The present application is a Continuation Application of International Application PCT/CN2014/086049, with an international filing date of Sep. 5, 2014, which is incorporated by reference in its entirety.
The present disclosure relates to a method for analyzing ionic structure, and more particularly, to a method which can be applied to a Fourier transform equipment, and analyze an ionic structure through determination of collision cross sectional areas.
Mass spectrometry refers to an analysis method that separates and detects compounds by different mass-to-charge ratios (m/z) to implement component and structure identification. The mass spectrometry technique has become increasingly prominent in the field of bioanalysis due to its high specificity and sensitivity. Bio-mass spectrometry (Bio-MS) is a mass spectrometry technique applied to analyze biomolecules, which is widely applied in protein and polypeptide researches such as relative molecular mass determination of protein, peptide mapping determination, peptide sequence determination technique, assignment of sulfhydryl and disulfide bond, posttranslational modification of protein, quantitative proteome analysis, protein-protein interaction research, and the like. Moreover, the bio-mass spectrometry is also applied to such fields as polysaccharose structure determination, oligonucleotide and nucleic acid analysis, microorganism identification, medicament research and development and the like.
The mass spectrometry can obtain the mass information of samples, but cannot give more information effectively for different samples having the same mass. For example, it is different to analyze an ionic structure via the mass spectrometry. Currently, ionic structures are generally analyzed through tandem mass spectrometry (tandem MS) and ion mobility spectrometry. The tandem MS applies energy to fragment an ion to be determined, and analyzes the fragment ion to reconstruct the ionic structure, while the ion mobility spectrometry analyzes the collision cross sectional area of the ion to be determined to analyze the ionic structure. The tandem MS usually works under a high vacuum condition (<1 mTorr), while the ion mobility spectrometry works under a high pressure condition (>1 Torr) and has lower resolution (usually lower than 1,000). These methods require a complicated instrument structure and increased vaccuum consumption due to the big difference between the working pressures. Meanwhile, experimental control conditions for the ion to be determined are harsh and ion loss is significant since migration movement of the ion between a plurality of vacuum cavities is involved.
In 2012, Fan Yang, Jacob E. Voelkel and David V. Dearden proposed to analyze collision cross sectional areas of ions from analysis of Fourier transform ion cyclotron resonance spectrum line width so as to analyze the ionic structure in “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 (11), pp 4851-4857). The method increases the pressure inside a Fourier transform ion cyclotron resonance ion trap to decay an image current of a dominant ion for ion-molecule collision. The rate of decay determines the half peak width (full width at half maximum, FWHM) of the spectrum line. The faster the rate of decay in time domain is, the wider the half peak in corresponding frequency domain is. The ion collision area can be calculated by measuring the half peak width, so that the ion collision area may be analyzed through the decay of the image current of the ion, and finally the ionic structure may be obtained.
Moreover, conventional ion mobility spectrometry can obtain the space size information of the samples, i.e. detect the collision cross sectional area (CCS) of the samples, while obtaining the mass of the samples, and then effectively identity various isomers having the same mass. However, the ion mobility spectrometry increases the analysis cost and reduces the analysis efficiency.
In order to at least partially solve the foregoing problems, the present disclosure provides a method for analyzing ionic structure which can analyze an ionic structure by analyzing a time-frequency signal of an ion to be measured from an ion mass analyzer.
According to an aspect of the present disclosure, there is provided a method for analyzing ionic structure, including the following steps.
Ion trapping and exciting step: applying a radio frequency electric field on an ion mass analyzer to trap sample ions in the ion mass analyzer, the applied radio frequency electric field having a high order component; and applying an an auxiliary alternating current electric field or applying a broadband excitation electric field on the ion mass analyzer to cause the sample ions to be excited to a motion amplitude not exceeding the trapping ability of the ion mass analyzer, the motion amplitude at this moment being recorded as a primary motion amplitude and the moment corresponding to the primary motion amplitude being recorded as a first moment.
Signal collecting step: continuously feeding carrier gas into the ion mass analyzer and keeping a degree of vacuum in the ion mass analyzer to be smaller than 1.333 Pa, the sample ions being collided with the carrier gas and the motion amplitude being decreased gradually till a second moment, and collecting a time domain signal of an image current generated by the sample ions during the process from the first moment to the second moment.
Signal processing step: analyzing the time domain signal through a time-frequency analysis method and respectively obtaining time-varying characteristic curves of the motion frequencies of ions having various sizes in the sample ions varied with time so as to distinguish among the ions having different sizes, each of the time-varying characteristic curves indicating a corresponding relation between the motion frequency of the ion having a corresponding size and the collision cross sectional area of the ion and the carrier gas.
According to an embodiment of the present disclosure, after the signal processing step, the method further includes a time domain signal filtering step: the time domain signal being filtered by a filter so as to obtain a less-interfered time domain signal.
According to an embodiment of the present disclosure, the time-frequency analysis method in the signal processing step is selected from Fourier transform method, fast Fourier transform method, short time Fourier transform method, fractional Fourier transform method or wavelet analysis method.
According to an embodiment of the present disclosure, in the signal processing step, the time-varying characteristic curve of the motion frequency (f) of the sample ion varied with time satisfies equation:
where a1 is the motion amplitude of the sample ion; f0 is the motion frequency of the ion without a high order field; and ∈3, ∈5 and ∈7 are even order field perturbation coefficients, and
where a0 is the primary motion amplitude of the sample;
is a constant;
is a Langevin decay coefficient;
is a hard-sphere decay coefficient; r is the effective radius of the sample ion; q is the charge of the sample ion; ap is a polarizability; ε0 is a permittivity of vacuum; p is a degree of vacuum; T is a thermodynamic temperature; k is a Boltzmann constant; M is the mass of the carrier gas; and m is the mass of the sample ion;
substituting equation (2) into equation (1), thus obtaining a relation equation of the effective radius of the sample ion and the motion frequency of the sample ion varied with time.
According to an embodiment of the present disclosure, the method further includes the following step of:
drawing a relation curve of the effective radius of the sample ion and the motion frequency of the sample ion varied with time through a numerical method according to the relation equation of the effective radius of the sample ion and the motion frequency of the sample ion varied with time.
According to an embodiment of the present disclosure, the method further includes a signal analysis step:
obtaining corresponding characteristic values by analyzing each time-varying characteristic curve from the signal processing step, and establishing a corresponding relation between each characteristic value and the collision cross sectional area of the ion having the corresponding size, thus determining a size relation between the ions having various sizes in the sample ions.
According to an embodiment of the present disclosure, in the signal analysis step, with respect to each time-varying characteristic curve, the characteristic value thereof is obtained through a following step:
connecting a motion frequency point of the sample ion at the first moment with a motion frequency point of the sample ion at the second moment to create a straight line, and integrating over a closed curve encircled by the straight line and the time-varying characteristic curve from the first moment to the second moment to obtain the characteristic value of the time-varying characteristic curve.
According to an embodiment of the present disclosure, in the signal processing step, the time-varying characteristic curve of the motion frequency varied with time satisfies equation:
where a1 is the motion amplitude of the sample ion; f0 is the motion frequency of the ion without a high order field; and ∈3, ∈5 and ∈7 are even order field perturbation coefficients, and
where a0 is the primary motion amplitude of the sample;
is a constant;
is a Langevin decay coefficient;
is a hard-sphere decay coefficient; r0 is the effective radius of the sample ion; q is the charge of the sample ion; ap is a polarizability; ε0 is a permittivity of vacuum; p is a degree of vacuum; T is a temperature; k is a Boltzmann constant; M is the mass of the carrier gas; and m is the mass of the sample ion;
substituting equation (2) into equation (1), thus obtaining a relation equation of the effective radius of the sample ion and the motion frequency of the sample ion varied the time; and under a same measuring condition, respectively integrating over time on both sides of the relation equation of the frequency and the effective radius of the sample ion, thus obtaining a corresponding relation between a characteristic value S and the effective radius of the ion having a corresponding size.
In the method for analyzing ionic structure according to the present disclosure, there is provided a collision cross sectional area determination method based on a Fourier transform equipment and time-frequency analysis, which enables ionic structure analysis, can be widely applied in an equipment based on Fourier transform, expands the detection ability of the Fourier transform equipment, and realizes non-destructive testing of the space size information of the sample ions. Based on the exact determination with respect to the space sizes of the sample ions, isomers such as different conformations of the same protein can be distinguished, or complicated mixed samples having the same mass but different sizes can be detected through this method at a time, thus the detection efficiency of the sample ions is improved, and the detection cost is reduced.
The accompanying drawings provide further understanding of the invention, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention. In the accompanying drawings:
The embodiments of the invention will be described in detail with reference to the accompanying drawings. It will be appreciated that the embodiments are only exemplary embodiments, and not intended to limit the present invention.
Referring to
Ion trapping and exciting step: a radio frequency electric field (RF) is applied on an ion mass analyzer in which there is a linear ion trap, for example, having a physical size of 5×5 mm and a distance from an electrode to the center of the trap is 5 mm; the radio frequency electric field applied has a high order component, and the high order component of the field may be obtained through a manner such as changing the shape of the electrode, so that sample ions are trapped in the linear ion trap; the frequency of the radio frequency electric field is 1 MHz, the amplitude of the radio frequency electric field is 400 V, and the q value of the radio frequency electric field is 0.3; moroever, an auxiliary alternating current electric field AC is applied on the 5×5 mm linear ion trap, so that the big and small ions of the sample ions are excited to a same motion amplitude not exceeding the trapping capacity of the linear ion trap, the motion amplitude being recorded as a primary motion amplitude a0 at this moment. The auxiliary alternating current electric field AC for exciting the sample ions may also be replaced by a broadband excitation electric field SWIFT. As shown in
The 5×5 mm linear ion trap in the step may also be replaced by other ion mass analyzers.
Signal collecting step: helium is continuously fed into the 5×5 mm linear ion trap and served as the carrier gas, a degree of vacuum in the linear ion trap is kept at 0.1333 Pa (1 mTorr), and the motion amplitude of the sample ion is gradually decreased from the primary motion amplitude a0; as the decay process shown in
Filtering step: the time domain signal obtained in the foregoing step is filtered twice by a low pass filter until a less-interfered time domain signal is obtained. The filter may also be configured with a band pass filter and the like.
Signal processing step: referring to
The small ion (light color portion in the middle) is taken as an example hereinafter to illustrate how to obtain the time-varying characteristic curve C1. Referring to
In the signal processing step, the short time Fourier transform method (STFT) may also be replaced by other time-frequency analysis methods such as Fourier transform method (FT), fast Fourier transform method (FFT), fractional Fourier transform method or wavelet analysis method and the like. Further, in the signal processing step, the time-varying characteristic curve C1 of the motion frequency f1 of the small ion varied with time satisfies following equation:
where a1 is the motion amplitude of the sample ion; f0 is the motion frequency of the ion without a high order field; and ∈3, ∈5 and ∈7 are even order field perturbation coefficients, and
where a0 is the primary motion amplitude of the sample;
is a constant;
is a Langevin decay coefficient;
is a hard-sphere decay coefficient; r1 is the effective radius of the sample ion; q is the charge of the sample ion; ap is a polarizability; ε0 is a permittivity of vacuum; p is a degree of vacuum; T is a thermodynamic temperature with a unit of Kelvin; k is a Boltzmann constant; M is the mass of the carrier gas; and m is the mass of the sample ion.
Equation (2) is substituted into equation (1), thus obtaining a relation equation of the effective radius r1 of the small ion and the motion frequency f1 of the small ion varied with time, and further obtaining a corresponding relation between the frequency f1 and the collision cross sectional area σ1 of the small ion according to a formula σ1=π(r1)2. Further, a relation curve of the effective radius r1 of the small ion and the motion frequency f1 of the small ion varied with time is drawn through a numerical method according to the relation equation of the effective radius r1 of the small ion and the motion frequency f1 of the small ion varied with time. Similarly, a relation equation of the effective radius r2 of the big ion and the motion frequency f2 of the big ion varied with time, and a relation curve of the effective radius r2 of the big ion and the motion frequency f2 of the big ion varied with time may be obtained.
The two isomers consisting of the big ion and the small ion as well as ions and isomers having other sizes included in the sample ions can be distinguished by using the method according to the present disclosure and through the foregoing ion trapping and exciting step, filtering step, signal collecting step and signal processing step. To further identify the relation between the isomers and ions, for example, size scale and the like thereof, the method according to the present disclosure may further include a signal analysis step: obtaining corresponding characteristic values S by analyzing time-varying characteristic curves of each isomer and ions having different sizes in the signal processing step, and establishing a corresponding relation between each characteristic value S and the collision cross sectional area of the corresponding isomer and ion having different sizes, thus determining a size scale relation.
The characteristic value of the time-varying characteristic curve with respect to the small ion is obtained through a method as follows.
As shown in
A corresponding relation between the characteristic value and the collision cross sectional area may be established through the method as follows.
As shown in
It should be noted that methods for creating the characteristic values are not exclusive. Rather, it may be feasible to obtain the characteristic values such as area, slope and radius of curvature through the time-frequency curves of ions by means of methods such as integral, differentiation and the like.
The method for analyzing ionic structure according to the present disclosure can be employed to quickly identify different isomers or ions having same mass but different space sizes in the sample ions, and exactly determine the scale relation between the effective radiuses of those isomers or ions having differentiation sizes.
In the method for analyzing ionic structure according to the present disclosure, there is provided a collision cross sectional area determination method based on a Fourier transform equipment and time-frequency analysis, which enables ionic structure analysis, can be widely applied in an equipment based on Fourier transform, expands the detection ability of the Fourier transform equipment, and realizes non-destructive testing of the space dimension information of the sample ions. Based on the exact determination with respect to the space dimensions of the sample ions, isomers such as different conformations of the same protein can be distinguished, or complicated mixed samples having the same mass but different sizes can be detected through this method at a time, thus the detection efficiency of the sample ions is improved, and the detection cost is reduced.
New embodiments may be obtained by splitting and combining the characteristics of each embodiment mentioned above, which may not depart from the scope of the present invention. Moreover, various changes and modifications apparent to those skilled in the art may not deviate from the principle and scope of the present invention. Therefore, these changes and modifications shall also be included in the present invention as long as the changes and modifications fall within the scope of the appended claims and equivalent schemes.
Filing Document | Filing Date | Country | Kind |
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PCT/CN2014/086049 | 9/5/2014 | WO | 00 |