth. Examples of computer executable instructions may include any suitable type of code, such as source code, compiled code, interpreted code, executable code, static code, dynamic code, object-oriented code, visual code, and the like. The examples are not limited in this context.

<FIG> illustrates an embodiment of an exemplary computing architecture <NUM> suitable for implementing various embodiments as previously described. In various embodiments, the computing architecture <NUM> may comprise or be implemented as part of an electronic device. In some embodiments, the computing architecture <NUM> may be representative, for example, of apparatus <NUM> and/or MS <NUM>.

As used in this application, the terms "system" and "component" and "module" are intended to refer to a computer-related entity, either hardware, a combination of hardware and software, software, or software in execution, examples of which are provided by the exemplary computing architecture <NUM>. For example, a component can be, but is not limited to being, a process running on a processor, a processor, a hard disk drive, multiple storage drives (of optical and/or magnetic storage medium), an object, an executable, a thread of execution, a program, and/or a computer. By way of illustration, both an application running on a server and the server can be a component. One or more components can reside within a process and/or thread of execution, and a component can be localized on one computer and/or distributed between two or more computers. Further, components may be communicatively coupled to each other by various types of communications media to coordinate operations. The coordination may involve the uni-directional or bi-directional exchange of information. For instance, the components may communicate information in the form of signals communicated over the communications media. The information can be implemented as signals allocated to various signal lines. In such allocations, each message is a signal. Further embodiments, however, may alternatively employ data messages. Such data messages may be sent across various connections. Exemplary connections include parallel interfaces, serial interfaces, and bus interfaces.

As shown in <FIG>, the computing architecture <NUM> comprises a processing unit <NUM>, a system memory <NUM> and a system bus <NUM>. The processing unit <NUM> can be any of various commercially available processors, including without limitation an AMD® Athlon®, Duron® and Opteron® processors; ARM® application, embedded and secure processors; IBM® and Motorola® DragonBall® and PowerPC® processors; IBM and Sony® Cell processors; Intel® Celeron®, Core (<NUM>) Duo®, Itanium®, Pentium®, Xeon®, and XScale® processors; and similar processors. Dual microprocessors, multi-core processors, and other multi-processor architectures may also be employed as the processing unit <NUM>.

The computer <NUM> may include various types of computer-readable storage media in the form of one or more lower speed memory units, including an internal (or external) hard disk drive (HDD) <NUM>, a magnetic floppy disk drive (FDD) <NUM> to read from or write to a removable magnetic disk <NUM>, and an optical disk drive <NUM> to read from or write to a removable optical disk <NUM> (e.g., a CD-ROM or DVD). The HDD <NUM>, FDD <NUM> and optical disk drive <NUM> can be connected to the system bus <NUM> by a HDD interface <NUM>, an FDD interface <NUM> and an optical drive interface <NUM>, respectively. The HDD interface <NUM> for external drive implementations can include at least one or both of Universal Serial Bus (USB) and IEEE <NUM> interface technologies, among others.

The drives and associated computer-readable media provide volatile and/or nonvolatile storage of data, data structures, computer-executable instructions, and so forth. For example, a number of program modules can be stored in the drives and memory units <NUM>, <NUM>, including an operating system <NUM>, one or more application programs <NUM>, other program modules <NUM>, and program data <NUM>. In one embodiment, the one or more application programs <NUM>, other program modules <NUM>, and program data <NUM> can include, for example, the various applications and/or components of apparatus <NUM> or MS <NUM>.

A user can enter commands and information into the computer <NUM> through one or more wire/wireless input devices, for example, a keyboard <NUM> and a pointing device, such as a mouse <NUM>. Other input devices may include microphones, infra-red (IR) remote controls, radio-frequency (RF) remote controls, game pads, stylus pens, card readers, dongles, finger print readers, gloves, graphics tablets, joysticks, keyboards, retina readers, touch screens (e.g., capacitive, resistive, etc.), trackballs, trackpads, sensors, styluses, and the like. These and other input devices are often connected to the processing unit <NUM> through an input device interface <NUM> that is coupled to the system bus <NUM>, but can be connected by other interfaces such as a parallel port, IEEE <NUM> serial port, a game port, a USB port, an IR interface, and so forth.

When used in a WAN networking environment, the computer <NUM> can include a modem <NUM>, or is connected to a communications server on the WAN <NUM>, or has other means for establishing communications over the WAN <NUM>, such as by way of the Internet. The modem <NUM>, which can be internal or external and a wire and/or wireless device, connects to the system bus <NUM> via the input device interface <NUM>. In a networked environment, program modules depicted relative to the computer <NUM>, or portions thereof, can be stored in the remote memory/storage device <NUM>. It will be appreciated that the network connections shown are exemplary and other means of establishing a communications link between the computers can be used.

The computer <NUM> is operable to communicate with wire and wireless devices or entities using the IEEE <NUM> family of standards, such as wireless devices operatively disposed in wireless communication (e.g., IEEE <NUM> over-the-air modulation techniques). This includes at least Wi-Fi (or Wireless Fidelity), WiMax, and Bluetooth™ wireless technologies, among others. Thus, the communication can be a predefined structure as with a conventional network or simply an ad hoc communication between at least two devices. Wi-Fi networks use radio technologies called IEEE <NUM>. 11x (a, b, g, n, etc.) to provide secure, reliable, fast wireless connectivity. A Wi-Fi network can be used to connect computers to each other, to the Internet, and to wire networks (which use IEEE <NUM>-related media and functions).

Some embodiments may be implemented, for example, using a machine-readable medium or article which may store an instruction or a set of instructions that, if executed by a machine, may cause the machine to perform a method and/or operations in accordance with the embodiments. Such a machine may include, for example, any suitable processing platform, computing platform, computing device, processing device, computing system, processing system, computer, processor, or the like, and may be implemented using any suitable combination of hardware and/or software. The machine-readable medium or article may include, for example, any suitable type of memory unit, memory device, memory article, memory medium, storage device, storage article, storage medium and/or storage unit, for example, memory, removable or non-removable media, erasable or non-erasable media, writeable or re-writeable media, digital or analog media, hard disk, floppy disk, Compact Disk Read Only Memory (CD-ROM), Compact Disk Recordable (CD-R), Compact Disk Rewriteable (CD-RW), optical disk, magnetic media, magneto-optical media, removable memory cards or disks, various types of Digital Versatile Disk (DVD), a tape, a cassette, or the like. The instructions may include any suitable type of code, such as source code, compiled code, interpreted code, executable code, static code, dynamic code, encrypted code, and the like, implemented using any suitable high-level, low-level, object-oriented, visual, compiled and/or interpreted programming language.

Claim 1:
A computer-implemented method of sample analysis, the method comprising:
accessing at least one product ion data set generated via mass analyzing a sample comprising at least one target precursor;
accessing precursor composition information for a plurality of constituent elements of the at least one target precursor, the precursor composition information comprising nominal mass information and mass defect information for at least a portion of the plurality of constituent elements;
determining nominal mass (NM)-mass defect (MD) relationship information comprising NM versus MD information for a plurality of ion fragments associated with the at least one target precursor, the NM-MD relationship information based on the precursor composition information;
determining at least one ion fragment upper boundary of the NM versus MD information for the plurality of ion fragments and at least one ion fragment lower boundary of the NM versus MD information for the plurality of ion fragments,
the at least one ion fragment upper boundary comprising a first regression line fit between a Bmax ion and a B1 ion,
the first regression line fit associated with a first upper boundary and a first lower boundary, each a threshold distance from the first regression line fit; and
the at least one ion fragment lower boundary comprising a second regression line fit between a Ymax ion and a Y1 ion,
the second regression line fit associated with a second upper boundary and a second lower boundary, each a threshold distance from the second firo regression line fit;
extracting a plurality of candidate ion fragments from the plurality of ion fragments by applying the at least one ion fragment upper boundary and the at least one ion fragment lower boundary to the NM versus MD information and selecting the candidate product ions that were captured between the at least one ion fragment upper boundary and the at least one ion fragment lower boundary; and
determining a plurality of target ion fragments from the plurality of candidate ion fragments based on fragmentation efficiency information associated with the plurality of candidate ion fragments, the plurality of target ion fragments belonging to the at least one target precursor.