CN211148673U - High-field asymmetric waveform ion mobility device for protein detection - Google Patents
High-field asymmetric waveform ion mobility device for protein detection Download PDFInfo
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- CN211148673U CN211148673U CN201921509165.6U CN201921509165U CN211148673U CN 211148673 U CN211148673 U CN 211148673U CN 201921509165 U CN201921509165 U CN 201921509165U CN 211148673 U CN211148673 U CN 211148673U
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Abstract
The utility model discloses a high-field asymmetric waveform ion mobility device for protein detection, characteristics are: including electrospray ion source, first group's plate electrode, second group's plate electrode, the mass spectrograph, sine wave power supply, high-field asymmetric waveform power supply, and DC power supply, one side of first group's plate electrode is provided with the introduction of a sample entry, coaxial parallel arrangement around first group's plate electrode and the second group plate electrode, constitute the annealing district in advance between the first group's plate electrode, constitute the disengagement zone between the second group's plate electrode, second group's plate electrode rear end is connected with the mass spectrograph, first group's plate electrode is connected with sine wave power supply and temperature control module, second group's plate electrode is connected with high-field asymmetric waveform power supply and DC power supply, the protein sample ion that electrospray ion source produced passes through annealing district in advance and disengagement zone under the carrier gas promotes, be applicable to the complicated structure molecule that has stereoisomer and: the resolution and sensitivity of detection are improved, and the repeatability is good.
Description
Technical Field
The utility model relates to an ion separation detects technical field, especially relates to a high-field asymmetric waveform ion mobility device for protein detects.
Background
High Field Asymmetric Ion Mobility Spectrometry (FAIMS) is a rapid gas phase Ion separation technique developed from conventional Ion Mobility techniques. The mobility of the ions with the high-field asymmetric waveform utilizes the characteristic that the periods of high and low electric fields of the ions are changed alternately, and different ions are separated in the polar plate gap according to the difference of the ion mobility under the conditions of the high and low electric fields. The technology has the advantages of simple structure, small core devices, easiness in continuous detection and the like, has great development potential in the field of field analysis and detection, and is applied to separation and detection of part of protein isomers at present.
The existing high-field asymmetric waveform ion mobility device generally comprises an ionization region, a separation region and a detection region, wherein a sample is ionized into sample ions in the ionization region and then is carried into the separation region by carrier gas, the separation region is used for separating target ions by applying high-field asymmetric waveform voltage, compensation voltage is applied to compensate high-field and low-field motion deviation of the target ions, and the target ions finally enter the detection region to be detected.
The technology is still lack of the following when used for detecting molecules with complex structures such as protein and the like: (1) the resolution is low: because the structure of the protein is complex, the isomers are numerous, and the difference of the three-dimensional structure exists, the resolution of the existing high-field asymmetric waveform ion mobility technology on protein analysis is insufficient, the spatial information is difficult to determine, and the separation and identification of the stereoisomer at the complete protein level are realized; (2) the sensitivity is low: the existing high-field asymmetric waveform ion mobility technology can only pass ions of corresponding compensation voltage at the same time, and other ions are neutralized by upper and lower electrode plates of a separation area, so that ions detected by a subsequent detector are greatly reduced, and the sensitivity is influenced; (3) poor repeatability: because the structure of the protein is easily affected by temperature, the excessive temperature in the separation zone during continuous working can easily cause the protein to generate a self-cleaning phenomenon in the separation process, namely, the intermediate between more stable isomers is eliminated, and simultaneously, the signal can be seriously inhibited, thereby affecting the repeatability of sample detection.
Disclosure of Invention
In order to solve the deficiencies existing in the prior art, the utility model provides a high-field asymmetric waveform ion mobility device for protein detection, can improve the resolution ratio and the sensitivity that protein detected, repeatability is good.
The utility model provides a technical scheme that above-mentioned technical problem adopted does: a high-field asymmetric waveform ion mobility device for protein detection comprises an electrospray ion source, a first group of electrode plates, a second group of electrode plates, a mass spectrometer, a sine wave power supply, a high-field asymmetric waveform power supply, a direct current power supply and a gas generator, wherein one side of the first group of electrode plates is provided with a sample inlet for protein sample ions generated by ionization of the electrospray ion source to enter, the first group of electrode plates and the second group of electrode plates are coaxially and parallelly arranged in front of and behind, a gap is reserved between the first group of electrode plates and the second group of electrode plates, a pre-annealing area is formed between the first group of electrode plates, a separation area is formed between the second group of electrode plates, the rear end of the second group of electrode plates is connected with a detection inlet of the mass spectrometer, and the first group of electrode plates is electrically connected with the sine wave power supply, the outer side of the first group of electrode plates is provided with a temperature control module for controlling the temperature of the pre-annealing area, the second group of electrode plates is electrically connected with the high-field asymmetric waveform power supply and the direct-current power supply, the gas generator is arranged at the front end of the first group of electrode plates, and the generated carrier gas pushes ionized protein sample ions to sequentially pass through the pre-annealing area and the separation area.
In some embodiments, the first group of electrode plates and the second group of electrode plates are both formed by two plate-type electrode plates which are symmetrical up and down, and the electrode plate spacing between the first group of electrode plates and the second group of electrode plates is equal. Therefore, the method has better protein ion pre-annealing and separating effects.
In some embodiments, the distance between the electrode plates of the first group of electrode plates and the second group of electrode plates is 0.1mm to 10cm, preferably 1mm to 5 mm. Therefore, the method has a better effect and is beneficial to improving the detection resolution.
In some embodiments, the sine wave power supply has a voltage amplitude of no less than 3 kV. Therefore, the annealing reaction of the protein sample ions in the pre-annealing area can be better realized, and the performance stability of the protein ions in the subsequent separation process is improved.
In some embodiments, the temperature of the pre-annealing zone is in the range of 0 ℃ to 500 ℃, preferably 100 ℃ to 300 ℃. Therefore, a stable temperature field is provided for protein ion pre-annealing, the performance of the protein ions after pre-annealing can be better achieved, the self-cleaning phenomenon during subsequent separation is reduced, and the ion signal intensity is ensured.
In some embodiments, the high-field asymmetric waveform power supply can adopt a square wave, a half sine wave or a double sine wave, the voltage amplitude of the high-field asymmetric waveform power supply is not less than 3kV, and the frequency is 1 kHz-100 MHz, preferably 100 kHz.
In some embodiments, the adjustable range of the compensation voltage of the dc power supply is-200V to 200V. Thereby having a superior separation effect.
In some embodiments, the protein sample ions include protein positive and negative ions and other ionizable particles. The electrospray ion source can ensure that the spatial structure of the generated protein sample ions is unchanged.
In some embodiments, the carrier gas is one of hydrogen, helium, nitrogen and sulfur hexafluoride, or a mixture of a plurality of hydrogen, helium, nitrogen and sulfur hexafluoride in any proportion, or a mixture of a plurality of hydrogen, helium, nitrogen and sulfur hexafluoride and a doping gas, the doping gas is selected from ethers, alcohols, ketones and aromatic hydrocarbon gases, and the flow rate of the carrier gas is 0.001L/min to 30L/min, preferably 1L/min to 5L/min.
Compared with the prior art, the utility model has the advantages of:
(1) the electrospray ionization source is arranged to generate protein sample ions, the protein sample ions sequentially pass through the two groups of electrode plates under the pushing of carrier gas, the protein sample ions oscillate under the action of a high-voltage sine wave power supply and a temperature field of the first group of electrode plates and collide with carrier gas molecules to convert heat energy, the temperature of the sample ions is raised to preheat, then the protein sample ions can automatically maintain the state of a potential low point, and an annealing effect is performed in a pre-annealing area, so that the space structure and the performance of protein can be stabilized, the generation of a protein self-cleaning phenomenon can be reduced, the uncontrollable self-cleaning phenomenon simultaneously occurring in the original separation process can be controlled, the subsequent separation effect is ensured, and the ion signal intensity is improved;
(2) compared with the traditional FAIMS structure, the distance between the electrode plates can be increased from 1.9mm to 5mm, the separation time can be increased from 0.2-0.4 s to 1-1.5 s, and the resolution can be increased from 200-400 to about 800 in a larger range on the premise of no sensitivity loss, so that the accurate identification of the stereoisomer at the complete protein level is realized;
(3) the duration of the protein ions in the pre-annealing stage and the separation stage can be reasonably controlled, and the protein ions after pre-annealing are not influenced by overhigh temperature in continuous working, so that further isomerization is avoided, and the repeatability of a detection map is improved.
Drawings
Fig. 1 is a schematic structural diagram of a high-field asymmetric waveform ion mobility device for protein detection according to the present invention.
The system comprises an electrospray ion source 1, a first group of electrode plates 2, a second group of electrode plates 3, a mass spectrometer 4, a sine wave power supply 5, a high-field asymmetric waveform power supply 6, a direct current power supply 7, carrier gas 8, a sample inlet 9, a pre-annealing area 10, a separation area 11 and a temperature control module 12.
Detailed Description
The high-field asymmetric waveform ion mobility device for protein detection according to the present invention will be described in further detail with reference to the accompanying drawings, but is not intended to limit the present invention.
Example one
As shown in the figure, the high-field asymmetric waveform ion mobility device for protein detection of the utility model comprises an electrospray ion source 1, a first group of electrode plates 2, a second group of electrode plates 3, a mass spectrometer 4, a sine wave power supply 5, a high-field asymmetric waveform power supply 6, a direct current power supply 7 and a gas generator, wherein one side of the first group of electrode plates 2 is provided with a sample inlet 9 for protein sample ions generated by ionization of the electrospray ion source, the first group of electrode plates 2 and the second group of electrode plates 3 are coaxially arranged in parallel, a gap is left between the first group of electrode plates 2 and the second group of electrode plates 3, a pre-annealing area 10 is formed between the first group of electrode plates 2, a separation area 11 is formed between the second group of electrode plates 3, the rear end of the second group of electrode plates 3 is connected with a detection inlet of the mass spectrometer 4, the first group of electrode plates 2 is electrically connected with the sine wave power supply 5, a temperature control, the second group of electrode plates 3 is electrically connected with a high-field asymmetric waveform power supply 6 and a direct current power supply 7, the gas generator is arranged at the front end of the first group of electrode plates 2, and the generated carrier gas 8 pushes ionized protein sample ions to sequentially pass through a pre-annealing area 10 and a separation area 11.
In this embodiment, the upper electrode plate of the first group of electrode plates 2 is provided with a sample inlet 9, and the sample introduction direction of the protein sample ions generated by the ionization of the electrospray ion source and the carrier gas direction form a certain angle, so that the loss of the sample ions can be reduced better.
In this embodiment, the temperature control module 12 includes a heating plate and a temperature sensor tightly disposed at the periphery of the first group of electrode plates, the heating plate is connected to an external power supply, and the temperature sensor is connected to the control system for accurately controlling the temperature of the pre-annealing region and ensuring a stable temperature field. The gas generator is also connected with a gas flow controller for controlling the flow rate of the carrier gas to be kept stable.
In this embodiment, the first group of electrode plates 2 and the second group of electrode plates 3 are both formed by two vertically symmetric flat plate-type electrode plates, the electrode plate spacing between the first group of electrode plates 2 and the second group of electrode plates 3 is equal to each other, and is 4mm, and in other embodiments, the electrode plate spacing may be 0.1mm, 1mm, 3mm, 5mm, 1cm, 10cm, and the like.
In this embodiment, the voltage amplitude of the sine wave power supply 5 is not less than 3 kV. The high-field asymmetric waveform power supply 6 is formed by superposing double sine waves, wherein the waveform formed by superposition meets the condition that the integral of one period is 0, namely the integral areas of a high-field part and a low-field part are the same, and the high-field asymmetric waveform power supply 6 can realize an approximate effect by adopting square waves, half sine waves or other high-order fitting waveforms in other embodiments. The amplitude of the voltage of the high-field asymmetric waveform power supply 6 is not less than 3kV, and the frequency is 100kHz, in other embodiments, the frequency of the high-field asymmetric waveform power supply 6 may be 1kHz, 10kHz, 500kHz, 10MHz, 100MHz, and the like.
The mass-to-charge ratio (m/z) of the mass spectrometer 4 is in the range of 1-1000000 amu, preferably 1000-200000 amu, and the mass resolution of the mass spectrometer 4 is in the range of 100-20000000. In this embodiment, the mass resolution of the mass spectrometer is not less than 10000 under the conditions of the mass-to-charge ratio of 1000amu and the highest sensitivity of the mass spectrometer, and the mass spectrometer has a better effect.
The protein sample ions produced by the electrospray ion source include protein positive and negative ions and other ionizable particles.
The carrier gas is one or a mixture of a plurality of hydrogen, helium, nitrogen and sulfur hexafluoride according to any proportion, or the mixture of a plurality of hydrogen, helium, nitrogen and sulfur hexafluoride and doping gas, wherein the doping gas is selected from ether, alcohol, ketone and aromatic hydrocarbon gases, so that the separation effect of a target sample can be improved, the flow rate of the carrier gas is 0.001L/min-30L/min, preferably 1L/min-5L/min, the voltage amplitude of the sine wave power supply in the step ② is not less than 3kV, and the temperature range of the temperature field is 0-500 ℃, preferably 100-300 ℃.
In the embodiment, pure nitrogen or a mixture of 75% of nitrogen and 25% of hydrogen is selected as the carrier gas, the flow rate is 2-3L/min, the pre-annealing temperature is 100-150 ℃, the adjustable range of the compensation voltage of the direct current power supply is-200V, based on the specific carrier gas flow rate, the pre-annealing temperature and the sine wave voltage, the protein is pre-annealed before separation, the uncontrollable self-cleaning phenomenon simultaneously occurring in the original separation process is controllable, and the signal intensity of protein detection is ensured.
Example two
The utility model relates to a method for detecting protein by a high-field asymmetric waveform ion mobility device, which comprises the following steps:
① using an electrospray ion source to ionize the target protein sample in the solution to generate protein sample ions, ensuring the spatial structure of the protein sample ions to be unchanged, and the protein sample ions enter the first group of electrode plates and are pushed by the carrier gas to move forwards;
② providing sine wave power supply for the first group of electrode plates, and providing stable temperature field for the pre-annealing region of the first group of electrode plates through the temperature control module, the protein sample ions oscillating under the combined action of carrier gas, sine wave power supply and temperature field in the pre-annealing region and completing pre-annealing;
③ providing high-field asymmetric waveform power supply and DC power supply for the second group of electrode plates, moving the protein sample ions forward to the separation region in the second group of electrode plates under the push of carrier gas, separating the target protein ions in the separation region and detecting in the mass spectrometer to obtain mass spectrum signals of scan compensation voltage, and neutralizing the rest ions in the separation region;
④ changing the compensation voltage of the DC power supply, repeating steps ① - ③ to obtain mass spectrum signals under different compensation voltages;
⑤ mass spectrum signals under different compensation voltages are integrated to obtain a signal spectrum of the high-field asymmetric waveform ion mobility of the target protein ions.
The utility model discloses a high-field asymmetric waveform ion mobility device for protein detection, the basic operating principle is as follows: protein sample ions are ionized by an electrospray ion source 1 to form sample ions, and then sequentially pass through two groups of flat plate electrode plates under the pushing of a carrier gas, the sample ions are subjected to the action of a stable temperature field of a high-voltage sine wave power supply 5 and a temperature control module 12 between a first group of electrode plates 2 (a pre-annealing area 10), the sample ions oscillate and collide with carrier gas molecules, partial kinetic energy is converted into heat energy, the temperature of the sample ions is increased to be preheated, and then the protein sample ions can be automatically maintained in a low-potential punctiform state to perform an annealing effect. The process can reduce the generation of protein 'self-cleaning', is favorable for stabilizing the spatial structure and performance of the protein, is favorable for subsequent separation, and improves the ionic signal intensity. Then the protein sample ions are separated by the action of the high-field asymmetric waveform power supply 6 between the second group of electrode plates 3 (separation area 11), the target ions fly out of the separation area by the compensation action of the direct current power supply 7 and are finally detected by the mass spectrometer 4, and the rest ions impact on the electrode plates to be neutralized.
Based on the characteristics, the utility model discloses a high field asymmetric waveform ion mobility device for protein detection, through introducing protein pre-annealing stage, adopt the higher sine wave of average voltage to make protein carry out pre-annealing before the separation, be favorable to stabilizing the spatial structure and the performance of protein, can reduce the production of protein "self-cleaning" phenomenon, with the original separation process simultaneously emergence uncontrollable "self-cleaning" phenomenon controllable to guarantee follow-up separation effect, improve ion signal intensity; compared with the traditional FAIMS structure, the utility model discloses an electrode plate interval can promote to 5mm from 1.9mm, and the separation time promotes to 1 ~ 1.5s from 0.2 ~ 0.4s, has prolonged the residence time of ion in the separation zone to under the prerequisite of not losing sensitivity, resolution ratio can promote to about 800 from 200 ~ 400 great amplitude, realize the accurate differentiation of complete protein level stereoisomer; the duration of the protein ions in the pre-annealing stage and the separation stage can be reasonably controlled through the length of the electrode plate and the flow rate of the carrier gas, and the protein ions are not affected by overhigh temperature in continuous working after the pre-annealing, so that the further isomerization can be avoided, and the repeatability of a detection map is improved.
The utility model discloses a high-field asymmetric waveform ion mobility device for protein detection still is applicable to the detection of other complex structure molecules that have stereoisomer and midbody.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, and the present invention can also be modified in materials and structures, or replaced by technical equivalents. Therefore, all structural equivalents which may be made by applying the present invention to the specification and drawings, or by applying them directly or indirectly to other related technical fields, are intended to be encompassed by the present invention.
Claims (9)
1. A high-field asymmetric waveform ion mobility device for protein detection is characterized by comprising an electrospray ion source, a first group of electrode plates, a second group of electrode plates, a mass spectrometer, a sine wave power supply, a high-field asymmetric waveform power supply, a direct current power supply and a gas generator, wherein one side of the first group of electrode plates is provided with a sample inlet for protein sample ions generated by ionization of the electrospray ion source to enter, the first group of electrode plates and the second group of electrode plates are coaxially and parallelly arranged in front of and behind, a gap is reserved between the first group of electrode plates and the second group of electrode plates, a pre-annealing area is formed between the first group of electrode plates, a separation area is formed between the second group of electrode plates, the rear end of the second group of electrode plates is connected with a detection inlet of the mass spectrometer, and the first group of electrode plates is electrically connected with the sine wave power supply, the outer side of the first group of electrode plates is provided with a temperature control module for controlling the temperature of the pre-annealing area, the second group of electrode plates is electrically connected with the high-field asymmetric waveform power supply and the direct-current power supply, the gas generator is arranged at the front end of the first group of electrode plates, and the generated carrier gas pushes ionized protein sample ions to sequentially pass through the pre-annealing area and the separation area.
2. The device of claim 1, wherein the first and second sets of electrode plates are each formed by two plate-type electrode plates that are symmetrical up and down, and the electrode plates of the first and second sets of electrode plates are spaced at equal intervals.
3. The device of claim 2, wherein the first and second sets of electrode plates are spaced apart by a distance of 0.1mm to 10 cm.
4. The device of claim 1, wherein the sine wave power supply has a voltage amplitude of no less than 3 kV.
5. The device of claim 1, wherein the pre-annealing region is at a temperature in the range of 0 ℃ to 500 ℃.
6. The device of claim 1, wherein the high-field asymmetric waveform power supply is a square wave, a half sine wave or a double sine wave, and the high-field asymmetric waveform power supply has a voltage amplitude of not less than 3kV and a frequency of 1 kHz-100 MHz.
7. The apparatus of claim 1, wherein the compensation voltage of the DC power supply is adjustable in a range of-200V to 200V.
8. The device of claim 1, wherein the protein sample ions comprise protein positive and negative ions.
9. The device of claim 1, wherein the carrier gas has a flow rate of 0.001L/min to 30L/min.
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| CN110596401A (en) * | 2019-09-11 | 2019-12-20 | 宁波盘福生物科技有限公司 | High-field asymmetric waveform ion mobility device and method for protein detection |
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| CN110596401A (en) * | 2019-09-11 | 2019-12-20 | 宁波盘福生物科技有限公司 | High-field asymmetric waveform ion mobility device and method for protein detection |
| CN110596401B (en) * | 2019-09-11 | 2024-05-14 | 宁波盘福生物科技有限公司 | High-field asymmetric waveform ion mobility device and method for protein detection |
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