WO2014200987A2

WO2014200987A2 - Ms1 gas-phase enrichment using notched isolation waveforms

Info

Publication number: WO2014200987A2
Application number: PCT/US2014/041686
Authority: WO
Inventors: Graeme Conrad MCALISTER; Steven P. Gygi; Brian Erickson
Original assignee: President And Fellows Of Harvard College
Priority date: 2013-06-10
Filing date: 2014-06-10
Publication date: 2014-12-18
Also published as: WO2014200987A3

Abstract

A method of performing mass spectrometry survey scans. The method includes using at least one isolation waveform of an ion trap, the isolation waveform with at least a first and second notch. Methods may include determining a first mass-to-charge ratio (m/z) spectrum of a first plurality of ions from a first plurality of discrete m/z ranges; determining a second m/z spectrum of a second plurality of ions from a second plurality of discrete m/z ranges different from the first plurality of discrete m/z ranges; and creating a combined m/z spectrum by combining the first and second m/z spectra.

Description

MSI GAS-PHASE ENRICHMENT USING NOTCHED ISOLATION

WAVEFORMS

RELATED APPLICATIONS

This patent application claims the benefit of U.S. provisional patent application No. 61/833,370, titled "MSI GAS-PHASE ENRICHMENT USING NOTCHED

ISOLATION WAVEFORMS," filed June 10, 2013, which is incorporated herein by reference in its entirety.

FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Contract No. HG3456 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF INVENTION

This application relates generally to mass spectrometry and specifically to a technique for enriching precursor ion populations in mass spectrometry analysis.

Mass spectrometry is a technique that analyzes one or more samples by identifying the mass-to-charge ratio of constituent parts of the sample. Mass spectrometry (MS) has many applications in the study of proteins, known as proteomics. MS may be used to characterize and identify proteins in a sample or to quantify the amount of particular proteins in one or more samples.

It is known to analyze proteins, peptides or other large molecules in a multistep process. In the example of a protein analysis, in a first portion of the process, the protein may be broken into smaller pieces, such as peptides. Certain of these peptides may be selected for further processing. Because the peptides are ions - or may be ionized by known processes such as electrospray ionization (ESI), matrix-assisted laser

desorption/ionization (MALDI), or any other suitable process - selection, manipulation, and analysis may be performed using an ion trap. Depending on their frequency of oscillation, ions of different mass-to-charge ratios (m/z - where m is the mass in atomic mass units and z is the number of elemental charges) may be excited by an excitation signal with sufficient energy to escape the ion trap. What remains in the trap following excitation are ions that did not have a mass-to-charge ratio corresponding to the excitation signal. To isolate ions with a particular mass-to-charge ratio, the ion trap may be excited with a signal that includes a range of frequencies except the frequency that excites the ions of interest. Such an excitation signal, also referred to as an isolation waveform, is said to have a frequency "notch" corresponding to the target ion that is to be isolated.

The selected ions remaining in the trap may be again broken into smaller pieces, generating smaller ions. These ions may then be further processed. Processing may entail selecting and further breaking up the ions. The number of stages at which ions are selected and then broken down again may define the order of the mass spectrometry analysis, such as MS2 (also referred to as MS/MS) or MS3.

In some forms of MS analysis, it may be useful to perform a "survey scan" of the ions entering the MS instrument. The results of the MSI survey scan may be useful on their own for particular analyses or the MS 1 survey scan results may be used as the basis for subsequent "data dependent" analyses.

BRIEF SUMMARY OF INVENTION

The inventors have recognized and appreciated that, though experimental, technical and computational advances have enabled deep sampling of complex proteomes, the wide dynamic range of protein abundance is an obstacle to performing accurate MS analysis. The inventors have also recognized and appreciated that standard gas-phase fractionation approaches, which enable sampling of very low abundance proteins, require careful selection of m/z ranges (based on either prior empirical or in silico analysis) and often result in unequal enrichment across both the MSI survey scan and across a chromatogram resulting from an MS analysis.

Accordingly, some embodiments are directed to a method of performing mass spectrometry (MS). The method may include performing a survey scan using a first isolation waveform of an ion trap, the first isolation waveform comprising at least a first and second notch.

In some embodiments, the survey scan may include at least a first stage and a second stage, the first isolation waveform being used during the first stage and the method may further include performing the second stage of the survey scan using a second isolation waveform of an ion trap, the second isolation waveform comprising at least two notches that are different from the first and second notch. The at least one portion of the first notch of the first isolation waveform may isolate the same m/z range as a portion of one of the at least two notches of the second isolation waveform. In some embodiments, the first stage is performed for a first time; the second stage is performed during a second time; and the first time corresponds to a first liquid chromatography process and the second time corresponds to a second liquid chromatography process. In some

embodiments, the survey scan may be a first survey scan, and the method may further include performing a second survey scan, wherein the second survey scan comprises: performing a first stage of the second survey scan using the first isolation waveform; and performing at least a second stage of the second survey scan using the second isolation waveform.

In some embodiments, the MS method may include obtaining a plurality of ions for analysis; and injecting the plurality of ions into the ion trap, wherein the first stage of the survey scan is performed on the plurality of ions. The method may also include performing liquid chromatography prior to obtaining the plurality of ions for analysis. In some embodiments, the plurality of ions may be obtained using matrix-assisted laser desorption/ionization (MALDI) or atmospheric sampling.

In some embodiments, the MS method may include performing a subsequent MS analysis based on the survey scan. The subsequent analysis may be a tandem MS analysis. Some embodiments are directed to a method of performing mass spectrometry (MS), the method comprising: simultaneously isolating a first plurality of ions within a first range of mass-to-charge ratio (m/z) and a second plurality of ions within a second range of m/z different from the first range of m/z; determining a first m/z spectrum of the first and second plurality of ions; isolating a third plurality of ions within a third range of m/z different from the first and second ranges of m/z; and determining a second m/z spectrum of the third plurality of ions. The method, according to some embodiments, may include generating a combined spectrum by combining the first and second m/z spectra.

Some embodiments are directed to a method of performing mass spectrometry (MS) using an ion trap MS apparatus. The method may include: determining a first mass- to-charge ratio (m/z) spectrum of a first plurality of ions from a first plurality of discrete m/z ranges; determining a second m/z spectrum of a second plurality of ions from a second plurality of discrete m/z ranges different from the first plurality of discrete m/z ranges; and creating a combined m/z spectrum by combining the first and second m/z spectra.

Some embodiments are directed to at least one computer readable medium encoded with instruction that, when executed by at least one processor, controls a mass

spectrometer to perform a survey scan method to obtain a survey scan spectrum, the survey scan method comprising: performing a number (N) of sub- scans, wherein each sub- scan of the N sub-scans comprises at least a number (n) of bins, wherein N is larger than one and n is larger than one; and combining results from the N subscans into the survey scan spectrum.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

FIG. 1 illustrates a known gas phase enrichment technique using a multiple stage

MSI survey scan;

FIG. 2 illustrates a multistage MSI survey scan with each stage using a different isolation waveform comprising a respective plurality of discrete notches according to some embodiments;

FIG. 3 illustrates an effect of applying a multiple notch isolation waveform to a sample of ions;

FIG. 4 illustrates a comparison of ion current as a function of time for an un- notched gas-phase enriched MS 1 survey scan and a notched gas-phase enriched MS 1 survey scan;

FIG. 5 illustrates the flux of ions using a 50 m/z notch relative to a conventional un-notched MS 1 scan;

FIG. 6 is a flow chart of a method of performing a multi-notched MS scan according to some embodiments;

FIG. 7 illustrates a flow chart of a method of performing an analysis based on multi-notched survey scan results according to some embodiments;

FIG. 8 is a schematic block diagram of a MS apparatus according to some embodiments; and

FIG. 9 is a schematic block diagram of a computing environment according to some embodiments.

DETAILED DESCRIPTION OF INVENTION

The inventors have recognized and appreciated that high quality MS 1 survey scans increase the quality of subsequent MS analysis. By way of example and not limitation, increased quality survey scans may result in more accurate MSI -based quantitation (e.g., Stable isotope labeling by amino acids in cell culture (SILAC)), improved discrimination for on-line data-dependent decisions (e.g., MS2 based interrogation of low intensity ions), and enhanced success of targeted methodologies. The inventors have also recognized and appreciated that MS instruments have a finite dynamic range, meaning, because only a limited number of total ions may be measured in a particular experiment, it may be difficult to detect low abundance ions due to the overwhelming presence of high abundance ions. Accordingly, the quality of survey scans may be increased by splitting a survey scan into a plurality of stages and combining the resulting m/z spectra from each stage into a single "combined m/z spectrum." A survey scan with a plurality of stages is herein referred to as a "multistage survey scan."

Known gas-phase fractionation techniques used in multistage survey scans enable sampling of very low abundance proteins. However, the inventors have recognized and appreciated that these known techniques require careful selection of m/z ranges (based on either prior empirical or in silico analysis) and often result in unequal enrichment across both the MSI survey scan and across the chromatogram. FIG. 1 illustrates a known technique of gas-phase enrichment, and how the problem of unequal enrichment arises. Generally, gas phase enrichment separates a sample to be analyzed into subsets to be analyzed by separate MSI survey scan stages, each with large and continuous m/z ranges. Such separation increases the likelihood that precursor ions of low concentrations will not be overwhelmed in the MS analysis by other precursor ions that occur in higher concentrations. The size of the bins for each stage is optimized such that the precursor ions are distributed evenly between the separate MSI survey scan stages.

FIG. 1A illustrates an m/z spectrum of precursor ions injected into a MS apparatus. The m/z values of the constitute ions ranges from about 300 m/z to 1500 m/z. The most abundant ions in the precursor ion distribution have a m/z value of approximately 500 m/z. The particular spectrum illustrated in FIG. 1A is shown by way of example and not limitation. Embodiments may use any suitable precursor ions with arbitrary precursor ion distributions.

FIG. IB illustrates a specific binning strategy according to an exemplary conventional multistage survey scan technique. The survey scan is separated into three stages, each stage isolating a particular range of ions using an isolation waveform with a single, continuous isolation notch. The first stage uses an isolation waveform with a single notch tailored to isolate ions with m/z values ranging from 300-500 m/z, as illustrated in the top spectrum of FIG. IB. The second stage uses an isolation waveform with a single notch tailored to isolate ions with m/z values ranging from 500-700 m/z, as illustrated in the middle spectrum of FIG. IB. The third stage uses an isolation waveform with a single notch tailored to isolate ions with m/z values greater than 700 m/z, as illustrated in the bottom spectrum of FIG. IB.

FIG. 1C illustrates how the precursor ion distribution injected into the MS apparatus at any particular time may change over time. Each circle in FIG. 1C represents the detection of an ion at a particular m/z value at a particular retention time. If vertical slices were taken through the figure, the data would represent ion distributions detected at a particular retention time. In the particular example of FIG. 1C, the mean m/z of ions injected into the MS apparatus increases over time. This change in distribution may occur for any of a number of reasons. For example, liquid chromatography may be used as a source of precursor ions to be injected into the MS apparatus. Liquid chromatography is a technique for separating and eluting complex mixtures of peptides slowly (over the course of 20 minutes to six hours) into simpler mixtures of peptides which may then be introduced to the MS apparatus. The m/z of the simplified peptide mixtures may correlate with time, such that the first peptides to be separated from the more complex mixture are the low m/z peptides, whereas the last peptides to be separated from the more complex mixture are the high m/z peptides. This correlation of m/z with time that occurs in liquid chromatography is the cause of the increase in m/z of the ions over time illustrated in FIG. 1C.

The problem of ion distributions changing over time is not limited to an m/z distribution that increases over time, as illustrated in FIG. 1C. In some embodiments, the m/z distribution of injected ions may decrease over time, randomly change over time, or vary in any other way. Moreover, any suitable method of preparing precursor ions may be used. By way of example and not limitation, gas chromatography, capillary

electrophoresis, Matrix-assisted laser desorption/ionization (MALDI), or atmospheric sampling are techniques that may be used to prepare ions for injection into an MS apparatus.

FIG. 1C also illustrates the problematic effect of using the aforementioned binning strategy of FIG. IB on precursor ions injected into an MS apparatus. Each of the large static bins of FIG. IB collects a set range of ions. The set range does not change over time. Thus, as the precursor ion distribution changes over time, each bin collects a different amount of ions. For example, the first stage of the survey scan initially (at early retention times) isolates a large amount of ions, whereas at later times, the first stage isolates fewer ions. On the other hand, the third stage initially isolates few ions, whereas at later times, the third stage isolates more ions.

It is desirable for each stage of the survey scan to isolate an equal amount of ions over time. Accordingly, it is known in the art to alter the position of the isolation notches over time for each subsequent survey scan. For example, the first stage may initially isolate ions with m/z values ranging from 300-500 m/z, while after 100 minutes of retention time, the first stage may be adjusted to isolate ions with m/z values ranging from 300-650 m/z. The other stages of the survey scan also have their m/z ranges adjusted accordingly such that each bin isolates a similar number of ions at each retention time value. Each bin of the multistage survey scan may be optimized in size and position based on the expected distribution of ions at a particular time. It is known to base these optimizations on the analysis of prior experimental data or in silico calculations.

The inventors have recognized and appreciated that optimizing the bins used in the different stages of the survey scan based on the retention time is a complex, tedious procedure that is not always successful. Accordingly, embodiments are directed to techniques for conducting multistage survey scans using gas-phase enrichment techniques that do not require a priori knowledge of the precursor ions to change the isolation waveforms over time. Embodiments are directed to a gas-phase enrichment based approach that significantly increases the detection of low abundance peptides using isolation waveforms with a plurality of discrete notches. By distributing the discrete notches across the m/z range, and by changing the set of notches used, embodiments are able to maintain consistent enrichment levels across the MSI survey spectra and across a chromatogram without any prior knowledge of the sample and without the need to adjust the isolation waveforms over time. The resulting improvement in MSI spectral quality affords opportunities for increased sampling depth, novel targeted identifications, and reproducible quantitation.

FIG. 2 illustrates a multistage MSI survey scan with each stage using a different isolation waveform comprising a respective plurality of discrete notches according to some embodiments. The mass range is divided into sets of multiple discontinuous notches. Consequently, the precursor ions are allowed to be distributed between the different MSI survey scan stages with greater regularity than the technique of FIG. 1.

FIG. 2A illustrates an m/z spectrum of precursor ions injected into a MS apparatus. The m/z values of the constitute ions ranges from about 300 m/z to 1500 m/z. The most frequently occurring ions in the precursor ion distribution have a m/z value of approximately 500 m/z. The particular spectrum illustrated in FIG. 2A is shown by way of example and not limitation. Embodiments may use any suitable precursor ions with arbitrary precursor ion distributions. For the purposes of comparing the multi-notch, multistage survey scan technique according to some embodiments with un-notched multistage survey scan techniques, the ion distribution in FIG. 2A is the same as the distribution in FIG. 1A.

FIG. 2B illustrates a specific binning strategy according to at least one

embodiment. The survey scan is separated into three stages, each stage isolating a plurality of discrete ranges of ions using an isolation waveform with a plurality of discrete isolation notches. The first stage uses an isolation waveform with a plurality of notches, each notch isolating a different m/z range with a width of approximately 50 m/z, illustrated in the top spectrum of FIG. 2B. The second stage uses an isolation waveform with a plurality of notches, each notch isolating a different m/z range with a width of approximately 50 m/z, illustrated in the middle spectrum of FIG. 2B. The third stage uses an isolation waveform with a plurality of notches, each notch isolating a different m/z range with a width of approximately 50 m/z, illustrated in the bottom spectrum of FIG. 2B. By way of example and not limitation, the experimental data shown in FIG. 2 uses six 50 m/z wide notches evenly distributed across the mass range of 300-1500 m/z.

In some embodiments, the plurality of notches in a first isolation waveform is different from the plurality of notches in at least a second isolation waveform. For example, the plurality of notches associated with the first stage of the survey scan in FIG. 2B is "orthogonal" to the other stages in that it isolates completely different m/z ranges of than the other stages. However, embodiments are not so limited. The ranges isolated in different stages may overlap in some embodiments, as is discussed in more detail below.

Embodiments are not limited to the particular number of stages or number of bins illustrated in FIG. 2B. Any suitable number of bins and stages may be used. By way of example and not limitation, 3-4 stages may be used with 4-8 bins per stage. The number of bins per stage may be related to the bin width. Any suitable bin width may be used. For example a bin width between 25-400 m/z may be used. Some embodiments have at least two stages with at least two bins in each stage. The number of bins per stage need not be equal for each stage. For example, the first stage may use 7 bins while the second stage uses 8 bins.

As in FIG. 1C, FIG. 2C illustrates the ion distribution injected into the MS apparatus changing over time. However, because each stage uses multiple bins spread across the total m/z range of the MS apparatus, the number of ions isolated during each stage remains approximately constant, unlike the bins used in FIG. 1C. The number of ions isolated by each stage remains constant because, as the ion distribution shifts to greater m/z values over time, the number of ions isolated by the lower m/z bins of each stage decreases at approximately the same rate as the number of ions isolated by the higher m/z bins of each stage.

Embodiments, according to the above description, eliminate the need to predict the ideal gas-phase fractions while still enriching ions in the MSI survey scans. By dividing the MSI survey scan mass range into a plurality of discrete slices, the isolated precursor ions are evenly distributed without any prior knowledge of the precursor ion distribution. In the particular experiment illustrated in FIG. 2 with six 50 m/z wide notches evenly distributed across range of 300-1500 m/z, the MSI spectral space is attenuated 4-fold, and consequently precursor intensity gains of approximately 4-fold are observed for those ions that fell within the enriched notches of the MSI mass range.

FIG. 3 illustrates the effect of using a multi-notch isolation waveform on an m/z spectrum by comparing a standard MSI survey scan (FIG. 3A) with a single stage multi- notch MSI survey scan of the same precursor ion distribution (FIG. 3B). The spectrum of FIG. 3B was acquired using eight evenly distributed 50 m/z notches across the mass range. The ability to detect low abundance ions using the multi-notch gas phase enrichment is clear when compared to the spectrum obtained by analyzing the full m/z range of the MS apparatus. As mentioned above, the dynamic range of MS apparatuses may be limited by the ability to detect a finite number of ions in a given experiment run. In both spectrums of FIG. 3, the same total number of ions was detected. However, because not all m/z ranges were analyzed in the multi-notch analysis, ions of low abundance that were not detected using standard MS 1 techniques were able to be detected. For example, many of the ions with high m/z values in FIG. 3B are not visible in the spectrum of FIG. 3A. Thus, across the m/z range - though particularly in the high m/z range - ion intensities may be increased by the notched waveform based gas-phase enrichment and the lower limit of ion detection is decreased. It is noted that the "x5" and the "xlO" labels across the top of FIG. 3 indicate that the ion intensities for those ranges are, after data acquisition, magnified five times and ten times, respectively, in order to adequately visualize the data.

As mentioned above, if the technique of FIG. 1, using multiple stages without multiple notches, is used, the number of ions isolated during each stage changes over time, whereas using the multi-notch, multi-stage gas phase enrichment of FIG. 2 results in a relatively constant number of ions being isolated as retention time increases. An overabundance of precursor ions in some spectra results in minimal increases in precursor ion intensities. FIG. 4 illustrates a comparison of the average MSI enrichment as a function of time for both the notched technique of FIG. 2 and the un-notched technique of FIG. 1. The comparison of the precursor ion current variations as a function of retention time for the standard, un-notched gas-phase enrichment (using the lowest m/z stage of FIG. 1) against the performance of the notched isolation waveform based gas-phase enrichment experiment (using the lower m/z set of multinotches from stage 1) illustrate the benefits of an isolation waveform with multiple notches. The vertical axis is an "average MS I enrichment," which is determined by taking the ratio of the number of ions isolated in the m/z range of interest for each technique with the number of ions isolated in the same m/z range for a standard MSI survey scan without multiple notches. For example, the average enrichment using the un-notched gas phase enrichment technique at early times is nearly unity, meaning there is no enrichment when compared to a standard MSI survey scan. This is because, as discussed previously, when using liquid chromatography, most low m/z ions elute early resulting in the un-notched gas phase enrichment technique providing little benefit during early retention times. As the retention time increases, the un-notched technique provides more enrichment due to the lower ion intensity for low m/z ions at higher retention times. On the other hand, the enrichment for the notched gas phase enrichment according to some embodiments is substantially constant as a function of retention time. This is because there is at least one portion of the m/z spectrum being enriched at any given retention time. Thus, there are no biases in precursor intensity enrichment as a function of retention time.

Ion enrichment may not be uniform across a given notch. By way of example, FIG. 5 compares the flux of ions (i.e., the rate at which ions are accumulated in the mass spectrometer) between the notched isolation waveform based MS 1 survey scan and a standard MSI scan with no notches. The vertical axis is a ratio of the ion flux using a notch versus the ion flux in the same range when a standard MS 1 survey scan is performed. In the middle of the 50 m/z bin, the ion flux is approximately identical to the ion flux resulting from the conventional MSI technique. However, near the boundaries of the bin, indicated in gray, the ion flux decreases. Accordingly, in some embodiments, when the notched waveform based gas-phase enrichment method is used prior to further data-dependent analyses (e.g., MS2, MSn, SIM, MRM), precursor ions from the central portions of the bin may be selectively used for the subsequent analysis. In other embodiments, the ion injection time for the subsequent data-dependent analyses may be dependent upon the location of the precursor ions relative the position of the bin.

In some embodiments, the location and the position of notches from other stages may be selected to negate the effects of decreased ion fluxes in the bracketed gray areas near the boundaries of the notch illustrated in FIG. 5. For example, instead of choosing bins for each stage that do not overlap with bins of other stages, bins may be selected to overlap between stages to compensate for the reduced ion flux in the gray regions. The overlap of bins may be selected in any suitable way. For example, a gray area of a first bin of a first stage may be selected to overlap with a gray area of a second bin of a second stage.

FIG. 6 illustrates a method 600 according to some embodiments. At act 602, an MS apparatus obtains a plurality of ions. The plurality of ions may be obtained in any suitable way. For example, the ions may be obtained from one or more protein samples using techniques such as liquid chromatography, gas chromatography, capillary electrophoresis, Matrix-assisted laser desorption/ionization (MALDI), and/or atmospheric sampling. These number and/or type of ions provided to the MS apparatus may change over time depending on the technique used to obtain the plurality of ions. In some embodiments, the plurality of ions may be referred to as precursor ions.

At act 604, at least a portion of the precursor ions are injected into the MS apparatus. This may be done in any suitable way. For example, one or more electric and/or magnetic fields may be used to guide the plurality of ions from outside the ion trap into the ion trap.

At act 606, a portion of the injected ions may be isolated with a first isolation waveform. As described above, the isolation waveform may comprise a plurality of notches, each notch having a m/z position and width. Any suitable number of notches may be used. By way of example and not limitation, 7-8 discrete notches may be used. Each notch may have any suitable width. By way of example and not limitation, each notch may be 50 m/z wide. In some embodiments, the width of each notch may be the same. In other embodiments, the width may be different for some or all of the notches. In some embodiments, act 604 and act 606 may be performed simultaneously such that the ions are isolated as they are being injected into the ion trap. However, embodiments are not so limited and isolation may occur after injection. At act 608, an m/z spectrum is determined for the isolated ions. The m/z spectrum may be determined in any suitable way. For example, the m/z spectrum may be obtained by detecting and analyzing the m/z values of the isolated ions in the MS apparatus.

Acts 602-608 may represent a first stage of a survey scan. At act 610, it is determined whether there are additional stages of the survey scan to be performed. In some embodiments, at least two stages are performed. Further, in some embodiments 7-8 stages are performed. Some embodiments may only use a single stage with a multiple notch waveform. Embodiments are not limited to any particular number of stages.

If it is determined at act 610 that there are additional stages, the method 600 returns to act 602 to repeat acts 602-608 for the subsequent stage. In some embodiments the isolation waveform used in act 606 may comprise a plurality of notches that differ from the notched used in the isolation waveform of other stages of the survey scan. In some embodiments, the multiple stages of the survey scan may be performed more than one time over the course of a liquid chromatography (LC) run. In other embodiments, a single stage may be analyzed for an entire LC run and the subsequent stages will be performed on a respective LC run. Embodiments are not limited to performing stages at any particular time or duration relative to the LC process used. Embodiments are also not limited to using LC. As previously mentioned, ions may be obtained for use in the MS apparatus in any suitable way.

If it is determined that there are no more stages of the survey scan to be performed, the method 600 continues to act 612, to generate a combined spectrum. The combined spectrum may be generated in any suitable way. For example, the individual m/z spectrums from each individual stage may be added together or averaged together to generated a combined spectrum.

At act 614, it is determined whether there are additional survey scans to be performed. For example, as illustrated above, multistage survey scans may be performed repeatedly over the course of tens of minutes up to several hours. In some embodiments, the isolation waveform used in each stage of the multistage survey scan is the same for each subsequent survey scan such that the notches are not changed as a function of retention time.

If it is determined at act 614 that there are additional survey scans to be performed, the method 600 returns to act 602. If it is determined that there are no more survey scans to be performed, the method 600 ends. In some embodiments, not every act of FIG. 6 is performed. For example, it may not be necessary to determine a spectrum at act 608 and/or it may not be necessary to generate a combined spectrum at act 612.

FIG. 7 illustrates a method 700 of performing an analysis based on multi-notched survey scan results according to some embodiments. At act 702, a multi-notch isolation waveform is determined for use in isolating ions injected into the MS apparatus. The multi-notch isolation waveform may be calculated by, for example, a controller or a processor. In some embodiments, the MS apparatus may store a plurality of available waveforms in memory and the multi-notch waveform may be determined by selecting a waveform from the options stored in memory.

At act 704, a multi-notch survey scan is performed. In some embodiments, this may be a single stage survey scan which is performed by injecting ions into the ion trap of the MS apparatus while the isolation wave form is being generated by the ion trap. Once the desired ions are isolated, the isolated ions are analyzed. The ions may be analyzed in any suitable way. For example, the m/z of each isolated ion may be detected. In addition, the absolute abundance and/or relative abundance of each ion may be determined. In other embodiments, a multi-stage survey scan may be performed, as described in connection with FIG. 6.

At act 706, the results obtained by the survey scan of act 704 may be used to perform subsequent analyses. In some embodiments, an MS2 analysis may be performed based, at least in part, on the results of the survey scan. The MS2 analysis may include, by way of example and not limitation, determining an isolation waveform based on the results of the survey scan, injecting ions into the ion trap while generating the determined isolation waveform, fragmenting the isolated ions and detecting the resulting product ions. The determined isolation waveform may be a single notch or a multi-notch isolation waveform. Embodiments are not limited to any particular type of subsequent analysis. By way of example and not limitation, a subsequent analysis that is based on the results of the survey scan of act 704 may include, tandem MS of any number of fragmentation steps (MSn), Secondary Ion MS (SIM), Multiple Reaction or Monitoring (MRM).

At act 708, it is determined whether there are additional analyses to perform. In some embodiments, the process of performing a survey scan followed by a data-dependent subsequent analysis may be performed multiple times over the course of performing liquid chromatography or any other process for presenting samples to the MS apparatus over a period of time. If it is determined that there is one or more additional analyses to be performed, the method 700 returns to act 702 for additional processing. If it is determined that there are no more additional analyses to be performed, the method 700 ends.

FIG. 8 illustrates a mass spectrometry (MS) apparatus 800 according to some embodiments. MS apparatus 800 comprises a controller 802, an ion trap 804, an isolation waveform generator 806, an ion injector 808 and an analyzer 810. MS apparatus 800 is not intended to suggest any limitation as to the scope of use or functionality of the invention. Neither should the MS apparatus 800 be interpreted as having any dependency or requirement relating to any one or combination of components illustrated in the exemplary MS apparatus 800.

MS apparatus 800 comprises at least one controller 802, which may be comprised of hardware, software, or a combination of hardware and software. In some embodiments, controller 802 determines one or more properties of the isolation waveforms, such as the number of notches and the position and width of the notches. The controller 802 may also determine the number of stages of each survey scan. The controller 802 may also instruct the ion trap, the isolation waveform generator 806, the ion injector 808 and the analyzer 810 to perform various acts. For example, controller 802 may perform, or instruct other components of MS apparatus 800 to perform, at least some of the acts described in FIG. 6 and FIG. 7. In some embodiments, MS apparatus 800 is not limited to a single controller - multiple controllers may be used.

Apparatus 800 comprises an ion trap 804 and an isolation waveform generator 806.

Controller 802 may be coupled to the ion trap 804 and/or isolation waveform generator 806 to allow communication. Any suitable form of coupling may be used. For example, the components may be coupled via a system bus. Alternatively, the components of apparatus 800 may be coupled via a communications network, such as an Ethernet network. Embodiments of the invention are not limited to any specific type of coupling.

The ion trap 804 may be any ion trap suitable for use in mass spectrometry. For example, ion trap 804 may be a quadrupole ion trap, a Fourier transform ion cyclotron resonance (FTICR) MS, or an orbitrap MS.

The isolation waveform generator 806 may be any suitable device for generating the isolation waveforms used to isolate ions in the ion trap 804. For example, isolation waveform generator 806 may be a radio frequency (RF) signal generator.

The analyzer 810 may analyze the results obtained from the ion trap. For example, it may determine the m/z spectrum for a given set of ions. In some embodiments, the controller 810 analyzes the results of the individual spectrums from survey scans performed by the MS device 800 and combines them into a combined spectrum. Though the analyzer 810 is shown separate from the controller 802 in FIG. 8, in some

embodiments, the analyzer 810 and the controller 802 may be a single physical computing device. Moreover, though the analyzer 810 is shown separate from the ion trap 804, in some embodiments they may be the same device.

FIG. 9 illustrates an example of a suitable computing system environment 900 on which the invention may be implemented. For example, the controller 802 and/or the analyzer 810 of FIG. 8 may include one or more aspects of the computing system environment 900 of FIG. 9. The computing system environment 900 is only one example of a suitable computing environment and is not intended to suggest any limitation as to the scope of use or functionality of the invention. Neither should the computing environment 900 be interpreted as having any dependency or requirement relating to any one or combination of components illustrated in the exemplary operating environment 900.

The invention is operational with numerous other general purpose or special purpose computing system environments or configurations. Examples of well-known computing systems, environments, and/or configurations that may be suitable for use with the invention include, but are not limited to, personal computers, server computers, handheld or laptop devices, multiprocessor systems, microprocessor-based systems, set top boxes, programmable consumer electronics, network PCs, minicomputers, mainframe computers, distributed computing environments that include any of the above systems or devices, and the like.

The computing environment may execute computer-executable instructions, such as program modules. Generally, program modules include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. The invention may also be practiced in distributed computing environments where tasks are performed by remote processing devices that are linked through a communications network. In a distributed computing environment, program modules may be located in both local and remote computer storage media including memory storage devices.

With reference to FIG. 9, an exemplary system for implementing the invention includes a general purpose computing device in the form of a computer 910. Components of computer 910 may include, but are not limited to, a processing unit 920, a system memory 930, and a system bus 921 that couples various system components including the system memory to the processing unit 920. The system bus 921 may be any of several types of bus structures including a memory bus or memory controller, a peripheral bus, and a local bus using any of a variety of bus architectures. By way of example, and not limitation, such architectures include Industry Standard Architecture (ISA) bus, Micro Channel Architecture (MCA) bus, Enhanced ISA (EISA) bus, Video Electronics Standards Association (VESA) local bus, and Peripheral Component Interconnect (PCI) bus also known as Mezzanine bus.

Computer 910 typically includes a variety of computer readable media. Computer readable media can be any available media that can be accessed by computer 910 and includes both volatile and nonvolatile media, removable and non-removable media. By way of example, and not limitation, computer readable media may comprise computer storage media and communication media. Computer storage media includes both volatile and nonvolatile, removable and non-removable media implemented in any method or technology for storage of information such as computer readable instructions, data structures, program modules or other data. Computer storage media includes, but is not limited to, RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital versatile disks (DVD) or other optical disk storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium which can be used to store the desired information and which can accessed by computer 910. Communication media typically embodies computer readable instructions, data structures, program modules or other data in a modulated data signal such as a carrier wave or other transport mechanism and includes any information delivery media. The term "modulated data signal" means a signal that has one or more of its characteristics set or changed in such a manner as to encode information in the signal. By way of example, and not limitation, communication media includes wired media such as a wired network or direct- wired connection, and wireless media such as acoustic, RF, infrared and other wireless media. Combinations of the any of the above should also be included within the scope of computer readable media.

The system memory 930 includes computer storage media in the form of volatile and/or nonvolatile memory such as read only memory (ROM) 931 and random access memory (RAM) 932. A basic input/output system 933 (BIOS), containing the basic routines that help to transfer information between elements within computer 910, such as during start-up, is typically stored in ROM 931. RAM 932 typically contains data and/or program modules that are immediately accessible to and/or presently being operated on by processing unit 920. By way of example, and not limitation, FIG. 9 illustrates operating system 934, application programs 935, other program modules 936, and program data 937.

The computer 910 may also include other removable/non-removable,

volatile/nonvolatile computer storage media. By way of example only, FIG. 9 illustrates a hard disk drive 941 that reads from or writes to non-removable, nonvolatile magnetic media, a magnetic disk drive 951 that reads from or writes to a removable, nonvolatile magnetic disk 952, and an optical disk drive 955 that reads from or writes to a removable, nonvolatile optical disk 956 such as a CD ROM or other optical media. Other

removable/non-removable, volatile/nonvolatile computer storage media that can be used in the exemplary operating environment include, but are not limited to, magnetic tape cassettes, flash memory cards, digital versatile disks, digital video tape, solid state RAM, solid state ROM, and the like. The hard disk drive 941 is typically connected to the system bus 921 through an non-removable memory interface such as interface 940, and magnetic disk drive 951 and optical disk drive 955 are typically connected to the system bus 921 by a removable memory interface, such as interface 950.

The drives and their associated computer storage media discussed above and illustrated in FIG. 9, provide storage of computer readable instructions, data structures, program modules and other data for the computer 910. In FIG. 9, for example, hard disk drive 941 is illustrated as storing operating system 944, application programs 945, other program modules 946, and program data 947. Note that these components can either be the same as or different from operating system 934, application programs 935, other program modules 936, and program data 937. Operating system 944, application programs 945, other program modules 946, and program data 947 are given different numbers here to illustrate that, at a minimum, they are different copies. A user may enter commands and information into the computer 910 through input devices such as a keyboard 962 and pointing device 961, commonly referred to as a mouse, trackball or touch pad. Other input devices (not shown) may include a microphone, joystick, game pad, satellite dish, scanner, or the like. These and other input devices are often connected to the processing unit 920 through a user input interface 960 that is coupled to the system bus, but may be connected by other interface and bus structures, such as a parallel port, game port or a universal serial bus (USB). A monitor 991 or other type of display device is also connected to the system bus 921 via an interface, such as a video interface 990. In addition to the monitor, computers may also include other peripheral output devices such as speakers 997 and printer 996, which may be connected through a output peripheral interface 995.

The computer 910 may operate in a networked environment using logical connections to one or more remote computers, such as a remote computer 980. The remote computer 980 may be a personal computer, a server, a router, a network PC, a peer device or other common network node, and typically includes many or all of the elements described above relative to the computer 910, although only a memory storage device 981 has been illustrated in FIG. 9. The logical connections depicted in FIG. 9 include a local area network (LAN) 971 and a wide area network (WAN) 973, but may also include other networks. Such networking environments are commonplace in offices, enterprise- wide computer networks, intranets and the Internet.

When used in a LAN networking environment, the computer 910 is connected to the LAN 971 through a network interface or adapter 970. When used in a WAN networking environment, the computer 910 typically includes a modem 972 or other means for establishing communications over the WAN 973, such as the Internet. The modem 972, which may be internal or external, may be connected to the system bus 921 via the user input interface 960, or other appropriate mechanism. In a networked environment, program modules depicted relative to the computer 910, or portions thereof, may be stored in the remote memory storage device. By way of example, and not limitation, FIG. 9 illustrates remote application programs 985 as residing on memory device 981. It will be appreciated that the network connections shown are exemplary and other means of establishing a communications link between the computers may be used.

Having thus described several aspects of at least one embodiment of this invention, it is to be appreciated that various alterations, modifications, and improvements will readily occur to those skilled in the art.

For example, in some embodiments, the resulting improved MS 1 spectral quality may be used to trigger an interrogation of precursors that are generally ignored. Due to the resulting enrichment, the properties of these precursor ions that determine

interrogation (charge state, intensity, monoisotopic peak assignment) are improved. This may result in the targeting of novel precursors that were either not present or of insufficient intensity using tradition MSI scans. For example, using the isolation waveform from FIG. 2 with six notches , each with a 50 m/z width, a set of 1,573 precursors were experimentally targeted in a yeast whole cell lysate that were never interrogated using a standard MSI technique. Correspondingly, an increase in the number of identifications was observed, the new identifications corresponded to low-copy number yeast proteins.

Some embodiments that utilize targeted approaches for reproducible identification and quantitation of desired precursors may benefit from the gain in MS 1 quality and enrichment of ions. An inclusion list may be used to direct the fragmentation towards specific precursor ions, which may or may not be visible in the standard MS 1 survey scans. Through applications of the notched isolation waveform based MSI enriched survey scan, precursors that are frequently limited in their identification may show enhanced recovery and reproducibility.

In some embodiments, the notched waveform based MSI survey scan may improve the quality of MSI -based quantitation methods. MSI based quantitation methods rely on accurate ion intensity measurements, including, but not limited to, spectral counting, N15 labeling, SILAC, re-ductive dimethylation, NeuCode and mTRAQ. The accuracy of the ion intensity measurements improve as the number of ions included in the measured population increases. As such, these methods may benefit from the inclusion of the notched isolation waveform based MSI survey scans according to some embodiments.

In some embodiments, the application of the notched isolation waveforms in an MSI survey scan could improve the depth and reproducibility of reporter ion based quantitation. Reporter ion quantitation relies on ions that are measured following fragmentation and include, but are not limited to, TMT and iTRAQ. These methods will likely benefit from the application of the notched MS 1 survey scan as the enrichment provides novel precursors for eventual interrogation, including isolation, fragmentation and quantification.

In some embodiments, the size and location of the notches may be adjusted during the analysis. That is, as the mass spectrometer is analyzing a sample, the size and location of the notches may be adjusted based on prior knowledge. This prior knowledge may take the form of earlier analyses, in silico analysis, or data-dependent analysis of recently acquired spectra. Additionally, the location and width of the notches may also be adjusted in order to, for example, exclude the top-N abundant species thereby further increasing the range of the enrichment for the low abundance precursors.

Also, while embodiments have been described that do not change the size or position of the isolation notches over time, embodiments are not so limited. The isolation waveforms used in various stages of the multistage survey scan of some embodiments may change the position and/or size of one or more notches over time. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the invention. Further, though advantages of the present invention are indicated, it should be appreciated that not every embodiment of the invention will include every described advantage. Some embodiments may not implement any features described as advantageous herein and in some instances. Accordingly, the foregoing description and drawings are by way of example only.

The above-described embodiments of the present invention can be implemented in any of numerous ways. For example, the embodiments may be implemented using hardware, software or a combination thereof. When implemented in software, the software code can be executed on any suitable processor or collection of processors, whether provided in a single computer or distributed among multiple computers. Such processors may be implemented as integrated circuits, with one or more processors in an integrated circuit component. Though, a processor may be implemented using circuitry in any suitable format.

Further, it should be appreciated that a computer may be embodied in any of a number of forms, such as a rack-mounted computer, a desktop computer, a laptop computer, or a tablet computer. Additionally, a computer may be embedded in a device not generally regarded as a computer but with suitable processing capabilities, including a Personal Digital Assistant (PDA), a smart phone or any other suitable portable or fixed electronic device.

Also, a computer may have one or more input and output devices. These devices can be used, among other things, to present a user interface. Examples of output devices that can be used to provide a user interface include printers or display screens for visual presentation of output and speakers or other sound generating devices for audible presentation of output. Examples of input devices that can be used for a user interface include keyboards, and pointing devices, such as mice, touch pads, and digitizing tablets. As another example, a computer may receive input information through speech recognition or in other audible format.

Such computers may be interconnected by one or more networks in any suitable form, including as a local area network or a wide area network, such as an enterprise network or the Internet. Such networks may be based on any suitable technology and may operate according to any suitable protocol and may include wireless networks, wired networks or fiber optic networks. Also, the various methods or processes outlined herein may be coded as software that is executable on one or more processors that employ any one of a variety of operating systems or platforms. Additionally, such software may be written using any of a number of suitable programming languages and/or programming or scripting tools, and also may be compiled as executable machine language code or intermediate code that is executed on a framework or virtual machine.

In this respect, the invention may be embodied as a computer readable storage medium (or multiple computer readable media) (e.g., a computer memory, one or more floppy discs, compact discs (CD), optical discs, digital video disks (DVD), magnetic tapes, flash memories, circuit configurations in Field Programmable Gate Arrays or other semiconductor devices, or other tangible computer storage medium) encoded with one or more programs that, when executed on one or more computers or other processors, perform methods that implement the various embodiments of the invention discussed above. As is apparent from the foregoing examples, a computer readable storage medium may retain information for a sufficient time to provide computer-executable instructions in a non-transitory form. Such a computer readable storage medium or media can be transportable, such that the program or programs stored thereon can be loaded onto one or more different computers or other processors to implement various aspects of the present invention as discussed above. As used herein, the term "computer-readable storage medium" encompasses only a computer-readable medium that can be considered to be a manufacture (i.e., article of manufacture) or a machine. Alternatively or additionally, the invention may be embodied as a computer readable medium other than a computer- readable storage medium, such as a propagating signal.

The terms "program" or "software" are used herein in a generic sense to refer to any type of computer code or set of computer-executable instructions that can be employed to program a computer or other processor to implement various aspects of the present invention as discussed above. Additionally, it should be appreciated that according to one aspect of this embodiment, one or more computer programs that when executed perform methods of the present invention need not reside on a single computer or processor, but may be distributed in a modular fashion amongst a number of different computers or processors to implement various aspects of the present invention.

Computer-executable instructions may be in many forms, such as program modules, executed by one or more computers or other devices. Generally, program modules include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. Typically the functionality of the program modules may be combined or distributed as desired in various embodiments.

Also, data structures may be stored in computer-readable media in any suitable form. For simplicity of illustration, data structures may be shown to have fields that are related through location in the data structure. Such relationships may likewise be achieved by assigning storage for the fields with locations in a computer-readable medium that conveys relationship between the fields. However, any suitable mechanism may be used to establish a relationship between information in fields of a data structure, including through the use of pointers, tags or other mechanisms that establish relationship between data elements.

Various aspects of the present invention may be used alone, in combination, or in a variety of arrangements not specifically discussed in the embodiments described in the foregoing and is therefore not limited in its application to the details and arrangement of components set forth in the foregoing description or illustrated in the drawings. For example, aspects described in one embodiment may be combined in any manner with aspects described in other embodiments.

Also, the invention may be embodied as a method, of which an example has been provided. The acts performed as part of the method may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments.

Use of ordinal terms such as "first," "second," "third," etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having," "containing," "involving," and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

What is claimed is:

Claims

1. A method of performing mass spectrometry (MS), the method comprising:

performing a survey scan using a first isolation waveform of an ion trap, the first isolation waveform comprising at least a first and second notch.

2. The method of claim 1, wherein the survey scan comprises at least a first stage and a second stage, the first isolation waveform being used during the first stage, wherein the method further comprises:

performing the second stage of the survey scan using a second isolation waveform of an ion trap, the second isolation waveform comprising at least two notches that are different from the first and second notch.

3. The method of claim 2, wherein at least one portion of the first notch of the first isolation waveform isolates the same m/z range as a portion of one of the at least two notches of the second isolation waveform.

4. The method of claim 2, wherein:

the first stage is performed for a first time;

the second stage is performed during a second time; and

the first time corresponds to a first liquid chromatography process and the second time corresponds to a second liquid chromatography process.

5. The method of claim 2, wherein the survey scan is a first survey scan, the method further comprising:

performing a second survey scan, wherein the second survey scan comprises: performing a first stage of the second survey scan using the first isolation waveform; and

performing at least a second stage of the second survey scan using the second isolation waveform.

6. The method of claim 1, the method further comprising:

obtaining a plurality of ions for analysis; and injecting the plurality of ions into the ion trap, wherein the first stage of the survey scan is performed on the plurality of ions.

7. The method of claim 6, the method further comprising:

performing liquid chromatography prior to obtaining the plurality of ions for analysis.

8. The method of claim 6, wherein obtaining the plurality of ions comprises matrix- assisted laser desorption/ionization (MALDI).

9. The method of claim 8, wherein an ion type of the plurality of ions changes over time.

10. The method of claim 6, wherein obtaining the plurality of ions comprises atmospheric sampling.

11. The method of claim 1, further comprising:

performing a subsequent MS analysis based on the survey scan.

12. The method of claim 11, wherein the subsequent MS analysis is a tandem MS analysis.

13. A method of performing mass spectrometry (MS), the method comprising:

simultaneously isolating a first plurality of ions within a first range of mass-to- charge ratio (m/z) and a second plurality of ions within a second range of m/z different from the first range of m/z;

determining a first m/z spectrum of the first and second plurality of ions;

isolating a third plurality of ions within a third range of m/z different from the first and second ranges of m/z; and

determining a second m/z spectrum of the third plurality of ions.

14. The method of claim 13, further comprising:

generating a combined spectrum by combining the first and second m/z spectra.

15. A method of performing mass spectrometry (MS) using an ion trap MS apparatus, the method comprising:

determining a first mass-to-charge ratio (m/z) spectrum of a first plurality of ions from a first plurality of discrete m/z ranges;

determining a second m/z spectrum of a second plurality of ions from a second plurality of discrete m/z ranges different from the first plurality of discrete m/z ranges; and creating a combined m/z spectrum by combining the first and second m/z spectra.

16. The method of claim 15, further comprising:

isolating the first plurality of ions with a first isolation waveform comprising a first plurality of notches; and

isolating the second plurality of ions with a second isolation waveform comprising a second plurality of notches.

17. The method of claims 16, wherein isolating the first plurality of ions occurs at a first time and isolating the second plurality of ions occurs at a second time different from the first time.

18. The method of claim 15, further comprising:

injecting the first plurality of ions and the second plurality of ions into the ion trap

MS apparatus.

19. The method of claim 15, further comprising:

obtaining the first plurality of ions and the second plurality of ions from at least one protein sample.

20. At least one computer readable medium encoded with instructions that, when executed by at least one processor, controls a mass spectrometer to perform a method to obtain a survey scan spectrum, the method comprising:

performing a number (N) of sub-scans, wherein each sub-scan of the N sub-scans comprises at least a number (n) of bins, wherein N is larger than one and n is larger than one; and

combining results from the N subscans into the survey scan spectrum.

21. The at least one computer readable medium of claim 19, wherein performing a single sub-scan of the number (N) of sub-scans comprises isolating ions using an isolation waveform .

22. The at least one computer readable medium of claim 19, wherein the method further comprises injecting ions into the mass spectrometer.

23. The at least one computer readable medium of claim 19, wherein the method further comprises performing a subsequent MS analysis based on the survey scan spectrum.

24. The at least one computer readable medium of claim 22, wherein the subsequent MS analysis is a tandem MS analysis.