DE102022128278A1 - Method of determining a measure of decay rate and mass spectrometry system - Google Patents

Abstract

A method for determining a measure of a decay rate of an ion sample undergoing mass analysis in a mass analyzer with image current detection, the method comprising: receiving a detection signal for the ion sample from a detection of the transient of the ion sample obtained using the mass analyser, wherein the ion sample detection signal has a rate of decay over time; and determining the measure of the decay rate of the ion sample based on an extrapolated resolution for the ion sample, the extrapolated resolution being an expected resolution of the detection signal under conditions where the decay rate of the detection signal for the ion sample is dominated by collision effects.

Description

Area

The present disclosure relates to methods for determining a measure of decay rate and to mass spectrometry systems

background

Measurements of collision cross-section areas (CCS areas) can provide valuable information about the structures of molecules such as proteins and protein complexes. It is known that in Fourier transform ion cyclotron resonance (FTICR) mass spectrometry, ion-molecule collision frequencies are related to signal transient decay rates and could be measured by analyzing these rates ( Parisod, G.; Comisarow, MB Chem. Phys. Latvia 1979, 62, 303-305; Guan, S.; Li, G.-Z.; Marshall, AG Int. J. Mass Spectrum. Ion Processes 1997, 167, 185-193 ). Measuring signal decay rates from peak widths can yield collision frequencies and hence collision CCS.

This effect has recently been increasingly used to measure CCS of both small molecules (“ Collision Cross Sectional Areas from Analysis of Fourier Transform Ion Cyclotron Resonance Line Width: A New Method for Characterizing Molecular Structure”, Anal. Chem. 2012, 84, 4851-4857 ) under the name CRAFTI (CRoss section Areas by FT ICR) as well as proteins ( Marshall et al., Phys Chem. Chem. Phys., 2016, 18, 713-717 ) used. It has also been used in orbital trap mass spectrometry (e.g. the Orbitrap™ analyzer manufactured by Thermo Fisher Scientific) ( Sanders JD et al. Anal.Chem., 2018; 90(9):5896-902 ). According to Guo et al. ( Phys.Chem.Chem.Phys., 2015, 17, 9060 ) it could be concluded that the signal decay in the orbitrap analyzer is described by an energetic hard-sphere collision model, in which each collision leads to the fragmentation of an ion and its removal from the detected ion packet.

In Fourier transform mass spectrometry (FTMS), the time-domain transient would theoretically be approximately sinusoidal except due to collisions, but it is actually more complex. Time domain signals in FTMS typically approximate a sine wave with an exponentially decreasing amplitude, with the decrease being attributed primarily to collisions with the background gas. The decay constant is therefore related to the CCS, since higher CCS ions are expected to collide with background gas more frequently and therefore the image signals provided by such ions are expected to decay faster. In general, considering two ions with the same mass-to-charge (m/z) ratio, the ion with the larger mass (and hence higher charge) would tend to have a larger CCS since it would be expected, by one to have such a large mass is physically larger and therefore would be expected to be expected to fall off more sharply.

Wobschall et al. (Phys. Rev. 131, 1565 (1963)) describe that the ion cyclotron resonance absorption line in weakly ionized gases can be interpreted to give the mass-to-charge ratio and the number and collision frequency of the ions. From the observed line widths, the collision frequencies of several ions are determined as a function of the pressure and the electric field strength. This data is used to determine the ion-molecule collision cross-section. Wobschall et al. (J. Chem. Phys. 47, 4091 (1967)) describe the determination of CCS of hydrogen and other ions using ion cyclotron resonance by determining the widths of ion cyclotron resonance lines as a function of pressure and electric field strengths.

For all FTMS instruments, collision cross-sections are calculated relative to a known ion, e.g. B. a calibration ion measured. However, these measurements show significant deviations in the decay time between isolated ion populations (e.g., a particular charge state of a protein) and populations with a large mass range. In addition, no method has been demonstrated on a time scale that is compatible with separation techniques such as liquid chromatography (LC) or gas chromatography (GC).

Existing methods usually rely on isolating specific ions of interest to obtain sufficiently high intensity for reliable determination of peak width and hence decay rate. Therefore they do not allow on-line determination of CCS over broad mass ranges and do not allow parallel determination of CCS together with m/z.

abstract

Against this background and according to a first aspect, a method for determining a measure of a decay rate of an ion sample according to claim 1 is provided. According to a second aspect, a method for determining a measure of a decay rate of an ion sample according to claim 2 is provided. A computer program according to claim 32, a computer readable medium according to claim 33 and a mass spectrometry system according to claim 34 are also provided. According to a third aspect, a mass spectrometry system according to claim 35 is provided.

The present disclosure provides methods and apparatus for determining decay constants and cross-sectional measurements in parallel with mass measurement and decay time correction. In particular, the disclosure relates to methods and apparatus for performing Fourier transform mass spectrometry (FTMS). The disclosure recognizes that existing methods for determining CCS typically rely on isolating particular ions for analysis to obtain sufficiently high intensities for reliable peak width determination. The disclosure recognizes that lower intensities lead to greater defocusing effects (e.g. due to space charge) and thus less reliable signal decay/CCS measurements. In practice, therefore, this does not permit the determination of the decay constant/CCS from the analysis of lower ion intensities, such as are encountered over a broad mass range of ions, and therefore does not permit a practical or routine CCS determination in parallel with conventional m/z determination to.

To address the limitations of existing solutions as outlined above, embodiments of the disclosure use a measure of signal intensity, such as signal-to-noise ratio (SNR), for intensity-dependent (e.g., SNR and /or m/z dependent) fall time corrections that conform to CCS. In some embodiments, signal resolution measurements are extrapolated from low SNR values, where measurements are likely to be inaccurate, to equivalent resolutions at higher SNR, where self-bundling of ions (resulting in reduced dephasing) can be exploited to improve measurement certainty . In some embodiments, the ion sample is generated by a separation technique, such as liquid chromatography (LC) or gas chromatography (GC), from which it has a temporal elution profile. The disclosure also recognizes that recording peak widths of each analyte across the entire elution profile can provide more information about the decay constant and CCS and can be used to improve the statistics of CCS measurements. For example, tracking the peak widths of each analyte throughout the elution profile can reduce the error of the CCS determination after corrections are applied.

In some embodiments, corrections are made for the signal-to-noise ratio (SNR) of mass peaks (which depends on the number of ions (intensity) corresponding to the peak) to improve the accuracy of the measured signal decay times and hence CCS determinations. The disclosure also provides corrections for m/z dependency. The method thus uses previously determined dependencies of the decay constants on the properties (e.g. ion intensity (SNR), m/z) for the calibration. Embodiments of the present disclosure therefore allow for the extraction of CCS values directly from high-resolution mass-accurate spectra of a broad mass range of ions, preferably "full MS" spectra. Such methods are fully compatible with high dynamic range scan methods (scanning methods with a large dynamic range). In some embodiments, peak widths for each analyte can be tracked throughout the analyte's elution profile, and the final decay constant for the analyte of interest can be calculated as a weighted average of all points over retention time, with lower intensity components receiving significantly lower weights than higher intensity components . The decay constant can also be determined in this way with weighting from all identified isotope/charge states of the analyte.

The disclosure also provides improved hardware for performing such methods. Decay constants depend on the gas pressure in the mass analyzer. A practical and safe means of varying gas pressure is also provided in the form of gas leakage from a vacuum region of a mass spectrometer into the region where the mass analyzer is located. This allows for a simpler and fail-safe design of a gas leak that, even in the event of a failure or human error, will not lead to unacceptable pressure rises in FTMS.

Thus, in general, the disclosure provides methods for performing mass spectrometry in which decay constant, m/z, and calibration are performed on-line. Such procedures Methods may involve generating a stream of ions, for example a stream of ions generated by an ion source, and injecting a set of ions from that stream into an ion optical trapping device, such as a mass analyzer, where ions exhibit periodic motion limited by electromagnetic fields. The image current is detected from the periodic motion of the ions for a period of time during which at least a portion of the ions undergo collisions with gas molecules in the trap. Properties of the periodic motion of the ions, such as one or more of intensities, phases, frequencies (ie, m/z), decay constants, can be determined for multiple ion species simultaneously within the set of ions. Values of decay constants or CCS for each species can be determined based on previously calibrated dependencies on the properties of the ions. The previously calibrated dependencies may include one or more of the following: an intensity of an ionic species of interest; an intensity and/or frequency of an ionic species of interest; and/or an intensity and/or abundance distribution of all ion species including the ion species of interest. Compared to existing methods, an improved dynamic range, improved accuracy and precision of the CCS measurement can be achieved and the signal decay can be used for quality control of detected mass peaks.

These and other advantages will become apparent from the following description of preferred embodiments.

character list

• 1a und 1b die Beziehung zwischen der Abfallkonstante, der Anzahl von Ionen in einem Paket und der Elutionszeit zeigen;
• 2A ein Verfahren zum Bestimmen eines Maßes einer Abfallrate einer lonenprobe gemäß einer ersten Ausführungsform zeigt;
• 2B ein Verfahren zum Bestimmen eines Maßes einer Abfallrate einer lonenprobe gemäß einer zweiten Ausführungsform zeigt;
• 3 Daten zeigt, die zum Implementieren der ersten und der zweiten Ausführungsform geeignet sind; 4A die Auflösung gegenüber der Peak-Intensität für die Daten von 3 zeigt;
• 4B die Daten von 4A im Hinblick auf die Auflösung gegenüber dem Signal-Rausch-Verhältnis zeigt;
• 5 beobachtete Auflösungs- und SNR-Werte, mit einer Anpassung zeigt, die dazu verwendet werden kann, extrapolierte Auflösungen zu erhalten;
• 6 ein Massenspektrometriesystem gemäß einer dritten Ausführungsform zeigt;
• 7 den Effekt des Drucks auf die Abfallkonstanten zeigt;
• 8 den Effekt des Drucks auf die Auflösung zeigt; und
• 9 eine Zusammenfassung der Daten in 8 zeigt.

The present disclosure will now be described by way of example with reference to the accompanying figures, in which:

• 1a and 1b show the relationship between decay constant, number of ions in a packet and elution time;
• 2A Figure 12 shows a method for determining a measure of a decay rate of an ion sample according to a first embodiment;
• 2 B Figure 12 shows a method for determining a measure of a decay rate of an ion sample according to a second embodiment;
• 3 Figure 12 shows data suitable for implementing the first and second embodiments; 4A the resolution versus peak intensity for the data from 3 shows;
• 4B the data from 4A in terms of resolution versus signal to noise ratio;
• 5 shows observed resolution and SNR values, with an adjustment that can be used to obtain extrapolated resolutions;
• 6 Figure 12 shows a mass spectrometry system according to a third embodiment;
• 7 shows the effect of pressure on the decay constant;
• 8th shows the effect of pressure on resolution; and
• 9 a summary of the data in 8th shows.

Detailed description

Based on a theoretical and experimental investigation of the signal decay in FTMS (and in particular Orbitrap MS) it has been recognized that ions are subject to a number of space charge related effects. Most striking seems to be the so-called “self-bunching”, where the “natural” broadening of an ion packet is suppressed at high ion populations due to the combined effect of space charge and electric field nonlinearity, as in US-7,714,283 described. By manufacturing an orbitrap analyzer with high accuracy and by optimizing its tuning, these effects can in many cases be reduced to a negligible level or at least to a tolerable level. However, space charge effects (e.g. self bunching) can still be evident when CCS is calculated from decay constants, as in 1a illustrated. In FTMS, decay constants can be determined from mass peak widths in mass spectra obtained from transients, for example using Fourier transform with apodization and zero-filling, as is known in the art. General equations for determining decay constants from Fourier transform mass spectral data are presented in Section "Theory and methods" by Marshall et al., Phy s.Chem.Chem.Phys., 2016, 18, 713. Likewise, methods from Sanders JD et al. Anal.Chem., 2018; 90(9):5896-902 also be used. CCS estimation errors depend on parameters of detected mass peaks, mainly SNR, with lower intensity peaks experiencing stronger space charge and other defocusing effects.

Although the actual number of ions in a packet is difficult to ascertain, it is proportional to the SNR of a peak at a given transient duration and decay constant, where the coefficient of proportionality depends only on the thermal noise of the detector's preamplifier and its frequency dependence. For example, the relationship between SNR and ion count is discussed in Section 2.1 of Eiler et al. (Analysis of molecular isotopic structures at high precision and accuracy by Orbitrap mass spectrometry ( International Journal of Mass Spectrometry, Volume 422, 2017, Pages 126-142, ISSN 1387-3806 ), which is incorporated herein by reference. The Eiler et al. The relationships described for determining ion count may be used in embodiments of the present disclosure whenever signal intensities or SNR are described as being used.

1a illustrates the dependencies of the decay constant on the number of ions in a packet for different m/z ratios. The error band caused by statistics and underlying thermal noise of a preamplifier is shown as grayed out bands. The hatched band on the left of 1a illustrates the lower limit of detection. It can be seen that at low ion counts the measurements of the decay constant (and hence the collision cross-section) are systematically higher than the measurement of the same sample when packets with a higher number of ions are analyzed. It is sometimes desirable to measure the collision cross-section to characterize a molecule as this is an inherent property of a molecule. Therefore, the present disclosure seeks to provide accurate measurements of decay constants and/or collision cross-sections that 1a Consider the variations shown.

The dependencies of 1a can be obtained first using a calibration mixture of compounds (also known as calibrants) and then refined for a particular sample or experiment. While decay constants are strongly dependent on m/z and/or the charge state, the shape of the curves is in 1a depends mainly on the number of ions in a packet and to a lesser extent on m/z. However, second-order effects can also occur, especially when very intense peaks appear near the ions of interest. The FT data processing method (e.g. size, absorbance, enhanced FT (eFT), etc.) may also affect the reported values, as each method may have its own dependence of peak width on decay constant and intensity. However, such dependencies are not necessarily problematic when the same FT processing technique is used for calibration and subsequent analysis. As long as the same method is used for the calibrant and the sample, any FT method can be used with appropriate statistics.

The dependencies of 1 can be used for post-acquisition correction of decay constant measurements via an elution profile of each mass peak and charge state, as in 1b shown, can be used for each isotope of the analyte of interest. The black circles in 1b indicate time points at which measurements are taken on the elution profile of mass peaks. It can be seen that these times correspond to different decay constants on the elution profile. The assignment of isotopes and charge states could be done using algorithms similar to those presented in US-10,593,530 , which is incorporated herein by reference. The final decay constant for the analyte of interest can be calculated as a weighted average of all points over retention time and/or for all isotopes, with lower intensity components receiving higher corrections as well as significantly lower weights than high intensity components. It might be preferable not to include all isotopic peaks but only the most intense ones, e.g. B. More than 30% or 50% of the most intense peak in a cluster. It may also be possible to only use points whose peak intensity is greater than a certain percentage (e.g. 10%, 20%, 30%, 40%, etc.). Definitely shows 1b that corrections can be made to the measured fall times for a given point in time on an elution profile for any m/z ratio.

For example, considering the first elution peak for m/z1, the first decay constant measurement (i.e. the bottom black circle in 1b ) obtained while the number of ions in the packet is relatively small. This causes the decay constant from the left side of the graph to change from 1a is measured, thus causing the measurement to have a large error. Over time, the next two black circles (i.e., the second and third black circles from the bottom in 1b ), that measurements of the decay constant are made for a relatively large number of ions in the bunch, which means that the decay constant varies from the right-hand side of the plot of 1a is measured, and thus results in a measurement with a relatively small error. Then the fourth black circle from the bottom comes from the trailing edge of the retention peak and again leads to a measure of the decay constant from the left of 1a . A similar pattern is repeated for the elution of m/z2. Thus, different measurements taken at different points in an elution profile can be corrected and/or weighted when determining an average decay constant for a given sample. Preferably, different measurements made at different points in an elution profile are corrected and weighted: first data is corrected using the data from calibration measurements, and then a weighted average is obtained for better statistics.

Corrections can be made to measured decay constants or CCS values based on m/z. As in 1b As can be seen, different m/z values have different decay constant vs. ion number profiles. Corrections based on m/z may be implicit since m/z affects decay rate in an indirect way: higher m/z values tend to have higher CCS for a given state of charge. Also, for a given transient length, the resolution is lower for higher m/z due to the lower frequency oscillations. Thus, when measures of the decay rate (e.g., decay constant or CCS) of an ion sample are corrected by adjusting a measure of intensity (e.g., SNR), these two m/z dependent effects become intertwined. In principle, more sophisticated fixes could be developed using machine learning based on big data collected on large datasets. These datasets could cover specific chemical classes such as tryptic peptides, lipids, glycans, etc. and/or use the similarity of mass spectra.

Out of 1b it is clear that the error in the decay constant and hence the CCS error of peak m/z ₂ is significantly larger than that of m/z1 because there are much larger errors in determining the decay constant at lower intensities. In order to improve the SNR and thus reduce the CCS error, high dynamic range scans are preferably used, as in US-10,224,193-B2 and WO-2018-134,346-A1 disclosed, both of which are incorporated herein by reference in their entirety.

1a and 1b illustrate that there is a limit to the resolution in FTMS signals that is dictated solely by the vacuum conditions in the detector for an ion species. As the number of oscillating ions in a given packet increases, essentially all space/image charge-related paths of signal coherence loss tend to shrink, resulting in the observed resolution gradually increasing to a certain point (known as self-bunching). Above this point, detection signals for ion samples typically show an exponential decay pattern dominated by collision effects. Above this point, the only decay mechanism is the ion collisions with background gas molecules. This is in through the plateau on the right 1a and to the right of the top panel of 1b shown. Some embodiments of the present disclosure attempt to ensure that the resolution values used to determine decay constant or CCS originate from these "plateau" regions.

2A FIG. 12 shows a method, described in generalized language, for determining a measure of a decay rate of an ion sample undergoing mass analysis in a mass analyzer according to a first embodiment of the present disclosure. The method can be used to determine a corrected measure of a decay rate of an ion sample using the methods described in FIGS 1a and 1b shown data serve. The method comprises a first step 201 of receiving a detection signal for the ion sample from a detection of transients of the ion sample obtained using the mass analyser, the detection signal for the ion sample having a decay rate with time. Such a detection signal can be an image stream detected by the mass analyzer. In a second step 202, the method includes determining a fitting function for the ion sample based on (e.g., comprising dependence on): a measure of intensity (e.g., SNR or total ion count) and/or a measure of mass to-charge ratio (e.g., frequency) of the detection signal for the ion sample; and a measure of intensity (e.g., SNR or total ion count) and/or a measure of mass-to-charge ratio (e.g., frequency) of a detection signal previously obtained for a calibrant using the mass analyzer. The method comprises a third step 203 of determining the measure of the decay rate of the sample ion by adjusting a measure of the decay rate over time of the sample ion detection signal using the matching function tion. In this way, an accurate measure of the decay rate of the ion sample can be obtained since the intensity and/or mass-to-charge ratio of a signal from a calibrant (e.g., having a decay constant and/or cross-section that/ which is precisely known) can be used to determine a fitting function which is used to fit the measured values for the ion sample. Thus, the in 1a and 1b shown dependence of the decay constant or CCS are taken into account.

In an alternative aspect, a weighted average can be calculated.

For example, a method for determining a weighted measure of a decay rate of an ion sample undergoing mass analysis in a mass analyzer can be provided, the method comprising: receiving a plurality of detection signals for the ion sample from a detection of transients of the ion sample obtained using the mass analyzer wherein the ion sample detection signals have decay rates over time; determining a plurality of measures of the decay rate of the ion sample; determining the weighted decay rate measure (and optionally correcting those decay rate measures) of the ion sample by taking a weighted average of the multiple ion sample decay rate measures, wherein relatively high measures of intensity of the detection signal for the ion sample are weighted to to provide a greater contribution to the weighted average than relatively low measures of the intensity of the detection signal for the ion sample. The multiple detection signals for the ion probe can be obtained at multiple times across the elution profile of the ion probe. This is dem in 2A similar to the process shown, and the various steps described in this disclosure may equally apply to this alternative aspect. However, this aspect recognizes that more accurate measurements can be made by taking a weighted average of the multiple measures of the decay rate of the ion sample. Weighted averaging may completely exclude certain decay rate measures (e.g. because they have extremely poor resolution) and therefore it may not be necessary to correct these values in the weighted averaging calculation (because their contribution would be zero) .

The methods of the present disclosure may include determining a weighted measure of the decay rate of the ion sample by taking a weighted average of the multiple measures of the decay rate of the ion sample. For example, where a measure of the decay rate of the ion sample is described, this may be a weighted measure. This can ensure that ion sample decay rate measures that are statistically likely to be accurate are prioritized, thereby improving the accuracy with which decay constants and CCS can be determined.

In such methods, the multiple measures of the decay rate of the ion sample may be one or more relatively high measures of the intensity of the detection signal for the ion sample (e.g., values on the right-hand side of 1a and 1b ) and one or more relatively low measures of intensity of the detection signal for the ion sample (e.g. values on the left side of 1a and 1b ) are equivalent to; and the one or more relatively high measures of intensity of the ion sample detection signal are weighted to provide a greater contribution to the weighted average than the one or more relatively low measures of intensity of the ion sample detection signal. In this way, values taken with relatively low ion counts and therefore poor accuracy contribute less to the overall average. In some cases, the weighted average may exclude one or more measures of the decay rate of the ion sample for which the measure of the intensity of the ion sample detection signal is below a threshold. For example, it may be desirable to ignore any values taken at particularly low ion counts when such values are known to produce erroneous results.

In the aspects described herein, and still using the generalized language previously used, the measure of the decay rate of the ion sample may be a decay constant or a collision cross-section. Since the CCS can be determined from a decay constant (and vice versa), any measure of the decay rate of an ion sample can be used in the algorithms of this disclosure.

As previously mentioned, it can be difficult to determine the actual number of ions in a packet, but it is proportional to the SNR of a peak at a given transient duration and decay constant. Thus, in the generalized language previously used, determining a fitting function (e.g., scaling factors) for the ion sample may be based on a ratio between the detection signal intensity measure for the ion sample and the detection signal intensity measure previously obtained for the calibrant. For example, under certain conditions, the curves in 1a and 1b have essentially the same shape but are roughly related by a constant of proportionality. Therefore, a measurement made on an ion sample can be corrected by utilizing this relationship. By establishing a relationship between two measurements, the need to consider various complex factors is reduced.

As a hypothetical example, consider a measurement made on an ion sample from a pack of, say, 100 ions (or equivalent SNR). Such a sample may have a data point toward the left of 1a and 1b (ie, near the lower limit of detection) so any decay constant or CCS calculation could be overstated due to space charge or other effects. However, from an experiment with the calibrant (which would of course have known composition, known decay constant and known CCS) it was known that an ion packet containing 100 ions of the calibrant resulted in a 10% overestimated decay constant. Thus, a decay constant for the ion sample can be determined using the data point from the packet with only 100 ions, and its accuracy can be increased by reducing the calculated decay constant by 10%. It is understood that a similar methodology can be implemented for CCS calculations. Therefore, in general terms, the disclosure provides ion sample adjustment functions that specify an amount by which the decay rate of the ion sample detection signal must be adjusted to compensate for errors in measuring the decay rate of the ion sample detection signal. The fitting functions may define an offset (e.g., an offset that can be added to or subtracted from a measured value) or a scaling factor that provides an indication of percent error. In such a case, adjusting the decay rate measure of the ion sample detection signal using the adjusting function may include scaling the decay rate measure of the ion sample detection signal by the scaling factor. The fitting functions described herein could be any program that receives a mass spectral signal and outputs a corrected CCS and/or decay constant by comparing the mass spectral data to the mass spectral data of a calibrant. Accordingly, a more accurate measure of the decay rate of the ion sample can be obtained.

definiert werden, deren Ausgabe eine korrigierte Abfallkonstante ist. Diese Funktion T_korr nimmt Eingänge von: einer unkorrigierten Abfallkonstante τ₀, die aus Massenspektraldaten bestimmt wird; einem Signal-Rausch-Verhältnis SNR von Massenspektraldaten; und einem Masse-zu-Ladung-Verhältnis m/z, das durch Massenspektraldaten angegeben wird. Sie könnte auch einen Satz anderer Eingänge X verwenden, wie etwa Gesamt-SNR für das gesamte Spektrum, Eigenschaften der nächsten oder intensivsten Massenpeaks usw. Daher kann X als optionale Variable angesehen werden, die weggelassen werden könnte.As an example, a multivariable function $\tau i_{{}_{kO\mathrm{right}\mathrm{right}}}={\tau }_{kO\mathrm{right}\mathrm{right}}\left({\mathrm{<figure-callout\; id="0"\; label="\tau "\; filenames="DE102022128278A1\_0019.png,DE102022128278A1\_0020.png"\; state="\{\{state\}\}">\tau </figure-callout>}}_0;SNR;\frac{m}{\mathrm{e.g}},X\right)$

can be defined, the output of which is a corrected decay constant. This function T _corr takes inputs from: an uncorrected decay constant τ ₀ determined from mass spectral data; a signal-to-noise ratio SNR of mass spectral data; and a mass-to-charge ratio m/z indicated by mass spectral data. It could also use a set of other inputs X, such as overall SNR for the entire spectrum, properties of the nearest or most intense mass peaks, etc. Therefore, X can be viewed as an optional variable that could be omitted.

The form of the function T _corr can be determined for a calibrant of known composition. Since the calibrant has a known composition with various known constituent species, a range of correct m/z values for the calibrant is known. In addition, a set of correct decay constants (ie the correct outputs of the function T _corr ) will be known for a good calibrant. Thus, the form of the function T _corr can be determined for a particular experimental setup using data obtained from the calibration means by solving an optimization problem. Then, when embodiments of the present disclosure perform a step of determining a fitting function for an ion sample, this may mean invoking the function T _corr as determined using the calibrant.

It is understood that other inputs to T _corr could be used. For example, the function T _corr could input the number of ions in a packet instead of SNR, and could input the frequency of the mass analyzer detection signal instead of m/z. These are simply coordinate transformations. In addition, some of the inputs to T _corr could be omitted. For example, if a full MS scan is desired, it may be preferable to apply both SNR and m/z (or frequency) based corrections, but for isolated scans corrections based solely on SNR (or some other measure of intensity) be sufficient. For an m/z-based correction, a calibrant mixture could be used that includes multiple calibrant ions with different m/z (preferably over a wide range) such that the relationships of 1 can be obtained for each m/z ion and then the derived fit values at each ion number can also vary with m/z so that a separate curve of fit value versus m/z at each ion number can be obtained and used for correction. Ideally should go to calibration a mixture of as many calibration tools as possible should be used. For example it could be our FlexMix that we use for instrument calibration, or alternatively it could be a mixture of peptides produced by digestion of bovine serum albumin (a standard quality control sample in proteomics) etc. Then we could choose between calibrated peaks e.g. B. interpolate with splines.

$${\mathrm{\tau }}_{Skalierung}\equiv {\tau }_{ikorr}\left({\mathrm{\tau }}_0;SNR;m/z\right)-{\tau }_0$$

In any event, the function T _corr , as defined above, provides an output that is a corrected decay constant. Of course, alternative functions could be defined and fall within the scope of the present disclosure, such as: $${\mathrm{\tau }}_{Offset}\equiv {\tau }_{ikO\mathrm{right}\mathrm{right}}\left({\mathrm{<figure-callout\; id="0"\; label="\tau "\; filenames="DE102022128278A1\_0019.png,DE102022128278A1\_0020.png"\; state="\{\{state\}\}">\tau </figure-callout>}}_0;SNR;m/\mathrm{e.g}\right)-{\mathrm{<figure-callout\; id="0"\; label="\tau "\; filenames="DE102022128278A1\_0019.png,DE102022128278A1\_0020.png"\; state="\{\{state\}\}">\tau </figure-callout>}}_0$$

$${\mathrm{\tau }}_{Skalie\mathrm{right}\mathrm{and}nG}\equiv {\tau }_{ikO\mathrm{right}\mathrm{right}}\left({\mathrm{<figure-callout\; id="0"\; label="\tau "\; filenames="DE102022128278A1\_0019.png,DE102022128278A1\_0020.png"\; state="\{\{state\}\}">\tau </figure-callout>}}_0;SNR;m/\mathrm{e.g}\right)-{\mathrm{<figure-callout\; id="0"\; label="\tau "\; filenames="DE102022128278A1\_0019.png,DE102022128278A1\_0020.png"\; state="\{\{state\}\}">\tau </figure-callout>}}_0$$

The outputs of T _offset would be an amount that would need to be added to an uncorrected decay constant to provide a corrected value. Likewise, the outputs of T _scale would be a scaling factor by which an uncorrected decay constant would need to be scaled to provide a corrected value. It is also understood that the function T _corr could be replaced with an equivalent function σ _corr that receives the same inputs and provides correct CCS values (or offsets/scale factors) as its outputs.

Another way in which accurate decay constants or CCS values can be calculated is described below. A curve like the curve in 1 can be obtained for a calibrant (ie the measure of the decay constant or CCS is found for a range of ion counts, S/N or intensities, etc.). Interpolation between points can be used, or a fitting function can be used to approximate the curve. From this curve, fit values for any ion count, S/N or intensity (based on deviation from the 'known' decay constant) can then be determined. Finally, the decay constant or CCS of the "ion sample" can be corrected using the fit value (from the calibration curve) that corresponds to the same ion number, S/N or intensity of the ion sample.

Throughout the present disclosure, the measure of intensity of the detection signal for the ion sample may include one or more of: total ion count; signal amplitude; and/or signal-to-noise ratio. In addition, the measure of intensity of the detection signal previously obtained for the calibrant may include one or more of the following: total ion count; signal amplitude; and/or signal to noise ratio. The measure of the mass-to-charge ratio of the detection signal for the ion sample may include a frequency (or, equivalently, a duration of the signal); and/or the measure of the mass-to-charge ratio of the detection signal previously obtained for the calibration means may comprise a frequency (or equivalently a duration of the signal).

Referring now to 2 B there is shown a method according to a second embodiment. The procedure of 2 B is similar to the method of Figure 2A in some respects. The procedure of 2 B is a method for determining a measure of a decay rate of an ion sample undergoing mass analysis in a mass analyzer with image current detection. The method comprises a first step 211 of receiving a detection signal for the ion sample from a detection of transients of the ion sample obtained using the mass analyzer, the detection signal for the ion sample having a decay rate with time. This can be essentially the same as step 201 of 2A be. The method may further comprise an optional second step 212 of determining whether the resolution of the ion sample detection signal indicates that the decay rate of the ion sample detection signal is dominated by collision effects (e.g. self bunching). This can be obtained, for example, by considering whether the resolution of the mass spectral detection signal is in the stable plateau of 1A and 1B lies. Whether or not self-bunching occurs does not necessarily matter, but whether self-bunching occurs affects which corrections are used. Thus, step 212 can be omitted, or step 212 can be used for quality control instead. For example, step 212 may include determining one or more of the following: whether a peak is baseline resolved (ie, whether both sides of a peak reach the baseline without interfering with other peaks); is the SNR high enough to even consider the peak; does the peak belong to an isotopic cluster; and then whether or not the peak is included in a weighted average.

Finally, the method includes a third step 213 for determining the measure of the decay rate of the ion sample. When the resolution of the ion sample detection signal indicates that the decay rate of the ion sample detection signal is dominated by collision effects, the determination of the measure of the ion sample detection signal is based on the decay rate of the ion sample detection signal. For example, if the data is from the plateau of stable values, no decay constant or CCS measurement correction may be required, and the decay constant of the signal can be used to calculate the CCS or decay constant of the ion sample. On the other hand, if the SNR of the ion sample detection signal indicates that the decay rate of the ion sample detection signal over time is not dominated by collision effects, determining the measure of the ion sample decay rate is based on an extrapolated resolution for the ion sample. The extrapolated resolution is an expected resolution of the detection signal under conditions where the decay rate of the detection signal for the ion sample is dominated by collision effects. Thus, if the value from the left side of 1A , 1B and 3(III) is taken, then the resolution can be determined by extrapolating to a region where conditions are collision-dominated and therefore more accurate. Thus, the disclosure provides a method that can automatically improve the accuracy of measurements of decay constants and/or CCS.

In the present disclosure, by “dominated” it is meant that the rate of decay of the detection signal can be fitted to a first-order exponential decay curve to a degree that exceeds a minimum accuracy threshold. This minimum threshold may be, for example, user set or factory set/defined (e.g., the minimum threshold could be programmed into the hardware during manufacture) and represents a point at which the rate of decay of the transients from exponential to an acceptable level transitions to the point where it is no longer exponential to an acceptable extent such that the matching methods described herein will not work. It is understood in this regard that there is no well-defined cut-off point in the transition from exponential to non-exponential decay. In some cases, a plot of resolution versus SNR may show an inflection point (e.g. where resolution begins to plateau after a constant increase), and this inflection point could also be viewed as the threshold above which conditions are said to be collision-dominated become. Alternatively, conditions can be said to be “collision-dominated” when the resolution (or other equivalent measurement) reaches a certain percentage (e.g. 80%, 90%, 95% or 99%) of its value in the stable plateau region.

3 shows an LC elution profile (I) of an isotopic shell with charge state Z = 3+ of a compound with a monoisotopic weight of 2200.09 Da (II). The distribution of the resolution values of their isotopes over the elution profile is plotted as a function of their signal-to-noise ratio as a log ₁₀ scale (III). Generally speaking, the multiple ion sample decay rate measures can be determined for multiple retention times of an elution profile. Such data can be used to ensure that measurements are made in the most informatively reliable regions (ie preferably near or within the self-bundling region). Although it is desirable to have high-intensity peaks that lie in the self-focusing region, there is usually a log-normal distribution of intensities in mass spectra, so most peaks are of lesser frequency. In an LC (or GC) experiment, some eluting samples have a very low abundance that it may not be possible to obtain measurements from the stable plateau.

An LC profile or, in the case of 3(I) , Selected Ion Monitoring (SIM) are used. Therefore, an initial step may be identifying the value of the decay parameter at the high ion count "plateau" level (e.g., the stable region in 1b ) include. Since resolution and decay are closely related (i.e. proportional in the case of a pure collision regime), the strategies for determining the pure collision component of the decay constant are identical when the decay parameter is explicitly known or resolution is used as an intermediate step.

For CCS analysis it is not necessary to limit the analysis to a single m/z species per compound. Since all isotopes within a given cluster are exactly the same size (and differ only in their mass), it can be advantageous to use the resolution data over the entire isotopic envelope as well. Therefore, the term "ionic species" can be generalized to refer to all isotopes for a given charge state of a given compound. 3(II) shows seven peaks 0.33 m/z apart, indicating isotopic variants of a triply charged ion. In general terms, therefore, in the methods described herein, the multiple measures for the Decay rate of the ion sample can be determined for several different isotopic compositions. Thus, the weighted averages described herein may be based on decay rate measures for multiple isotopic variants of the ion sample. This can allow analysis of multiple isotopic variants of a single analyte, which can improve error statistics.

Because higher intensity peaks tend to provide more accurate estimates of the resolution value, when calculating decay constants it may be advantageous to only use values that are near or at the apex of the SIM peak crest (ie, near the plateau). However, this is not necessary in all embodiments. Such as referring to 5 As described in more detail, some embodiments of the present disclosure determine decay constants using the following method. First, a record, such as that of 3(III) , receive. A line of best fit is then determined to try to relate SNR to resolution. In 3(III) this line of best fit would plateau at a constant resolution of about R=63000 above SNR values of about 50. If a measure of the decay constant or CCS is then determined for a species with an SNR of, say, 10 and a corresponding R=55000, it would be apparent that that particular species is not experiencing self-bundling since R=55000 is significantly below the plateau at where self-bundling occurs. Therefore, this species would be expected to have a large error in the decay constant and CCS since the decay constant and CCS can be calculated directly from R and this measured value of R = 55000 indicates suboptimal experimental conditions. This large error in the decay constant and CCS could be corrected by using a value of R=63000 instead of R=55000 when determining the decay constant or CCS. This is because if it had been possible to get a higher SNR (e.g. by using a large amount of analyte and thus more ions in the packet) the resolution would have been R=63000. Such a line of best fit can include all points in 3(III) take into account.

wobei R _m/z der gewichtete Wert für die Auflösung ist, R_i die gemeldeten Auflösungswerte jedes einzelnen Peaks sind, C; die Korrektur dieses Werts unter Verwendung von Kalibrierungen aus 1a, 1b, 3(III) (C_i≥1) ist, I_i das Intensitätsmaß eines einzelnen Peaks ist, und W(I_i) die Gewichtung ist, die dem Auflösungswert eines Peaks in Abhängigkeit von seiner Intensität zugewiesen wird. Gewichtete Durchschnittswerte können dazu verwendet werden, gewichtete Auflösungswerte für verschiedene SNR-Bereiche zu bestimmen. Beispielsweise könnte ein gleitender Durchschnitt der Daten in 3(III) genommen werden, um eine Trendlinie zu erhalten.Alternatively, to improve statistics, one can use weighted averages of resolution for all observed peaks for a particular ion species, as in: $${\tilde{R}}_{m/\mathrm{e.g}}=\raisebox{1ex}{${\displaystyle \sum {}_i\left(W\left(I_i\right)R_i{C}_i\right)}$}\!\left/ \!\raisebox{-1ex}{${\displaystyle \sum {}_{i}W\left(I_i\right)}$}\right.$$

whereby R _m/z is the weighted resolution value, R _i are the reported resolution values of each individual peak, C; correction of this value using calibrations 1a , 1b , 3(III) (C _i ≥1), I _i is the intensity measure of a single peak, and W(I _i ) is the weight assigned to a peak's resolution value depending on its intensity. Weighted averages can be used to determine weighted resolution values for different SNR ranges. For example, a moving average of the data in 3(III) be taken to obtain a trend line.

wobei p eine Konstante sein kann). Andere Gewichtungsstrategien sind ebenfalls möglich. In jedem Fall kann eine genauere grafische Darstellung (die zur Korrektur von Abfallkonstanten und CCS verwendet werden kann) von SNR (oder einem gleichwertigen Maß) gegenüber der Auflösung erhalten werden, indem Peaks von niedrigerer Intensität niedrigere Gewichtungen zugewiesen werden, um zu verhindern, dass Peaks mit großen Fehlern die ermittelte Trendlinie verzerren.Alternatively, could R _m/z be a weighted resolution weighted only over peaks that are in the self-focusing region (ie excluding peaks whose intensities are not sufficiently high for self-focusing). The intensity measure can be in au, such as an absolute value of the apex of the peak in magnitude or in the absorption FT spectrum, SNR, etc. The weighting function can use fixed values for each intensity value (e.g. W(I _i ) = 1 as on average) can be linear with respect to the weights (e.g. W(I _i ) = const * I _i or could be the non-linearity of the curves in 1b with more complex functionality (e.g. $W\left(I_i\right)=kOnst*I_i^P,$

where p can be a constant). Other weighting strategies are also possible. In any case, a more accurate plot (which can be used to correct decay constants and CCS) of SNR (or equivalent measure) versus resolution can be obtained by assigning lower weights to peaks of lower intensity to prevent peaks distort the determined trend line with large errors.

wobei Y_Plateau, der gesuchte Wert des Plateaus ist und die Koeffizienten K und y die Anstiegsrate der Funktion vor Erreichen von Y_Plateau beschreiben. Es ist wichtig anzumerken, dass eine derartige Funktion nicht glatt sein muss. Für die Zwecke der Offenbarung könnte eine derartige Funktion eine stückweise Funktion sein, z. B. $$Y=\{\begin{array}{cc}Y=a\ast x^2+b\ast x+c& forx<x_{kritischl}\left(Y_{Plateau}\right)\\ Y=Y_{Plateau}& forx>x_{kritisch}\end{array},$$

wobei die Koeffizienten a, b, c einen parabelförmigen Anstieg beschreiben, bevor sie an einem Punkt x_kritisch ein Plateau erreichen. Die Funktion könnte auch eine stückweise lineare Funktion sein, die sich einer glatten Funktion annähert. Diese Funktionen sind lediglich Beispiele, und Ausführungsformen dieser Offenbarung können andere Funktionen verwenden.It is also possible to find parameters that describe the curves accurately enough to allow reliable extraction of the resolution values. For this strategy to work, a model describing the phenomenon is required. The preferred choice for such a model is a function that plateaus on the y-axis (here representing the decay constant or resolution). The most preferred example of a smooth function with such a property is an exponential plateau: $$Y=Y_{Platea\mathrm{and}}-K\ast e^{-g\ast x}$$

where Y _plateau is the desired value of the plateau and the coefficients K and y describe the rate of increase of the function before reaching Y _plateau . It is important to note that such a function need not be smooth. For the purposes of the disclosure, such a function could be a piecewise function, e.g. B. $$Y=\{\begin{array}{cc}Y=a\ast x^2+b\ast x+c& fO\mathrm{right}x<x_{k\mathrm{right}itiscHl}\left(Y_{Platea\mathrm{and}}\right)\\ Y=Y_{Platea\mathrm{and}}& fO\mathrm{right}x>x_{k\mathrm{right}itiscH}\end{array},$$

where the coefficients a, b, c describe a parabolic slope before reaching a _critical plateau at a point x. The function could also be a piecewise linear function that approximates a smooth function. These functions are just examples, and embodiments of this disclosure may use other functions.

wobei β ein Satz gesuchter Parameter ist, y(x_i,β) ein Wert der Modellfunktion an einem Punkt x_i,ŷ_i ist - eine Beobachtung an einem Punkt x_i, und w_i ein ŷ_i zugeordnetes Gewicht ist. Die Gewichtungen können abhängig von der Intensität oder einigen statistischen Eigenschaften sein (z. B. lokal oder Streuung). Es kann auch von Vorteil sein, die Daten vor der Anpassung vorzuverarbeiten, indem Ausreißer entfernt oder mit einem Filter geglättet werden. Gute Beispiele für eine derartige Vorverarbeitung wären die Anwendung verschiedener Varianten (z. B. exponentiell modifizierte) der Filterung des gleitenden Durchschnitts mit variierender Periode je nach der lokalen statistischen Landschaft der Daten.The parameters for these functions can be obtained in a number of ways, including minimizing the discrepancy between observed values and the values predicted by the function. An optimization problem can be specified in terms of minimizing a norm of this discrepancy, e.g. in Lp space (at p=2 this optimization method is known as least squares). It is worth noting that it might be advantageous to consider other peculiarities of the data in the optimization procedure (e.g. the spread of values tends to be larger for lower intensity peaks, as in 3(III) shown). For example, one can consider introducing weights as in weighted least squares: $$\underset{\beta }{\text{argmin}}{\displaystyle \sum {}_i{w}_i{\left|{\widehat{y}}_i-y\left(x_i,\beta \right)\right|}^2}$$

where β is a set of parameters sought, y(x _i ,β) is a value of the model function at a point x _i ,ŷ _i - an observation at a point x _i , and w _i is a weight associated with ŷ _i . The weights can depend on the intensity or some statistical property (e.g. local or scatter). It can also be beneficial to preprocess the data before fitting, by removing outliers or smoothing them with a filter. Good examples of such pre-processing would be the application of different variants (e.g. exponentially modified) of the moving average filtering with varying period depending on the local statistical landscape of the data.

wobei die Intensität a eines Oszillierens mit der Frequenz wo Abnahmen gegenüber seinem Anfangswert von a_o mit der Zeit t signalisiert, wobei die Rate t dem exponentiellen Trend folgt. Nach der Fourier-Transformation weist das Spektrum der Absorptionsmoden einen Lorentz-Peak $$A\left(\omega \right)=\frac{A_0}{1+{\left(\frac{\left(\omega -{\omega }_0\right)}{2\tau }\right)}^{2_1}}$$

mit der Amplitude A_o auf, die bei ω₀ liegt und die Halbwertsbreite (FWHM) von 2T aufweist.Regardless of the way resolution values are extrapolated, a relationship between resolution and decay constant is required to express the in 1A , 1B and 3(III) to be able to use the plateau shown. The collision-induced decay in an FTMS generally follows an exponential law, ie $$a\left(t\right)=a_0\text{cos}\left({\omega }_{0}t\right)\ast e^{-\tau t},$$

where the intensity a of an oscillation with frequency wo signals decreases from its initial value of a _o with time t, the rate t following the exponential trend. After the Fourier transformation, the spectrum of the absorption modes has a Lorentz peak $$A\left(\omega \right)=\frac{A_0}{1+{\left(\frac{\left(\omega -{\omega }_0\right)}{2\tau }\right)}^{2_1}}$$

with the amplitude A _o which is at ω ₀ and has the full width at half maximum (FWHM) of 2T.

Since Equation (VI) is the result of the Fourier transform result of Equation (V) for a signal of infinite length, the peak width (which can be used to determine CCS) of 2 _T can be a good approximation for fast decaying be signals. In other cases it can be advantageous to consider the finite duration of the transient FTMS signals.

wobei T die Dauer des transienten Signals (das aus den Geräteeinstellungen bekannt ist) ist und Δω' die beobachtete spektrale FWHM (d. h. die FWHM des Peaks, der dazu verwendet wird, die Abfallkonstanten zu bestimmen) ist. Die Gleichung (VII) kann nummerisch aufgelöst werden, um eine genauere Beziehung zwischen der Peakbreite (d. h. FWHM im Frequenzbereich) und der Abfallkonstanten zu bestimmen als die vereinfachte Annäherung, die die FWHM 2T für einen Lorenzschen Peak ist.A way of correction was proposed by Li et al. (Dayu Li, Yang Tang and Wei Xu, Analyst, 2016, 141.3554) using the following expression: $$\frac{e^{-T/\tau }\tau \left(e^{T/\tau }-\text{cos}\left(T\Delta \omega \text{'}\right)+\tau \Delta \omega \text{'}\text{sin}\left(T\Delta \omega \text{'}\right)\right)}{1+{\left(\tau \Delta \omega \text{'}\right)}^2}=\frac{e^{-T/{\tau }_{\tau }}\left(e^{-T/\tau }-1\right)}{2},$$

where T is the duration of the transient signal (which is known from the instrument settings) and Δω' is the observed spectral FWHM (ie the FWHM of the peak used to determine the decay constant). Equation (VII) can be solved numerically to determine a more accurate relationship between peak width (ie, FWHM in the frequency domain) and roll-off constant than the simplified approximation, which is the FWHM 2T for a Lorenzian peak.

One way to determine whether the correction in Equation (VII) is beneficial is to compare Δω' to the limitation imposed on the resolution by length of a time-domain signal. Each FTMS transient can be expressed as the multiplication of an oscillating signal of infinite length by a square function of duration T, which corresponds in the frequency domain to the convolution of an FT image of a signal by that of a window function, ie $$e^{-T\omega }sinc\left(\frac{\omega }{T}\right).$$

Therefore, under the conditions that the FWHM of equation (VIII) is comparable to Δω', it may be advantageous to use correction equation (VII), whereas when Δω' is much larger, the advantages may not be as pronounced, particularly with noisy signals. In the generalized terms used above, the step of determining a measure of the decay rate over time of the detection signal for the ion sample is based on a peak width of the detection signal for the ion sample (e.g., a width of a peak in the frequency domain Δω that can be used to determine a decay constant ). It is preferable that the step of determining the measure of the rate of decay of the detection signal for the ion sample takes duration into account (e.g. includes some correction for errors introduced by the use of a discrete Fourier transform on a finite-wavelength signal). Duration are caused) during which the detection of transients of the ion sample takes place. For example, a correction of the form of equation (VII) can be useful and advantageous.

CL, wobei Δm die FWHM eines Peaks (im Massenbereich) in der Position m ist. In 4A wird die Intensität in willkürlichen Einheiten (AU) angegeben, könnte aber auch als Signal-Rausch-Verhältnis (SNR) oder in anderen Einheiten dargestellt werden. Die angegebenen Werte stammen von einer lonenspezies bei 739,03 Th und dem Ladungszustand Z = 3+. Wenn die Anzahl der Ionen zunimmt, nimmt auch die Auflösung zu, bis sie ein Plateau erreicht, auf dem die Ionen zur „Selbstbündelung“ neigen. An diesem Punkt endet das „Dephasieren“ der lonenwolke und die einzige Quelle für den Abfall ist der Tod einzelner Ionen infolge von Kollisionen mit Hintergrundgasmolekülen. Die Daten von 4A sind die Ergebnisse von Orbitrap-FTMS-Versuchen, die mit demselben System unter verschiedenen Druckbedingungen durchgeführt wurden: (I) niedriger Druck, bei ausgeschaltetem HCD-Gasventil; und (II) hoher Druck, Gas eingeschaltet. In 4B ist effektiv dieselbe grafische Darstellung wie in 4A gezeigt. In 4B ist die Auflösung gegen logio(SNR) bei Aus- und Einschalten des Gases aufgetragen. Das SNR ist ein bevorzugtes Maß zum Quantifizieren der Anzahl von Ionen in einem Paket, und die logarithmische Darstellung von 4B dient dazu, das Auflösungsplateau mit zunehmender lonenzahl deutlicher zu veranschaulichen. 4A provides experimental data showing the effect of self-bundling. In 4A the horizontal axis is the total ion count of a peak detected from oscillating ion clouds. The Y-axis are the observed values of the resolution of selected peaks. The resolution values are defined as $\frac{m}{\Delta m},$

CL, where Δm is the FWHM of a peak (in the mass domain) at position m. In 4A Intensity is given in arbitrary units (AU), but could also be presented as signal-to-noise ratio (SNR) or other units. The values given are from an ionic species at 739.03 Th and the charge state Z = 3+. As the number of ions increases, so does the resolution, until it reaches a plateau where the ions tend to “self bunch”. At this point the ion sheath stops "dephasing" and the only source of decay is the death of individual ions due to collisions with background gas molecules. The data from 4A are the results of Orbitrap FTMS tests performed on the same system under different pressure conditions: (I) low pressure, with HCD gas valve off; and (II) high pressure, gas on. In 4B is effectively the same graphical representation as in 4A shown. In 4B the resolution is plotted against logio(SNR) when the gas is switched off and on. The SNR is a preferred measure for quantifying the number of ions in a packet, and the logarithmic representation of 4B serves to more clearly illustrate the dissolution plateau with increasing ion number.

5 provides an example of how data such as the in 4A and 4B data shown, can be used numerically to obtain improved CCS estimates using embodiments of this disclosure. All data in the example from 5 were collected on a commercial Orbitrap Exploris™ mass spectrometer. The resolution was set to 120k, which corresponds to a transient length of T = 256 ms. Equation (IX) provides the mass calibration parameters B and C in a mass versus frequency (kHz) dependence relationship: $$\raisebox{1ex}{$m$}\!\left/ \!\raisebox{-1ex}{$\mathrm{e.g}$}\right.=\frac{\mathrm{<figure-callout\; id="2"\; label="B\; ƒ"\; filenames="DE102022128278A1\_0022.png,DE102022128278A1\_0023.png"\; state="\{\{state\}\}">B</figure-callout>}}{{ƒ}^{}}+\frac{\mathrm{<figure-callout\; id="4"\; label="C\; ƒ"\; filenames="DE102022128278A1\_0023.png"\; state="\{\{state\}\}">C</figure-callout>}}{{ƒ}^{}}$$

In the example of 5 the values were B = 169449512.441 and C = 72890453.4333. As previously described, the first step in evaluating the CCS is to determine only the decay caused by the collisions with the residual gas. One approach is to assess the resolution at which self-bundling is initiated. 5 shows the fit of an exponential plateau function as in Equation (III) to the observed distribution of resolutions 3(III) by minimizing the L ₂ --norm.

definiert ist, kann Δω' oder Δf (die FWHM der Frequenz) unter Verwendung der Gleichung (IX) erhalten werden. Insbesondere kann die Gleichung (IX) dazu verwendet werden, nachzuweisen, dass bei hohen Frequenzen, wobei die Gleichung (IX) durch B/f² dominiert ist, den Term C/f⁴ dominiert, Δω' proportional ist zu: der Frequenz (in diesem Fall 478,840 kHz); und der Auflösung auf dem Plateau, die 63070,3159 beträgt. In diesem speziellen Fall, Δf = 3,7961 Hz, was direkt gemessen werden kann: zur Frequenz FWHM aus der Auflösung im Massenbereich können Werte für Frequenzen beim halben Maximum des Peaks berechnet werden, d. h. für die Auflösung von 63070,32 @ 739,0237 Da beträgt das Δm/z beim halben Maximum 0,011717 und die Massen selbst betragen 739,0237 - 0,011717/2 = 739,0178 und 739,0237 + 0,011717/2 = 739,0296, wobei die entsprechenden Frequenzen (die sich aus der Gleichung (IX) ergeben) 478838,7 und 478842,5 Hz betragen, was Δf= 3,796085 ergibt. Da die Massenauflösung nahe an der Grenze liegt, die durch die Dauer der Transienten vorgegeben ist (in diesem Fall sind es ~80k im Massenbereich, was Δf = 2,9907Hz) entspricht, können wir nicht einfach 2τ = Δω' verwenden, um t zu schätzen, sondern sollten stattdessen die Gleichung (VII) für eine genauere Bestimmung verwenden. Das nummerische Auflösen der Gleichung (VII) ergibt einen Wert für den erhaltenen Abfallparameter von τ = 0,3798s^-1.The resolution plateau is at Y _Plateau = 63070.3159 and is reached at SNR values of ~50. Since the resolution in the mass domain as $\frac{m/{\mathrm{e.g}}_{\mathrm{e.g}ent\mathrm{right}e}}{\Delta {\text{m/z}}_{fWHM}}$

is defined, Δω' or Δf (the FWHM of frequency) can be obtained using equation (IX). In particular, equation (IX) can be used to prove that at high frequencies, where equation (IX) is dominated by B/f ² , the term C/f ⁴ dominates, Δω' is proportional to: the frequency (in in this case 478.840 kHz); and the resolution on the plateau, which is 63070.3159. In this particular case, Δf = 3.7961 Hz, which can be measured directly: at the frequency FWHM from the resolution in the mass domain, values can be calculated for frequencies at half maximum of the peak, ie for the resolution of 63070.32 @ 739.0237 Since the Δm/z at half maximum is 0.011717 and the masses themselves are 739.0237 - 0.011717/2 = 739.0178 and 739.0237 + 0.011717/2 = 739.0296, the corresponding frequencies ( resulting from equation (IX)) are 478838.7 and 478842.5 Hz, giving Δf= 3.796085. Since the mass resolution is close to the limit dictated by the duration of the transients (in this case it is ~80k in the mass domain, which corresponds to Δf = 2.9907Hz), we cannot simply use 2τ = Δω' to get t estimate, but should instead use Equation (VII) for a more accurate determination. Solving equation (VII) numerically gives a value for the obtained decay parameter of τ = 0.3798s ^-1 .

wobei P der Druck im Inneren des Orbitrap-Analysators ist, L die Länge einer lonenflugbahn pro Schwingung entlang der Z-Achse (hier 65,1 mm) ist, f die axiale Frequenz der Ionen (hier 478,840 kHz) ist, Tdie Temperatur (295K) ist und k die Boltzmann-Konstante ist. Für diese lonenspezies ist der resultierende Wert für CCS 424 Ų, was ein vernünftiger Querschnitt für ein dreifach geladenes Peptid in diesem Massenbereich ist. Somit ist ersichtlich, dass die Verfahren der vorliegenden Offenbarung dazu verwendet werden können, genaue CCS-Werte zu bestimmen. Insbesondere zeigt dieses Beispiel, wie das Bestimmen des Maßes für die Abfallrate der lonenprobe auf einer extrapolierten Auflösung für die lonenprobe basieren kann, wobei die extrapolierte Auflösung eine erwartete Auflösung des Detektionssignals unter Bedingungen ist, bei denen die zeitliche Abfallrate des Detektionssignals für die lonenprobe von Kollisionseffekten dominiert wird.Once a decay value has been obtained, the determination of CCS can be made as in Sanders JD et al. (Sanders JD et al. Anal. Chem., 2018; 90(9):5896-902 ) described. Based on the fact that the pressure displayed on the UHV manometer (for the data presented here it is 5.85 × ^10-10 mbar) is about 2 times lower than the residual vacuum in the Orbitrap analyzer, the cross-section a be obtained as $$\sigma =\raisebox{1ex}{$\tau $}\!\left/ \!\raisebox{-1ex}{$\frac{PLƒ}{kT}$}\right.$$

where P is the pressure inside the orbitrap analyzer, L is the length of an ion trajectory per oscillation along the Z axis (here 65.1 mm), f is the axial frequency of the ions (here 478.840 kHz), T is the temperature (295K ) and k is the Boltzmann constant. For this ionic species, the resulting value for CCS is 424 Å ² , which is a reasonable cross section for a triply charged peptide in this mass range. Thus, it can be seen that the methods of the present disclosure can be used to determine accurate CCS values. In particular, this example shows how determining the measure of the ion sample decay rate can be based on an extrapolated resolution for the ion sample, where the extrapolated resolution is an expected resolution of the detection signal under conditions where the decay rate of the detection signal for the ion sample over time is affected by collision effects is dominated.

Returning to the generalization used previously, in embodiments of the disclosure, determining whether the resolution of the detection signal for the ion sample indicates that the rate of decay of the detection signal for the ion sample is dominated by collision effects may include: determining whether the resolution of the detection signal is stable (e.g. constant, such as in the stable plateau region of 5 ) in relation to an increasing number of sample ions in the sample ion cloud used to generate the detection signal for the ion sample. Of course, as discussed in detail herein, SNR is just a measure against which resolution can be plotted, and ion count or signal intensity could also be used. In the stable region, the resolution gradient versus SNR can be below a threshold or close to zero. Such a threshold could be set by a user or set at the factory.

Determining whether the resolution of the sample ion detection signal indicates that the rate of decay of the sample ion detection signal over time is dominated by collision effects may be accomplished by determining whether the sample ion cloud used to generate the sample ion detection signal contains a sufficient amount of sample ions to cause self-bundling. This can be identified by looking at the mass spectral signal for signs of it identifying that there is a sufficiently large number of ions in the ion packet, or by observing that dephasing ceases and an exponential decay is observed as collision effects dominate.

The extrapolated resolution for the ion sample can be determined from an extrapolated function that provides an expected resolution of the detection signal under conditions dominated by collision effects. This can be done as in relation to 5 described. For example, the extrapolated function can be determined based on multiple measures of resolution and multiple measures of signal intensity that were not obtained under conditions dominated by collision effects. Therefore, the extrapolated function can be based on values corresponding to the left-hand side of 1A , 1B , 3(III) and 5 are taken. Such values can be used to draw conclusions about the properties of the plateau on the right-hand side by analyzing the data accordingly. In some cases (and particularly when the data used for extrapolation does not cover the entire stable plateau) it is advantageous if the extrapolated function is based on multiple measures of resolution and multiple measures of signal intensity for one or more calibrators. In this way, the behavior of the sample ions at higher SNR can be more accurately characterized by considering the behavior of the calibrant.

The extrapolated resolution for the ion sample is determined from: a fitted function for multiple measures of resolution and multiple measures of signal intensity that provides an expected resolution of the detection signal under conditions dominated by collision effects. This can be done as described in relation to equation (II) or (III). Additionally or alternatively, an average resolution, preferably a moving average or a weighted average, for multiple measures of resolution and multiple measures of signal intensity may be used that provides an expected resolution of the detection signal under conditions dominated by collision effects. This can be done as described in relation to equation (I).

If the extrapolated resolution for the ion sample is determined from a weighted average resolution for multiple measures of resolution and multiple measures of signal intensity, the multiple measures of resolution may include one or more relatively high measures of resolution and one or more relatively low measures of resolution; and the one or more relatively high resolution measures are weighted to provide a greater contribution to the weighted average resolution than the one or more relatively low resolution measures. The multiple measures of resolution could be corrected by using: a fitted function for multiple measures of resolution and multiple measures of signal intensity that provides an expected resolution of the detection signal under conditions dominated by collision effects; and/or an average resolution, preferably a moving average or a weighted average, for multiple measures of resolution and multiple measures of signal intensity, providing an expected resolution of the detection signal under conditions dominated by collision effects. Thus, the statistics of the analysis can be improved by ensuring that low-intensity peaks with large errors are not assigned high weights and do not contribute significantly to errors.

As previously described, the weighted average resolution may be based on resolution for multiple isotopic variants of the ion sample and/or the multiple measures of resolution are determined for multiple different isotopic compositions. Analysis of multiple isotopes gives a wide range of data points, which can reduce statistical errors. It may be unavoidable to analyze isotopes as isotopes will always be present in a sample unless intentionally isolated to exclude isotopic variants. Because isotopic variants of species tend to behave similarly and elute from a GC or LC at about the same time, mass spectral analysis of a sample eluting from a GC or LC is likely to record data for multiple isotopic variants of a given sample. Thus, as previously described, it is preferred that multiple measures of resolution are determined for multiple retention times of an elution profile, such as a gas chromatography (GC) and/or a liquid chromatography (LC) elution profile.

Given the effect that ion count has on resolution, the data can be 1a and 1b used to determine confidence intervals for CCS (or decay constant) measurements and thus define the usefulness of CCS (or decay constant) values. For example, with relatively large error ranges, e.g. >10-20%, CCS could only be used to discriminate between very different chemical classes (e.g. linear molecules versus polyaromatic molecules or lipids versus peptides, etc.) or to analyze the collision-induced decay tion of intact proteins or protein complexes can be used. For small errors on the order of 1%, CCS could be used to reduce the search space within a single charge state of a single class of compounds, e.g. B. in lipidomics or bottom-up proteomics. Additionally or alternatively, as a quality control measure, CCS can be used to flag peaks that are likely to mask interference of multiple peaks, as indicated by unexpected values of CCS. Interfering peaks are for example in 4 by Lyutvinskiy Y et al. (Adding color to mass spectra: Charge Determination Analysis (CHARDA) assigns charge state to every ion peak. ChemRxiv. Cambridge: Cambridge Open Engage; 2021). Interference effects could be identified via irregularities in the CCS values (and could manifest themselves, for example, as erratic changes in the CCS during the CCS determination). Thus, in general, the methods described herein may further comprise using a measure of the decay rate of an ion sample to: classify the ion sample; and/or to detect interfering species in the ion sample.

6 Figure 1 shows a third embodiment of the present disclosure, which is a preferred mass spectrometry system for implementing the methods described herein. The mass spectrometry system is a modified Thermo Scientific Exploris™ 480 mass spectrometer. The mass spectrometry system includes a high performance transmission tube 601, an electrodynamic ion funnel 602, an EASY-IC internal calibrant source 603, an advanced active beam guide (AABG) 604, advanced quadrupole technology ( advanced quadrupole technology, AQT) 605, an independent charge detector 606, a C-trap 607, an ion guide multipole 608, and an ultra high field orbitrap mass analyzer 609, all found in existing Thermo Scientific Exploris 480 mass spectrometers. The mass spectrometry system of this embodiment includes a pressure control in the orbitrap chamber.

The mass spectrometry system also includes pressure regulation components for the hybrid quadrupole/orbital trap instrument. The mass spectrometry system includes a first vacuum region 621 in which the orbital trap mass analyzer 609 is located. The first vacuum region 621 is at a first vacuum level, typically ultra high vacuum. The mass spectrometry system includes a second vacuum region 622 at a second vacuum level that is a lower vacuum level (i.e., higher pressure) than the first vacuum level. The quadrupole mass filter 605, the independent charge detector 606, the C-trap 607 and the ion guide multipole are located in the second vacuum region 622.

The mass spectrometry system includes a bellows 610 connected to the second pressure region 622 near the ion guide multipole 608 . The bellows 610 is connected to a valve 611 which is connected to a ConFlat (CF) tee 612 . The tee 612 is connected to the first pressure region 621 . A heater seal ion gauge 613 is connected to a tube 614 terminating within the first vacuum region 621 so that the ion gauge 613 can measure the pressure within the first vacuum region. The bellows 610, the valve 611 and the tee 612 can form a first fluid connection between the first and second vacuum regions. The fluid connection could, of course, be any means to allow gas to be transferred from one vacuum region to another. Various combinations of hoses, tubing, and/or conduits could be used.

Gas is introduced into the mass analyzer 609 from the quadrupole chamber 605 of the hybrid quadrupole/orbital trap instrument at a pressure of 3-5 x 10 ^-5 mbar by nitrogen (about 20%) coming from the ion source and from the ion guide multipole 610 via the C-trap 607 exiting nitrogen is formed. Even a catastrophic incursion of such pressures into the UHV region of the 609 mass analyzer will not result in high voltage breakdown.

In contrast to a conventional leak line of atmospheric or even higher pressure, this gas leak arrangement allows the use of relatively simple valves 611 with larger cross-sections without long and narrow capillaries to protect the system from accidental venting. For example, a Parker 9 series HV compatible miniature calibration valve controlled by pulse width modulation (PWM) can be used. This achieves a higher dust and error tolerance. Preferably, the PWM works with feedback loop correction based on the resulting UHV. For example, the readings of the ion gauge 613 can be used to control the amount of gas that has exited the valve 611 and thus control the pressure within the mass analyzer 609 . By controlling the pressure under which ions are analyzed in mass analyzer 609, their decay constants can be controlled to obtain accurate decay constant and collision cross-section measurements. The mass spectrometry system can measure the pressure within the mass analyzer constantly monitor and can keep the pressure substantially constant using such a feedback loop. However, if the pressure drifts away from the chosen constant value, using the techniques disclosed herein it may be possible to introduce corrections into the calculation for CCS and/or the decay constant to account for the pressure drift.

Thus, returning to the general terms used previously, the mass spectrometry system may include a controller configured to operate the valve to control the pressure within the first vacuum region based on: the pressure within the first vacuum region (e.g. to ensure that the pressure in the first vacuum region is suitable for the analysis); and/or the measure of the temporal decay rate of the detection signal for the ion sample (e.g. to ensure that an appropriate detection time for transients is used). The mass spectrometry system may further include a pressure sensor (e.g., ion gauge 613, although other types of pressure sensors could be used, such as a Bayard-Alpert gauge) for measuring the pressure within the first vacuum region and for providing the measured pressure the controller is configured. The pressure in the trap could also be estimated based on transient decay rates, as in US-9,460,905-B2 which is incorporated herein by reference in its entirety. The methods described herein may include controlling the pressure within the mass analyzer, and the mass spectrometry systems may be configured to control the pressure within the mass analyzer to control: the rate of decay over time of the detection signal for the ion sample; and/or the time decay rate of the detection signal for the ion sample. The first vacuum region may be less than 3x10 ^-8 mbar (3x10 ^-6 Pa) and/or the second vacuum region is 1x10 ^-5 mbar (1x10 ^-3 Pa) to 5x10 ^-5 mbar (5 × 10 ^-3 Pa). For an orbitrap region of a mass spectrometry system, the vacuum should be sufficient for long transients, so the vacuum level could be adjusted to provide a long mean free path, such as more than 1000 meters or even longer, for example. Other pressures could be used. For example, in some cases the first vacuum region could be less than 5 _× 10 ^-9 mbar and/or the second vacuum region could be 2×10 ^-5 mbar, 3×10 ^-5 mbar, or 4×10 ^-5 mbar.

Preferably, gas is introduced through the same port used for the UHV pressure measurement. However, a precaution can be taken to maintain correct operation of the UHV meter. In the embodiment of 6 this can be achieved by connecting the ion gauge 613 to the UHV chamber by a long tube 614 which is not directly connected to the gas leak as it could pick up gas molecules at least after one or more reflections from the chamber walls. Thus, in general, the pressure sensor may be connected to the first vacuum region through a second fluid connection that is not directly connected to the second vacuum region.

While a particular advantage of the system of 6 is that due to the specific gas leakage, relatively cheap and simple valves 611 can be used, different valves can be used. For example, a high quality valve (e.g., a sapphire valve) could be used. In such cases, a gas leak of atmospheric pressure or even higher could be accommodated.

In the general terms used above, the mass analyzers of the present disclosure are preferably Fourier transform mass analyzers and preferably an orbital trap mass analyzer or a Fourier transform ion cyclotron resonance mass analyzer. Various other electrostatic multi-reflection traps could also be used, such as one or more of: a Cassini trap, a linear electrostatic trap, a ConeTrap, etc. The second vacuum region (e.g., the vacuum region used for the gas leak) can one or more (and optionally all) of: a quadrupole; a charge detector; an ion trap such as a C-trap; and/or an ion guide multipole. The mass spectrometry systems may further include an ion guide multipole and/or C-trap, wherein the fluid connection allows gas to be introduced from the second vacuum region into the first vacuum region via the ion guide multipole and/or C-trap. The gas introduced into the first vacuum region from the second vacuum region may have leaked from a quadrupole of the mass spectrometry system.

In the methods and mass spectrometry systems described herein, it may be preferable to ensure that the pressure within the mass analyzer for analytical measurements of a sample is the same as for analysis of the calibrant. Nonetheless, the fitting functions and scaling factors described herein can account for the effects of pressure. If the pressure is kept constant, it is generally not necessary to correct measured values to account for changes in pressure. However, the pressure can change randomly (e.g. due to temperature changes), so in some cases it may be preferable to inject calibrants (e.g. Calmix, which includes about 10 species) periodically to ensure reliable operation . Regardless of which calibrant is used, the calibrant is likely to have isotopes, so peaks from isotopic variants can also be used for calibration. Different isotopes have different intensities and therefore would have different decay rates. Thus, if three isotopes are present, analysis of one isotope will result in values occurring at three different points on the curves of 1a and 1b be taken. Preferably, the pressure is adjusted to achieve a desired signal drop for a given set of analytes (eg, multiply charged peptides) for a given transient length. For example, the signal decay (i.e. the ratio of final intensity to initial intensity) for +2, +3, +4 tryptic peptides at a MS1 resolution setting of 60K (corresponding to a 128 ms transient for high-field orbitrap analyzer) would preferably be the 2nd - be up to 4 times. This would correspond to a certain drop in the calibration ion (e.g. 1.5x over the same transient since it has a lower m/z and +1 charge) as determined from previous experiments. Thus, the pressure is increased (e.g., by modulation, such as PWM control of valve 611) until a desired drop in calibrant is achieved, and then the pressure is held approximately constant for all trials. During the duration of the experiments it may be preferable to occasionally (e.g. at the beginning of each LC/MS run) measure the calibrant and adjust the m/z and CCS of the analytes according to any drift encountered.

There are many modifications of the arrangement of 6 apparent. For example, the vacuum regions can have various different pressures and can accommodate various different components. Similarly, while a Thermo Scientific Exploris™ 480 mass spectrometer is shown, other Fourier transform mass spectrometry systems may be used. For example, a Q Exactive™ GC hybrid quadrupole orbitrap mass spectrometer system could be used in a similar manner. Various types of fluid connections can be provided to vent gas from one vacuum region to another, and the disclosure is not limited to the specific ones in 6 shown examples of bellows and valves.

7 shows the transient signal (in arbitrary units, au) of different species (195 m/z, 262 m/z, 524 m/z and 1421 m/z) from a calibration mixture at different pressure settings (4 × 10 ^-9 mbar ), 1 × 10 ^-9 mbar and 4 × 10 ^-10 mbar). Such data can be obtained by the mass spectrometry system of 6 using the regarding 6 described pressure control are provided. 7 shows the decay of the ion signal at different m/z for a calibration mixture over a transient duration of 512 ms. It can be seen that the +2 charge state of the MRFA peptide shows an accelerated decay compared to the +1 charge state of the same peptide.

7 shows the accelerated decay from higher charge states of the same compound, which is believed to be due to higher center of mass collision energy. This trend is particularly visible in a larger scale study of HeLa digestion tryptic peptides as shown below.
Faster signal decay results in somewhat reduced resolution after Fourier transform and could therefore be extracted directly from RAW files stored by the mass spectrometer data acquisition system.

The chart of 8th shows the recorded resolutions of thousands of tryptic peptides with different masses for different charge states at elevated pressure compared to standard low pressure operating conditions. In particular is 8th a comparison of the recorded resolutions at elevated pressure (“high UHV” of 4 × 10 ^-9 mbar) with standard operating conditions at low pressure (“low UHV” of ^4-10 mbar) for different states of charge. If there were no reduction in resolution, all points would lie on the diagonal (which would indicate that the low and high pressure resolutions are the same). The records in 8th however, do show data points that fall on a bottom line parallel to the diagonal, suggesting that resolution is lower on average at high pressures than at low pressures.

Since the resolution is m/z dependent, it covers the range from 40000 to 100000 for the entire mass range of the measured peptides. The relative resolution reduction is higher at lower recorded resolutions, simply reflecting the fact that higher mass results in faster decay for any given charge state.

provides an overview of this dependency 9 ready, comparing the relative reduction in resolution for each charge state. Decreases in resolution are shown relative to the case with no gas added. 9 shows a summary of the data in 8th (without mass dependence correction) for different pressures. 9 does not contain any mass dependency, but already shows the diagnostic value of such measurements.

In the embodiments described herein, the ion sample detection signal may be obtained for an isolated ion sample or as a partial or full range MS scan. An advantage of the present disclosure is that intensity dependent (e.g., SNR dependent) corrections can allow full online determination of decay constants and CCS. While broad mass ranges (e.g. hundreds of mass units) tend to give lower intensities and therefore less self-focusing, such values may still need to be corrected (e.g. due to space charge effects). When a peak is isolated, there are more ions of a given species, so more self-focusing (and higher resolution) occurs and thus less correction may be needed. In any event, the methods of the present disclosure are fully compatible with both isolated mass analysis and full MS scans.

It is understood that embodiments of the disclosure may be implemented using a variety of different information handling systems. Specifically, while the figures and discussion thereof provide example computing systems and methods, they are presented solely to provide a useful reference in discussing various aspects of the disclosure. Embodiments may be executed on any suitable data processing device, such as a personal computer, laptop, personal digital assistant, server computer, etc. Of course, the description of the systems and methods has been simplified for discussion purposes and they are just one of many different types of systems and Procedures that can be used. It should be understood that the boundaries between logic blocks are merely illustrative, and that in alternative embodiments, logic blocks or elements may be merged or different logic blocks or elements provided with an alternative breakdown of functionality.

It is understood that the functionality mentioned above can be implemented as one or more corresponding modules in the form of hardware and/or software. For example, the functionality noted above may be implemented as one or more software components for execution by a processor of the system. Alternatively, the aforementioned functionality may be implemented in hardware, such as on one or more Field Programmable Gate Arrays (FPGAs) and/or one or more Application Specific Integrated Circuits (ASICs) and/or one or more Digital Signal Processors (DSPs) and/or other hardware configurations. Method steps implemented in flowcharts contained herein or as described above may each be implemented by corresponding respective modules. Additionally, multiple method steps implemented in flowcharts, as contained herein or as described above, may be implemented together by a single module. Such modules and hardware can be integrated into a mass spectrometry system.

It is understood that if embodiments of the disclosure are implemented by a computer program, then a storage medium and a transmission medium with the computer program form aspects of the disclosure. The computer program may comprise one or more program instructions or program code that, when executed by a computer, causes an embodiment of the disclosure to be carried out. The term "program" as used herein can be a sequence of instructions designed to be executed on a computer system, and can be a subroutine, function, procedure, module, object method, object implementation, executable An application, an applet, a servlet, source code, object code, a shared library, a dynamically linked library, and/or other sequence of instructions designed to be executed on a computer system. The storage medium may be a magnetic disk (such as a hard disk or a floppy disk), an optical disk (such as a CD-ROM, a DVD-ROM, or a BluRay disc), or a memory (such as a ROM, RAM, EEPROM, EPROM, flash memory or a portable/removable storage device), etc. The transmission medium can be a communication signal, a data broadcast, a communication link between two or more computers, and so on. More particularly, this disclosure relates to methods and apparatus for performing Fourier transform mass spectrometry (FTMS). In this disclosure, FTMS refers to mass spectrometry in which ions are caused to undergo periodic motion which generates an image current whose frequency depends on the m/z of the ions. Mass spectra can be obtained by Fourier transforming the image current signals, or transients as they are often called. However, other mathematical transforms or deconvolution methods can be used as an alternative to the Fourier transform to obtain mass spectra from the image stream signals, as is known in the art. Examples of FTMS include Fourier transform ion cyclotron resonance and orbital trap mass spectrometry, the latter of which can be performed using an Orbitrap instrument. FTMS can also generally be performed using electrostatic Kingdon ion traps.

Each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose, unless otherwise specified. Thus, unless otherwise noted, each feature disclosed is one example only of a generic set of equivalent or similar features.

In addition, a number of variations can be made to the described embodiments and will become apparent to a skilled reader upon reading this specification. For example, while orbital traps have been primarily described, the mass analyzers described herein may be one or more of: an orbital trap mass analyser; or a Fourier transform ion cyclotron resonance mass analyser; and/or an electrostatic trap with an open (e.g. using only one multireflection passage of ions along one of the spatial dimensions) or a closed one (e.g. where ions pass along all dimensions multiple times over the duration of the m/z separation change direction) configuration.

As used in this document, including the claims, singular forms of terms used in this document should be construed to include the plural form and vice versa, where the context permits. For example, herein, including the claims, unless the context dictates otherwise, a singular reference such as "a" or "an" (such as an ion or a detection signal) means "one or more" (e.g., one or more ions or one or more detection signals). Throughout the specification and claims of this disclosure, the words "comprise," "include," "have," and "include" and variants thereof, such as "comprising" and "comprises" or the like, mean that the feature described means the additional features below, and is not intended to exclude (or exclude) the presence of other components. Additionally, when a first feature is described as "based" on a second feature, it may mean that the first feature is based entirely on the second feature or that the first feature is based at least in part on the second feature.

The use of any and all examples provided herein, or exemplary phrases (“for example,” “such as,” “such as,” and the like) is intended only to further illustrate the disclosure and is not intended to be a limitation on the scope of the disclosure unless otherwise claimed. In no case should any language in the specification be construed as referring to a non-claimed element as relevant to the practice of the disclosure.

All of the steps described in this specification can be performed in any order or simultaneously, unless otherwise noted or unless the context otherwise dictates. Furthermore, if a step is described as being performed after a step, that does not preclude intervening steps from being performed.

Any of the aspects and/or features disclosed in this specification may be combined in any combination, except for combinations where at least some of those features and/or steps are mutually exclusive. In particular, the preferred features of the disclosure apply to all aspects and embodiments of the disclosure and can be used in any combination. Likewise, features described in non-essential combinations may be used separately (not combined with each other).

QUOTES INCLUDED IN DESCRIPTION

This list of documents cited by the applicant was generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

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Claims (47)

A method of determining a measure of a decay rate of an ion sample undergoing mass analysis in an image current detection mass analyzer, the method comprising: receiving an ion sample detection signal from a detection of transients of the ion sample obtained using the mass analyzer, the ion sample detection signal having a decay rate over time; and determining the measure of the decay rate of the sample ion based on an extrapolated resolution for the sample ion, the extrapolated resolution being an expected resolution of the detection signal under conditions where the decay rate of the sample ion detection signal over time is dominated by collision effects. A method of determining a measure of a decay rate of an ion sample undergoing mass analysis in an image current detection mass analyzer, the method comprising: receiving an ion sample detection signal from a detection of transients of the ion sample obtained using the mass analyzer, the ion sample detection signal having a decay rate over time; Determine a fitting function for the ion sample based on: a measure of intensity and/or a measure of the mass-to-charge ratio of the detection signal for the ion sample; and a measure of intensity and/or a measure of mass-to-charge ratio of a detection signal previously obtained for a calibration means using the mass analyser; and determining the measure of the rate of decay of the ion sample by adjusting a measure of the rate of decay over time of the detection signal for the ion sample using the adjustment function. procedure after claim 1 or claim 2 comprising determining the measure of the rate of decay of the ion sample only if one or more conditions are met, the one or more conditions including one or more of: whether a peak of the detection signal is resolved after baseline; a signal-to-noise ratio of the detection signal that satisfies a threshold condition; and/or a peak of the detection signal belonging to an isotope cluster. A method according to any one of the preceding claims dependent on claim 1 , wherein the extrapolated resolution for the ion sample is an expected resolution of the detection signal under conditions in which the resolution of the detection signal is stable with respect to increasing the number of sample ions in the sample ion cloud used to generate the detection signal for the ion sample is. A method according to any one of the preceding claims dependent on claim 1 , wherein the extrapolated resolution for the ion sample is determined from an extrapolated function that provides an expected resolution of the detection signal under conditions dominated by collision effects, the extrapolated function being based on multiple measures of resolution and multiple measures of signal intensity that are not under conditions dominated by collision effects were obtained. procedure after claim 5 , where the extrapolated function is based on multiple measures of resolution and multiple measures of signal intensity for one or more calibration means. A method according to any one of the preceding claims dependent on claim 1 wherein the extrapolated resolution for the ion sample is determined from: a fitted function for multiple measures of resolution and multiple measures of signal intensity that provides an expected resolution of the detection signal under conditions dominated by collision effects; and/or an average resolution, preferably a moving average or a weighted average, for multiple measures of resolution and multiple measures of signal intensity, providing an expected resolution of the detection signal under conditions dominated by collision effects. procedure after claim 7 wherein the extrapolated resolution for the ion sample is determined from a weighted resolution for multiple measures of resolution and multiple measures of signal intensity, wherein: the multiple measures of resolution include one or more relatively high measures of resolution and one or more relatively low measures of resolution ; and the one or more relatively high measures of resolution are weighted to provide a greater contribution to the weighted resolution than the one or more relatively low measures of resolution. procedure after claim 7 or claim 8 , where the weighted resolution is based on a resolution for multiple isotopic variants of the ion sample. procedure after claim 6 until 9 , where the multiple measures of resolution are determined for several different isotopic and charge compositions. Procedure according to one of Claims 6 until 10 , wherein the multiple measures of resolution are determined for multiple retention times of an elution profile, preferably wherein the elution profile is a gas chromatography (GC) and/or a liquid chromatography (LC) elution profile). A method according to any one of the preceding claims, wherein the measure of the decay rate of the ion sample is a decay constant or a collision cross-section. The method of any preceding claim, wherein the step of determining the magnitude of the decay rate of the ion sample detection signal is based on a peak width of the ion sample detection signal, preferably wherein the step of determining the magnitude of the decay rate of the ion sample detection signal has a duration taken into account during which the detection of the transients of the ion sample takes place. A method according to any one of the preceding claims, comprising determining a plurality of measures of the decay rate of the ion sample. procedure after Claim 14 comprising determining a weighted measure of the decay rate of the ion sample by taking a weighted average of the plurality of measures of the decay rate of the ion sample. procedure after claim 15 wherein: the plurality of measures of decay rate of the ion sample comprises one or more relatively high measures of intensity of the ion sample detection signal and one or more relatively low measures of intensity of the ion sample detection signal; and the one or more relatively high measures of intensity of the ion sample detection signal are weighted to provide a greater contribution to the weighted average than the one or more relatively low measures of intensity of the ion sample detection signal. procedure after claim 15 or Claim 16 wherein the weighted average excludes one or more measures of the decay rate of the sample ion for which the measure of the intensity of the sample ion detection signal is below a threshold. Procedure according to one of Claims 14 until 17 wherein the multiple measures of decay rate of the ion sample are determined for multiple different isotopic compositions. procedure after Claim 18 , where the weighted average is based on decay rate measures for several isotopic variants of the ion sample. Procedure according to one of Claims 14 until 19 wherein the plurality of measures of decay rate of the ion sample are determined for a plurality of retention times of an elution profile, the elution profile preferably being a gas chromatography (GC) and/or a liquid chromatography (LC) elution profile. Method according to the preceding claim depending on claim 2 wherein: the measure of intensity of the detection signal for the ion sample comprises one or more of the following: total ion count; signal amplitude; and/or signal to noise ratio; and/or the measure of intensity of the detection signal previously obtained for the calibrant comprises one or more of the following: total ion count; signal amplitude; and/or signal-to-noise ratio. A method according to any one of the preceding claims dependent on claim 2 , where: the measure of the mass-to-charge ratio of the detection signal for the ion sample includes a frequency; and/or the measure of the mass-to-charge ratio of the detection signal previously obtained for the calibration means comprises a frequency. A method according to any one of the preceding claims, wherein: the ion sample detection signal is obtained for an isolated ion sample; or the detection signal for the ion sample is obtained as a full MS scan. A method according to any one of the preceding claims, wherein the mass analyzer is one or more of the following: an orbital trap mass analyser; or a Fourier transform ion cyclotron resonance mass analyser; and/or an electrostatic trap and preferably an electrostatic trap with an open configuration or a closed configuration. The method of any preceding claim, further comprising using the measure of the decay rate of the ion sample to: classify the ion sample; and/or to detect interfering species in the ion sample. The method of any preceding claim, further comprising controlling the pressure within the mass analyzer to control: the rate of decay of the detection signal for the ion sample; and/or the time decay rate of the detection signal for the ion sample. A method according to any one of the preceding claims, wherein the mass analyzer is in a first vacuum region at a first vacuum level, the method further comprising selectively introducing gas into the first vacuum region from a second vacuum region at a second vacuum level having a lower vacuum level than the first vacuum level is included, thereby controlling the pressure within the first vacuum region. A method according to any one of the preceding claims dependent on claim 2 wherein the fitting function for the ion sample is based on a measure of intensity of the detection signal for the ion sample and a measure of intensity of a detection signal previously obtained for a calibrant. A method according to any one of the preceding claims dependent on claim 2 wherein the fitting function for the ion sample is based on a ratio between the measure of intensity of the detection signal for the ion sample and the measure of intensity of the detection signal previously obtained for the calibrating means. A method according to any one of the preceding claims dependent on claim 2 wherein the ion sample adjustment function specifies an amount by which the decay rate of the ion sample detection signal must be adjusted to compensate for errors in measuring the decay rate of the ion sample detection signal. A method according to any one of the preceding claims dependent on claim 2 , where: the fitting function for the ion sample is a scaling factor; and adjusting the measure of the rate of decay of the sample ion detection signal using the adjustment function comprises scaling the measure of the rate of decay of the sample ion detection signal with the scaling factor. A computer program comprising instructions which, when executed by a processor, cause the processor to perform the method of any preceding claim. Computer-readable medium on which the computer program Claim 32 is saved. A mass spectrometry system comprising a mass analyzer and a processor configured to perform the method of any one of Claims 1 until 31 performs. A mass spectrometry system for performing a mass analysis on an ion sample, comprising: a first vacuum region comprising a mass analyzer configured to provide a detection signal for the ion sample, the first vacuum region being on a first vacuum level; a second vacuum region at a second vacuum level, the first vacuum level being a higher vacuum level than the second vacuum level; and a fluid connection between the first vacuum region and the second vacuum region, the fluid connection configured to selectively supply gas from the second vacuum region to the first vacuum region to control the pressure within the first vacuum region. mass spectrometry system Claim 35 , wherein the first vacuum region has a pressure of less than 3 × 10 ^-8 mbar (3 × 10 ^-6 Pa). mass spectrometry system Claim 35 or Claim 36 , wherein the second vacuum region has a pressure of 1 x 10 ^-5 mbar (1 x 10 ^-3 Pa) to 5 x 10 ^-5 mbar (5 x 10 ^-3 Pa). Mass spectrometry system according to any one of Claims 35 until 37 , wherein the fluid connection comprises a valve. Mass spectrometry system according to any one of Claims 35 until 38 , further comprising a controller configured to operate the valve to control the pressure within the first vacuum region based on: the pressure within the first vacuum region; and/or the measure of the time decay rate of the detection signal for the ion sample. mass spectrometry system Claim 39 , further comprising a pressure sensor configured to measure the pressure within the first vacuum region and provide the measured pressure to the controller. mass spectrometry system Claim 39 or Claim 40 wherein the controller is configured to modulate the valve, and preferably modulates the valve using pulse width modulation. Mass spectrometry system according to any one of Claims 39 until 41 wherein the pressure sensor is connected to the first vacuum region by a second fluid connection that is not directly connected to the second vacuum region. Mass spectrometry system according to any one of Claims 35 until 42 wherein the mass analyzer is a Fourier transform mass analyser, and preferably wherein the mass analyzer is an orbital trap mass analyzer or a Fourier transform ion cyclotron resonance mass analyser. Mass spectrometry system according to any one of Claims 35 until 43 wherein the second vacuum region comprises one or more of the following: a quadrupole; a charge detector; a C-trap; and/or an ion guide multipole. Mass spectrometry system according to any one of Claims 35 until 44 , further comprising an ion guide multipole and/or a C-trap, wherein the fluid connection allows gas to be introduced into the first vacuum region from the second vacuum region via the ion guide multipole and/or the C-trap. Mass spectrometry system according to any one of Claims 35 until 45 , wherein the gas introduced from the second vacuum region into the first vacuum region exits a quadrupole of the mass spectrometry system. Mass spectrometry system according to any one of Claims 35 until 46 , which is used to carry out the method according to one of Claims 1 until 31 is configured.

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