CN109037024B - Electrospray ion source for source-internal disulfide bond fragmentation - Google Patents
Electrospray ion source for source-internal disulfide bond fragmentation Download PDFInfo
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- CN109037024B CN109037024B CN201810566170.4A CN201810566170A CN109037024B CN 109037024 B CN109037024 B CN 109037024B CN 201810566170 A CN201810566170 A CN 201810566170A CN 109037024 B CN109037024 B CN 109037024B
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/02—Details
- H01J49/10—Ion sources; Ion guns
- H01J49/16—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission
- H01J49/165—Electrospray ionisation
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/02—Details
- H01J49/10—Ion sources; Ion guns
- H01J49/16—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission
- H01J49/165—Electrospray ionisation
- H01J49/167—Capillaries and nozzles specially adapted therefor
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Abstract
The invention discloses the field of analytical instruments, and particularly relates to an electrospray ion source generation instrument for in-source disulfide bond fragmentation. The invention comprises a hollow capillary tube emission needle and a hollow capillary tube emission needle bracket, wherein the middle of the hollow capillary tube emission needle bracket is a through cavity, a hollow capillary tube emission needle is arranged in the cavity at the front end of the hollow capillary tube emission needle bracket, ultraviolet lamps are arranged on the periphery of the hollow capillary tube emission needle, and the hollow capillary tube emission needle and an ion beam cluster are collected in an ionization chamber. The invention has the advantages that the invention has more intuitive disulfide bond fragmentation efficiency, and the molecular ions before and after the polypeptide biomacromolecule containing disulfide bonds is fragmented appear in the same mass spectrogram, thus being an ideal electrospray ion source for rapidly analyzing disulfide bonds at present.
Description
Technical Field
The invention relates to the field of analytical instruments, in particular to an electrospray ion source generation instrument for in-source disulfide bond fragmentation.
Background
Since the mid-80 s, one of the 2002 nobel prize winners, John b.fenn, applied electrospray Ion sources to macromolecular mass spectrometry, over 20 years, it was left to two modes for the electrospray Ion source mechanism, Ion Evolution Model (IEM) Ion Evaporation, and Charged Residue Model (CRM) charge retention mechanism. The basic structure of an electrospray ion source was not substantially different from that of the 90 s.
The basic structure of an electrospray ion source is that a hollow metal or glass capillary is filled with liquid, positive or negative high voltage is applied to the liquid opposite to an ion inlet of a mass spectrometer, positive and negative ions are formed in the atmosphere, and a vacuum system of the mass spectrometer sucks a part of the ions into a mass analyzer of the mass spectrometer.
For electrospray ion sources, the effective ions to transport molecular ions to the mass spectrometer mass detector is only between 0.01% and 0.1% of the total ion population, although the ionization probability is high, almost 100%.
In order to solve the above problems, various mechanisms of electrospray ion sources have been invented by many inventors, see U.S. Pat. Nos. 4861988, 5412208, 5432343, US6992299 and US 5504329. However, in all the inventions, the emission needle tip of the electrospray ion source is positioned in the atmosphere, the ion flow is closely related to the liquid flow, stable ion emission cannot be obtained within the large flow range of 100 nanoliters/min to 100 microliters/min, and the ion transmission efficiency is not obviously improved.
Disulfide bond is a common protein posttranslational modification, and plays an important role in stabilizing the spatial structure of protein and maintaining and regulating the biological activity of the protein. Determining the position of disulfide bonds in proteins is a necessary condition for comprehensive resolution of the chemical structure of proteins containing disulfide bonds. The current method for analyzing the position of disulfide bonds in proteins is to compare the difference between peptide fragments obtained by enzymatically digesting proteins with trypsin before and after the opening of disulfide bonds. The disulfide bond must first be opened with a reducing agent such as dithiothreitol, tris (2-carboxyethyl) phosphine, etc., and then the labile SH group must be alkylated with an alkylating agent such as iodoacetamide to increase the mass of each cysteine by 57 daltons. The secondary structure of the protein after the two steps of treatment is opened, which is favorable for further trypsin enzymolysis. Because the two peptide fragments after the opening of the disulfide bond generally have different retention times in the chromatogram, and the two peptide fragments without the opening of the disulfide bond also have different retention times, the comparison of the peptide fragments in the two cases needs to be carried out in the full-chromatogram time, thereby greatly increasing the difficulty of calculation.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides an electrospray ion source which is high in efficiency, reasonable in design, simple in structure and high in ionization probability and has stable ion emission. Rate and ion emission are stable.
The invention aims to provide an electrospray ion source which is reasonable in design, simple in structure, high in ionization probability and stable in ion emission.
The purpose of the invention is realized by adopting the following technical scheme: it comprises a hollow capillary emission needle; the emission needle support forms a laminar flow gas hollow capillary tube which flows forwards around the emission needle and is exposed in ultraviolet light, medical micromolecules and biological macromolecules containing disulfide bonds are irradiated by the ultraviolet light to crack the disulfide bonds, fragment molecules after the disulfide bonds are cracked form ion beam clusters after passing through the tip of the emission needle, the signal-to-noise ratio of ion signals of an ion source is improved through auxiliary gas, stable ion current is finally obtained, and the molecular ions before and after the polypeptide biological macromolecules containing the disulfide bonds are cracked appear in the same mass spectrogram.
An electrospray ion source for breaking an in-source disulfide bond comprises a hollow capillary tube emission needle and a hollow capillary tube emission needle support, and is characterized in that a through cavity is formed in the middle of the hollow capillary tube emission needle support, a hollow capillary tube emission needle is arranged in a cavity at the front end of the hollow capillary tube emission needle support, an ultraviolet lamp is placed on the periphery of the hollow capillary tube emission needle, and the hollow capillary tube emission needle and an ion beam cluster are collected in an ionization chamber.
Preferably, the ionization chamber of the electrospray ion source for intrasource disulfide bond fragmentation is connected at its outlet to a vacuum introduction capillary, which is connected to the inlet of the mass spectrometer.
Preferably, the bottom surface of the vacuum introduction capillary in the electrospray ion source for source-internal disulfide bond fragmentation is an arc-shaped surface, and a gap is formed between the bottom surface and the end plane of the hollow capillary emission needle bracket; vacuum is introduced into the secondary gas channel of the capillary.
Preferably, the tip of the hollow capillary emission needle in the electrospray ion source for breaking the intraclass disulfide bonds exceeds the end plane of the hollow capillary emission needle support by 10 mm and extends into the vacuum introduction capillary by 5 mm.
Preferably, the gas introduced into the auxiliary gas channel in the electrospray ion source for intra-source disulfide bond fragmentation is nitrogen, oxygen, argon, hydrogen, air, or a mixture of several of the above gases, and an atmosphere for liquid evaporation, specifically is one or more of organic acid, methanol, ethanol, acetone, isopropanol, and organic solvent.
Preferably, the auxiliary gas channels on the side surface of the vacuum introduction capillary in the electrospray ion source for intrasource disulfide bond fragmentation are uniformly distributed around the emission support of the hollow capillary, and the number of the auxiliary gas channels is 1-16.
Preferably, the inner wall of the cavity of the hollow capillary emission needle support in the electrospray ion source for breaking the intracorporeal disulfide bonds is provided with an ultraviolet light reflector;
ultraviolet lamps are arranged between the hollow capillary emission needle and the ultraviolet light reflector, and the number of the ultraviolet lamps is 1-16.
Preferably, the hollow capillary emission needle in the electrospray ion source for intrasource disulfide bond fragmentation is a hollow glass capillary or a hollow metal capillary.
Preferably, in the electrospray ion source for breaking the intraclass disulfide bonds, the inlet of the hollow capillary emission needle holder is connected with a high-voltage electric connector, and the vacuum introduction capillary, the mass spectrometer inlet, the vacuum introduction capillary, the high-voltage electric connector and the hollow capillary emission needle holder are arranged on the same axis.
Preferably, the pressure in the ionization chamber of the electrospray ion source for intrasource disulfide bond fragmentation is less than atmospheric pressure.
Compared with the background technology, the invention has the advantages that the emission tip and the vacuum lead-in capillary are perfectly coaxial by reasonable design, the brand-new airflow design is adopted, the gas flow rate at the outlet of the emission needle of the hollow capillary is ensured to be zero, and the reasonably designed vacuum lead-in capillary enables the ion clusters emitted in the fan shape to be focused towards the center of the vacuum lead-in capillary and leads the ions into the mass spectrometer. The invention has the advantages of stable ion emission, high ion transmission efficiency and the like, and is an ideal electrospray ion source at present.
Drawings
FIG. 1 is a schematic cross-sectional view of the present invention
FIG. 2 is a schematic diagram of the molecular structure of the sample
FIG. 3 comparison of results
Detailed Description
The following detailed description of the invention
Example 1
According to the structure shown in fig. 1, an electrospray ion source for source internal disulfide bond fragmentation is manufactured, and comprises a hollow capillary emission needle 10 and a hollow capillary emission needle support 40, wherein the middle of the hollow capillary emission needle support 40 is a through cavity, a hollow capillary emission needle 10 is arranged in the cavity at the front end of the hollow capillary emission needle support 40, an ultraviolet lamp 30 is placed on the periphery of the hollow capillary emission needle 10, and the hollow capillary emission needle 10 and an ion beam cluster 6 are collected in an ionization chamber 90.
The implementation process comprises the following steps: the medicinal micromolecules and organisms containing disulfide bonds are mainly shunted into the hollow capillary tube to be emitted 10, and because the hollow capillary tube emission needle is made of ultraviolet light transmitting materials, the polypeptide biomacromolecules containing disulfide bonds in the emission needle are irradiated by ultraviolet light emitted by the ultraviolet lamp 30, namely the hollow capillary tube emission needle 10, the ultraviolet light intensity at the position is enhanced, and the fragmentation efficiency of the disulfide bonds is improved. The ultraviolet light breaks partial disulfide bonds of the polypeptide biomacromolecules, the polypeptide biomacromolecules with broken disulfide bonds and without breaking are subjected to electrospray ionization through the tip of the emission needle to form an ion beam cluster 60, and auxiliary gas enters the ionization chamber 90 through the auxiliary gas channel 50 to improve ionization efficiency, so that the ion density of the ion beam cluster and the signal-to-noise ratio of ion signals are improved.
Samples for the examples: the molecular formula of the sample is: c107H185N27O43S2The molecular weight is: 2601.9230, the sample molecule C is composed of polypeptide molecule A (1066.2360) and polypeptide molecule B (1537.7030) hinged by a disulfide bond, and the molecular structure is shown in FIG. 2.
Fig. 3 shows the result of this example, the upper part of fig. 3 is the ion spectrum of the sample molecule when the ultraviolet lamp is turned off, the middle ion spectrum is obtained after the ultraviolet lamp is turned on, and the sample molecule C is shown in the figure, and the polypeptide molecule a and the polypeptide molecule B appear in the same spectrum. The lower part of the graph shows that the signal intensity is increased from 3.04E7 to 5.30E7 after the auxiliary gas is added, which shows that the auxiliary gas has the function of enhancing the signal.
Example 2
According to the structure shown in the attached figure 1, an electrospray ion source for source internal disulfide bond fragmentation is manufactured, and the electrospray ion source comprises a hollow capillary emission needle 10 and a hollow capillary emission needle support 40, wherein the middle of the hollow capillary emission needle support 40 is a through cavity, a hollow capillary emission needle 10 is arranged in the front end cavity of the hollow capillary emission needle support 40, an ultraviolet lamp 30 is placed on the periphery of the hollow capillary emission needle 10, the hollow capillary emission needle 10 and an ion beam cluster 60 are gathered in an ionization chamber 90, the outlet of the ionization chamber 90 is connected with a vacuum introduction capillary 70, and the vacuum introduction capillary 70 is connected with a mass spectrometer inlet 80. The bottom surface of the vacuum introduction capillary 70 is an arc surface, and a gap is formed between the bottom surface and the end plane of the hollow capillary emission needle support 40; a vacuum is introduced into the secondary gas channel 50 of the capillary 70.
The samples and the implementation process used in the present embodiment are the same as those in the present embodiment, and the obtained results are also shown in fig. 3, but the corresponding signal intensity values are different, which is more advantageous.
Example 3
According to the structure shown in the attached figure 1, an electrospray ion source for source internal disulfide bond fragmentation is manufactured, and the electrospray ion source comprises a hollow capillary emission needle 10 and a hollow capillary emission needle support 40, wherein the middle of the hollow capillary emission needle support 40 is a through cavity, a hollow capillary emission needle 10 is arranged in the front end cavity of the hollow capillary emission needle support 40, an ultraviolet lamp 30 is placed on the periphery of the hollow capillary emission needle 10, the hollow capillary emission needle 10 and an ion beam cluster 60 are gathered in an ionization chamber 90, the outlet of the ionization chamber 90 is connected with a vacuum introduction capillary 70, and the vacuum introduction capillary 70 is connected with a mass spectrometer inlet 80. The bottom surface of the vacuum introduction capillary 70 is an arc surface, and a gap is formed between the bottom surface and the end plane of the hollow capillary emission needle support 40; a vacuum is introduced into the secondary gas channel 50 of the capillary 70. The needle point of the hollow capillary emission needle 10 exceeds the end plane of the hollow capillary emission needle support 40 within 10 mm, and extends into 70 vacuum leading-in capillaries within 5 mm; the gas introduced into the auxiliary gas channel 50 is nitrogen, and the applicant takes oxygen, argon, hydrogen, air or the mixture of several gases in the gases as a contrast test at the same time, and the obtained result has similar good effect; and the atmosphere of the liquid evaporation is methanol, and is also replaced by one or more of organic acid, ethanol, acetone, isopropanol and organic solvent, and the similar good effect is also achieved.
The samples and the implementation process used in the present embodiment are the same as those in the present embodiment, and the obtained results are also shown in fig. 3, but the corresponding signal intensity values are different, which is more advantageous.
Example 4
According to the structure shown in the attached figure 1, an electrospray ion source for source internal disulfide bond fragmentation is manufactured, and the electrospray ion source comprises a hollow capillary emission needle 10 and a hollow capillary emission needle support 40, wherein the middle of the hollow capillary emission needle support 40 is a through cavity, a hollow capillary emission needle 10 is arranged in the front end cavity of the hollow capillary emission needle support 40, an ultraviolet lamp 30 is placed on the periphery of the hollow capillary emission needle 10, the hollow capillary emission needle 10 and an ion beam cluster 60 are gathered in an ionization chamber 90, the outlet of the ionization chamber 90 is connected with a vacuum introduction capillary 70, and the vacuum introduction capillary 70 is connected with a mass spectrometer inlet 80. The bottom surface of the vacuum introduction capillary 70 is an arc surface, and a gap is formed between the bottom surface and the end plane of the hollow capillary emission needle support 40; a vacuum is introduced into the secondary gas channel 50 of the capillary 70. The needle point of the hollow capillary emission needle 10 exceeds the end plane of the hollow capillary emission needle support 40 within 10 mm, and extends into 70 vacuum leading-in capillaries within 5 mm; the gas introduced into the auxiliary gas channel 50 is nitrogen, and the applicant takes oxygen, argon, hydrogen, air or the mixture of several gases in the gases as a contrast test at the same time, and the obtained result has similar good effect; and the atmosphere of the liquid evaporation is methanol, and is also replaced by one or more of organic acid, ethanol, acetone, isopropanol and organic solvent, and the similar good effect is also achieved.
The auxiliary gas channels 50 on the side surface of the vacuum introduction capillary 70 are uniformly distributed around the hollow capillary emission support 40, and the number of the auxiliary gas channels is 4, 6, 8, 12 and 16 respectively, so that corresponding effects can be obtained, and only when the auxiliary gas channels are uniformly distributed, the auxiliary gas channels have better effects; the inner wall of the cavity of the hollow capillary emission needle bracket 40 is provided with an ultraviolet reflector 20; ultraviolet lamps 30 are arranged between the hollow capillary tube emitting needle 10 and the ultraviolet reflector 20, and the number of the ultraviolet lamps is respectively 4, 6, 8, 12 and 16, so that corresponding effects can be achieved, and the ultraviolet lamps have better effects only when the hollow capillary tube emitting needle is uniformly distributed.
The samples and the implementation process used in the present embodiment are the same as those in the present embodiment, and the obtained results are also shown in fig. 3, but the corresponding signal intensity values are different, which is more advantageous.
Example 5
According to the structure shown in the attached figure 1, an electrospray ion source for source internal disulfide bond fragmentation is manufactured, and the electrospray ion source comprises a hollow capillary emission needle 10 and a hollow capillary emission needle support 40, wherein the middle of the hollow capillary emission needle support 40 is a through cavity, a hollow capillary emission needle 10 is arranged in the front end cavity of the hollow capillary emission needle support 40, an ultraviolet lamp 30 is placed on the periphery of the hollow capillary emission needle 10, the hollow capillary emission needle 10 and an ion beam cluster 60 are gathered in an ionization chamber 90, the outlet of the ionization chamber 90 is connected with a vacuum introduction capillary 70, and the vacuum introduction capillary 70 is connected with a mass spectrometer inlet 80. The bottom surface of the vacuum introduction capillary 70 is an arc surface, and a gap is formed between the bottom surface and the end plane of the hollow capillary emission needle support 40; a vacuum is introduced into the secondary gas channel 50 of the capillary 70. The needle point of the hollow capillary emission needle 10 exceeds the end plane of the hollow capillary emission needle support 40 within 10 mm, and extends into 70 vacuum leading-in capillaries within 5 mm; the gas introduced into the auxiliary gas channel 50 is nitrogen, and the applicant takes oxygen, argon, hydrogen, air or the mixture of several gases in the gases as a contrast test at the same time, and the obtained result has similar good effect; and the atmosphere of the liquid evaporation is methanol, and is also replaced by one or more of organic acid, ethanol, acetone, isopropanol and organic solvent, and the similar good effect is also achieved.
The auxiliary gas channels 50 on the side surface of the vacuum introduction capillary 70 are uniformly distributed around the hollow capillary emission support 40, and the number of the auxiliary gas channels is 4, 6, 8, 12 and 16 respectively, so that corresponding effects can be obtained, and only when the auxiliary gas channels are uniformly distributed, the auxiliary gas channels have better effects; an ultraviolet reflector 20 is arranged on the inner wall of the cavity of the hollow capillary tube emission needle support 40, and the ultraviolet reflector 20 reflects and focuses ultraviolet light radiated outwards to the center of a circle; ultraviolet lamps 30 are arranged between the hollow capillary tube emitting needle 10 and the ultraviolet reflector 20, and the number of the ultraviolet lamps is respectively 4, 6, 8, 12 and 16, so that corresponding effects can be achieved, and the ultraviolet lamps have better effects only when the hollow capillary tube emitting needle is uniformly distributed.
The hollow capillary tube emitting needle 10 is a hollow glass capillary tube or a hollow metal capillary tube; the inlet of the hollow capillary emission needle 10 of the hollow capillary emission needle support 40 is connected with a high-voltage electric connector, and the vacuum introduction capillary 70, the mass spectrometer inlet 80, the vacuum introduction capillary 70, the high-voltage electric connector and the hollow capillary emission needle support 40 are arranged on the same axis. The pressure of the ionization chamber 90 is less than atmospheric pressure.
The samples and the implementation process used in the present embodiment are the same as those in the present embodiment, and the obtained results are also shown in fig. 3, but the corresponding signal intensity values are different, which is more advantageous.
Claims (9)
1. An electrospray ion source for source internal disulfide bond fragmentation comprises a hollow capillary tube emission needle (10) and a hollow capillary tube emission needle support (40), and is characterized in that a through cavity is arranged in the middle of the hollow capillary tube emission needle support (40), a hollow capillary tube emission needle (10) is arranged in the cavity at the front end of the hollow capillary tube emission needle support (40), an ultraviolet lamp (30) is placed on the periphery of the hollow capillary tube emission needle (10), and the hollow capillary tube emission needle (10) and an ion beam group (60) are converged in an ionization chamber (90); the outlet of the ionization chamber (90) is connected with a vacuum introduction capillary (70), and the vacuum introduction capillary (70) is connected with the inlet (80) of the mass spectrometer;
the in-source disulfide bond is characterized in that in an ionization cavity inside an electrospray ion source, medicinal small molecules and biological large molecules containing disulfide bonds are irradiated by ultraviolet light to fragment the disulfide bonds, fragment molecules after the disulfide bonds are fragmented form ion beam groups after passing through the tip of an emission needle, and the signal-to-noise ratio of ion signals of the ion source is improved through auxiliary gas, so that stable ion current is finally obtained.
2. An electrospray ion source for intrasource disulfide bond fragmentation according to claim 1, characterized in that the bottom surface of the vacuum introduction capillary (70) is an arc-shaped surface having a gap with the end plane of the hollow capillary emission needle holder (40); vacuum is introduced into the auxiliary gas channel (50) of the capillary (70).
3. An electrospray ion source for intrasource disulfide fragmentation according to claim 2, characterized in that the tip of the hollow capillary emission needle (10) is within 10 mm of the end plane of the hollow capillary emission needle holder (40) and protrudes (70) into the vacuum introduction capillary within 5 mm.
4. An electrospray ion source for intrasource disulfide bond fragmentation according to claim 2, characterized in that the gas introduced into the auxiliary gas channel (50) is nitrogen, oxygen, argon, hydrogen, air, or a mixture of these gases, and the liquid evaporation atmosphere is one or more of organic acid, methanol, ethanol, acetone, isopropanol, and organic solvent.
5. An electrospray ion source for intrasource disulfide bond fragmentation according to claim 4, characterized in that the auxiliary gas channels (50) on the sides of the vacuum introduction capillaries (70) are uniformly distributed around the hollow capillary emission support (40) in a number of 1-16.
6. An electrospray ion source for intrasource disulfide bond fragmentation according to claim 1, characterized in that the inner wall of the cavity of the hollow capillary emission needle holder (40) has an ultraviolet light reflector (20);
ultraviolet lamps (30) are arranged between the hollow capillary tube emitting needle (10) and the ultraviolet light reflector (20), and the number of the ultraviolet lamps is 1-16.
7. An electrospray ion source for intrasource disulfide bond fragmentation according to claim 1, characterized in that the hollow capillary emission needle (10) is a hollow glass capillary or a hollow metal capillary.
8. An electrospray ion source for intrasource disulfide bond fragmentation according to claim 1, characterized in that the inlet of the hollow capillary emission needle (10) of the hollow capillary emission needle holder (40) is connected to a high voltage electrical connector, and the vacuum introduction capillary (70), the mass spectrometer inlet (80), the vacuum introduction capillary (70), the high voltage electrical connector, and the hollow capillary emission needle holder (40) are coaxial.
9. An electrospray ion source for intrasource disulfide fragmentation according to claim 1, characterized in that the pressure of the ionization chamber (90) is less than atmospheric pressure.
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| US6294780B1 (en) * | 1999-04-01 | 2001-09-25 | Varian, Inc. | Pulsed ion source for ion trap mass spectrometer |
| US7919746B2 (en) * | 2007-10-16 | 2011-04-05 | Perkinelmer Health Sciences, Inc. | Atmospheric pressure ion source performance enhancement |
| CN102142352B (en) * | 2011-02-12 | 2013-05-08 | 浙江好创生物技术有限公司 | Electro-spray ion generator |
| CN102800554B (en) * | 2012-09-02 | 2015-08-19 | 王利兵 | A kind of multimode ionization method |
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