CN111965292A - Multidimensional dual-channel liquid chromatogram-mass spectrum combined device - Google Patents
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Abstract
The invention relates to a multidimensional double-channel liquid chromatogram-mass spectrum combined device which comprises a first liquid phase pump, a second liquid phase pump, a six-way valve, a primary analytical column, a first secondary analytical column, a second secondary analytical column and liquid chromatogram components of an analytical column position switching device. The liquid chromatogram-mass spectrum combined device can prevent the damage of specific mobile phase components in the multidimensional liquid chromatogram to a mass spectrum system, and particularly has good effect in the research of proteomics.
Description
Technical Field
The invention belongs to the technical field of liquid chromatography-mass spectrometry (LC-MS) in chemical analysis, and particularly relates to a multidimensional dual-channel LC-MS device and an application method of the device, in particular to application in proteome identification.
Background
Proteins are the translation products of genetic information and the major contributors to biological functions in living organisms, catalyzing and controlling most processes in the organism. The traditional protein research mainly focuses on the structure and function of a single protein, and the result is matched with the existing gene sequence information, so as to explain the physiological or pathological phenomena of the organism. However, due to the complexity of life activities, most physiological or pathological processes often require the participation of multiple biomolecules (especially protein molecules), and the research on a single protein does not reflect the kinds and interaction relationships among multiple proteins involved in physiological or pathological processes. Therefore, with the development of technology, proteomics targeting the entire proteome of a specific biological system has begun to appear and have rapidly developed in recent years. Proteomics is an emerging discipline for the large-scale systematic analysis of a whole set of proteins within a living body, and its scope includes not only large-scale qualitative and quantitative analysis of proteins, but also post-translational modification, interaction, structural and functional studies of proteins.
Mass Spectrometry (MS) is an important tool in proteomics research. Mass spectrometry has sufficient analytical sensitivity and breadth of analysis to match the complexity of the information in proteomic studies compared to other proteomic study tools (e.g., 2D PAGE). In Large scale bottom-up proteomics, the high complexity of peptide mixtures resulting from protease digestion requires an efficient separation method prior to mass spectrometer analysis, and therefore two orthogonal separation methods are commonly employed, such as the developed multidimensional protein identification technology (multidimensional protein identification technology, MudPIT) (Washburn, m.p. et al, Large-scale analysis of the surface protein by multidimensional protein identification technology, nat biotechnology, (19), 242-7, 2001). This revolutionary technology has evolved the model of sample preparation in proteomics from 2D gel to direct digestion in solution, and has led to proteomics operating from a low throughput off-line (off-line) mode to a high throughput on-line (on-line) mode.
Mass spectrometry technology is a central part of proteomics research tools, and mass spectrometers become more advanced with increasing precision of the internal components of the instrument, but the compatibility of the MudPIT is poorer, in part because, during classical elution of the MudPIT peptide, the peptide will move from the strong cation exchange (SCX) portion to the Reversed Phase (RP) portion through a salt gradient and then elute from the RP to the mass spectrometer. This process will repeat for several cycles as the elution salt concentration increases from 1% to 100% (Washburn, m.p., et al, supra). Although mass-compatible volatile salts (e.g., ammonium acetate) are widely used in proteomic analysis, the ultra-high sensitivity characteristic greatly reduces salt tolerance in modern mass spectrometry instruments, even for volatile salts. Introduction of high concentrations into mass spectrometry instruments can rapidly contaminate ion optics, thereby greatly reducing sensitivity and performance. In addition, in the form of cationsNH in subform4 +The ions also increase the polydispersity of the peptide ions, thereby reducing the mass of the mass spectrum. Therefore, a desalting step must be performed before injecting the sample into the mass spectrometer. Two representative methods are desalting by a C18 trapping column (trapping column) and separating the peptides in an SCX column using a pH gradient instead of a salt gradient. However, both of these representative methods have inherent drawbacks: the method of desalting by the C18 trap column is generally by adding a portion of C18 downstream of SCX as a biphasic trap column, which requires the liquid phase to be broken off so that the salt composition therein is completely washed into waste before the peptides retained in the analytical column can continue to be eluted using an organic mobile phase gradient; while the pH gradient method, although it can use the traditional MudPIT setup, requires the use of a pH gradient instead of a salt gradient in the SCX column, or the addition of a new loop-trapping column to complete the acid-salt-pH gradient of the MudPIT, such orthogonality is not satisfactory or the dead volume is too high to result in the loss of a large amount of sample, and these methods are too complex to be widely used. In order to realize intensive research on proteomics without contaminating the mass spectrometer, offline operation has been recently attempted, however, the offline mode is not only inefficient, but also causes a large amount of sample loss.
Therefore, how to optimize the classical MudPIT method to maintain high throughput analysis capability while avoiding the introduction of any salt species into the mass spectrometry system is a technical problem that those skilled in the art expect to solve.
Disclosure of Invention
In order to solve the technical problems, the inventor provides the technical scheme of the invention.
The invention relates to a multidimensional dual-channel liquid chromatography-mass spectrometry combined device, wherein the liquid chromatography component comprises: the device comprises a first liquid phase pump, a second liquid phase pump, a six-way valve, a primary analytical column, a first secondary analytical column, a second secondary analytical column and an analytical column position switching device; wherein the first secondary analytical column and the second secondary analytical column are identical; wherein the connection state of the component is selected from one of a state A and a state B, and the state A and the state B are switched by the six-way valve; the state a is composed of a first path and a second path, wherein the first path includes the first liquid phase pump, the primary analytical column, the six-way valve, and the first secondary analytical column in this order, and the second path includes the second liquid phase pump, the six-way valve, the second secondary analytical column in this order; the state B is composed of a third path and a fourth path, wherein the third path sequentially includes the first liquid phase pump, the primary analytical column, the six-way valve, and the second secondary analytical column, and the fourth path sequentially includes the second liquid phase pump, the six-way valve, and the first secondary analytical column; wherein the analytical column position switching device is for switching the alignment of the outlet of the first or second secondary analytical column with the ion source inlet of the mass spectrum such that the outlet of the first secondary analytical column is aligned with the ion source inlet of the mass spectrum and the outlet of the second secondary analytical column is not aligned with the ion source inlet of the mass spectrum, or such that the outlet of the second secondary analytical column is aligned with the ion source inlet of the mass spectrum and the outlet of the first secondary analytical column is not aligned with the ion source inlet of the mass spectrum.
In the present invention, the term "multidimensional dual channel" refers to a liquid chromatography structure having a primary analytical column and a secondary analytical column with two Parallel Channels (PC), for example, when this type of multidimensional dual channel liquid chromatography-mass spectrometry apparatus is used in a multidip protocol, it may be referred to as "PC-multidip" (Parallel Channels-multidimensional protein identification technology, dual channel). In contrast, prior art MudPIT may alternatively be referred to herein as "classical MudPIT" or "traditional MudPIT".
In the present invention, the term "liquid chromatography-mass spectrometry" is used interchangeably herein with the terms "liquid chromatography-mass spectrometry", LC-MS, LC/MS, and the like, and means an analysis system of a chemical substance using liquid chromatography as a separation system and mass spectrometry as a detection system. In the invention, the mass spectrum included in the multidimensional dual-channel LC-MS device can be a 1-level mass spectrum, and can also be a multi-level tandem mass spectrum, such as a 2-level tandem mass spectrum LC-MS/MS. The mass spectrometer of the invention can be of a type known to those skilled in the art, except for the sample injection system. For example, types of ion sources for mass spectrometry of the present invention include, but are not limited to, electron ionization, chemical ion, field ionization, Fast Atom Bombardment (FAB), Matrix Assisted Laser Desorption Ionization (MALDI), pass through (ESI), atmospheric pressure chemical ionization source (APCI), preferably electrospray ionization source (ESI) or atmospheric pressure chemical ionization source (APCI), more preferably electrospray ionization (ESI); the mass analyzer of the mass spectrometer of the present invention includes, but is not limited to, a single focus mass analyzer, a double focus mass analyzer, a quadrupole mass analyzer, an ion trap mass analyzer, a fourier transform ion cyclotron resonance (FT-ICR) or a time of flight mass analyzer (TOF), and when the mass spectrometer is in the form of a multi-stage tandem mass spectrometer, the mass analyzers used therein may be the same, for example, triple quadrupole mass analyzers are used, and the mass analyzers used therein may also be any two or more of the mass analyzers described above, for example, a tandem mass spectrometer composed of a quadrupole mass spectrometer and a time of flight mass spectrometer.
The invention relates to a multidimensional dual-channel liquid chromatogram-mass spectrum coupling device, which is one of multidimensional liquid chromatogram coupling technologies. Multidimensional liquid chromatography may employ two or more analytical columns of different dimensions (i.e., different separation principles) in the liquid chromatography portion (used interchangeably herein with "chromatographic columns"). Preferably, the present invention employs two analytical columns of different dimensions in the liquid chromatography section, namely a primary analytical column and a secondary analytical column, wherein the secondary analytical column has two analytical columns, divided into a first secondary analytical column and a second secondary analytical column. In one embodiment, the first and second secondary analytical columns are identical. In another embodiment, the first and second secondary analytical columns are different. In one embodiment, the primary analytical column is a cation exchange chromatography (SCX) column. In another embodiment, the secondary analytical column is a reverse phase chromatography (RP) column. In a preferred embodiment, the primary analytical column is an SCX column, and the first and second of the secondary analytical columns are both RP columns.
The "liquid phase pump" or "first liquid phase pump" or "second liquid phase pump" referred to in the present invention may use the type of liquid phase pump known to those skilled in the art, including but not limited to a constant flow pump or a constant pressure pump, or a reciprocating pump, an accumulation type reciprocating pump, a syringe pump, a pneumatic amplification pump divided from other angles. In the present invention, the first liquid-phase pump and the second liquid-phase pump are the same or different. In one embodiment, the first or second liquid phase pump of the present invention may be obtained by modifying a known liquid phase pump, for example by increasing the pressure, or by offline loading using an air pump.
The "six-way valve" referred to in the present invention may use a six-way valve as used in the art of liquid chromatography, which is known to those skilled in the art. The six-way valve has 6 interfaces for connecting other liquid-phase components (e.g., a liquid-phase pump or an analytical column), and can switch the connection state between the components connected thereto, and the function of the six-way valve can be alternatively described as "for switching flow paths" in the present invention. The connection mode of the six-way valve in the multidimensional liquid chromatography technology and the technology of switching the connection state of the liquid phase assembly are known to those skilled in the art (see for example CN110346478A, CN 101169391A). In one embodiment of the present invention, the six-way valve of the present invention can be connected to other components (e.g., a liquid phase pump, a primary analytical column, or a secondary analytical column) through a capillary tube, preferably a 50 μm 365 μm capillary tube.
Through the six-way valve, the multi-dimensional dual-channel LC-MS can be switched between two connection states (namely, the state A and the state B), and the multi-dimensional dual-channel LC-MS can be only in one of the state A and the state B at one time point, namely, the state A and the state B cannot exist at the same time. Wherein, as described above, state a is composed of a first path and a second path, wherein the first path includes, in order, a first liquid phase pump, a primary analytical column, a six-way valve, and a first secondary analytical column, and the second path includes, in order, a second liquid phase pump, a six-way valve, and a second secondary analytical column; the state B is composed of a third path and a fourth path, wherein the third path includes a first liquid phase pump, a primary analytical column, a six-way valve, and a second secondary analytical column in this order, and the fourth path includes a second liquid phase pump, a six-way valve, and a first secondary analytical column in this order. In the context of the present invention, in relation to the LC MS of the present invention, the meaning of "sequentially" means that, starting from the liquid pump of any one of the above-mentioned channels, the order of the components passing from upstream to downstream in the direction of flow of the mobile phase in the liquid chromatograph is fixed, rather than being combinable at will, for example, the order of the connection of the individual liquid components in the first channel from upstream to downstream as described above must be in the order of "first liquid pump → primary analytical column → six-way valve → first secondary analytical column" rather than in other opposite or different order, and the order of the connection of the individual liquid components in the second channel from upstream to downstream as described above must be in the order of "second liquid pump → six-way valve → second secondary analytical column"; likewise, the order of connection of the respective liquid-phase modules in the third passage from upstream to downstream as described above must be in the order of "first liquid-phase pump → primary analytical column → six-way valve → second secondary analytical column" and cannot be in other opposite or different orders, and the order of connection of the respective liquid-phase modules in the fourth passage from upstream to downstream as described above must be in the order of "second liquid-phase pump → six-way valve → first secondary analytical column" and cannot be in other different orders. In one embodiment of the invention, the switching of the six-way valve is manual. In another embodiment of the invention, the switching of the six-way valve is automatic, for example by being automated in a computer-controlled manner.
In accordance with the principles of the present invention, the liquid chromatography portion of the present invention separates the sample and then transfers the sample to mass spectrometry for analysis. Therefore, in the multi-dimensional dual channel LC MS of the present invention, one of the first and second secondary analytical columns is aligned with the ion source inlet of the mass spectrometer, in other words, at the same time point, one and only one of the first and second secondary analytical columns is aligned with the mass spectrometer ion source inlet and the other is not aligned with the mass spectrometer ion source inlet. The alignment of the first or second secondary analytical column with the inlet of the mass spectrometry ion source is switched by an analytical column position switching device, which may be switched manually or automatically, for example by a computer program.
In the multidimensional dual-channel LC/MS device, a hydraulic electric device is arranged between the first secondary analytical column and the six-way valve, and a hydraulic electric device is arranged between the second secondary analytical column and the six-way valve. Preferably, the hydraulic device is a PEEK micro tee, a first end or a second end of the PEEK micro tee is connected with the six-way valve and the first secondary analytical column or the six-way valve and the second secondary analytical column, respectively, and a third end of the PEEK micro tee is connected with the hydraulic device.
A second aspect of the present invention relates to a method for analyzing proteome, wherein the method employs the multi-dimensional dual channel liquid chromatography-mass spectrometry apparatus according to the first aspect of the present invention, and comprises the steps of:
(1) the multidimensional dual-channel liquid chromatography-mass spectrometry combined device is in the state A through a six-way valve, and a first secondary analysis column is not aligned with an ion source inlet of a mass spectrum and a second secondary analysis column is aligned with the ion source inlet of the mass spectrum through an analysis column position conversion device;
(2) injecting the pretreated proteome sample by liquid chromatography;
(3) passing the sample through the primary analytical column, the six-way valve, and into the first secondary analytical column for separation by a first liquid phase pump and using a first mobile phase;
(4) switching the six-way valve from state a to state B, flushing the sample in the first secondary analytical column by the second liquid phase pump and using the second mobile phase;
(5) aligning the first secondary analytical column with an ion source inlet of a mass spectrometer and the second secondary analytical column with an ion source inlet of the mass spectrometer by an analytical column position translation device such that proteins in the first analytical column enter the mass spectrometer through the ion source inlet of the mass spectrometer for mass spectrometric detection and, at the same time, separating a sample through the primary analytical column, a six-way valve, and into the second secondary analytical column by a first liquid phase pump and using a first mobile phase;
(6) switching the six-way valve from state B to state a, flushing the sample in the second secondary analytical column by the second liquid phase pump and using the second mobile phase;
(7) aligning the second secondary analytical column with an ion source inlet of the mass spectrometer and the first secondary analytical column with no ion source inlet of the mass spectrometer by an analytical column position translation device such that protein in the second analytical column enters the mass spectrometer through the ion source inlet of the mass spectrometer for detection and, at the same time, separating the sample through the primary analytical column, the six-way valve, and into the first secondary analytical column by a first liquid phase pump and using a first mobile phase;
(8) and (5) circularly repeating the step (4) to the step (7) until the analysis of the proteome is finished.
In one embodiment of the present invention, for the purpose of cyclic repetition in step (8), the time used for step (4) and step (6) is the same, and the time used for step (5) and step (7) is the same.
In one embodiment of the invention, the binding of the protein in the sample to the stationary phase in the first secondary analytical column or the second secondary analytical column is stronger in the first mobile phase than in the second mobile phase.
In one embodiment of the invention, the sample is a sample comprising two or more proteins. For example, the sample of the present invention may be a naturally occurring proteome derived from a human body, an animal, a plant, a microorganism, or the like, or may be an artificially produced proteome.
In one embodiment of the present invention, the time for washing the sample in the first secondary analytical column with the second mobile phase in step (4) is required to be such that the components in the first mobile phase do not affect the mass spectrometric detection in step (5); similarly, the time for washing the sample in the second secondary analytical column with the second mobile phase in step (6) needs to be such that the components in the first mobile phase do not affect the mass spectrometric detection in step (7); the time can be appropriately determined by those skilled in the art, for example, by detecting whether the second mobile phase used for washing in step (4) or (6) further contains a component in the first mobile phase which may affect the mass spectrometric detection.
In one embodiment of the invention, the six-way valve or the analytical column position switching device is controlled manually or automatically, for example by means of a computer program.
In one embodiment of the invention, the first mobile phase or the second mobile phase is gradient or non-gradient.
In one embodiment of the invention, the first mobile phase contains ammonium acetate and the second mobile phase does not contain ammonium acetate.
A third aspect of the present invention relates to the use of a combined liquid chromatography-mass spectrometry device as described above in proteomics studies, wherein the proteomics studies comprise qualitative or quantitative studies on proteomics.
By applying the technology of the invention, the high-throughput analysis capability of classical MuDPIT can be retained at a high level, the high-efficiency characteristic of an online mode is retained, and the sample loss is reduced; meanwhile, the two channels are arranged, so that the salt of the mobile phase is prevented from being used for mass spectrometry in the liquid chromatography process, the risk of mass spectrometry pollution is reduced, and the service life of the mass spectrometry instrument is prolonged.
Drawings
It is to be noted, however, that the appended drawings, which form a part of this specification, are provided solely to provide a further understanding of the invention in connection with the detailed description thereof, and are not intended to limit the scope of the invention. Wherein:
fig. 1 shows a schematic structure of a classical MudPIT.
Fig. 2A shows a schematic structure of a PC-MudPIT according to an embodiment of the invention.
Fig. 2B shows an operational state of a schematic structure of the PC-MudPIT according to an embodiment of the present invention.
Fig. 2C shows another operational state of the schematic structure of the PC-MudPIT according to an embodiment of the invention.
FIG. 3 shows a comparison of the analytical effects of classical and PC-MudPIT at the proteome level (FIG. 3A) and at the peptidyl fragment level (FIG. 3B). Wherein, the MudPIT refers to a MudPIT experiment in a classical mode; PC-MudPIT refers to the MudPIT experiment in dual-pass mode. Three replicates in the classical mode identified 7479 proteomes in total, and 6153 on average per experiment, and three replicates in the double-pathway mode identified 6203 proteomes in total, and 5136 on average per experiment. The three repeated experiments under the classical mode totally identify 38354 peptide segments, and the three repeated experiments under the double-channel mode totally identify 25570 peptide segments.
FIG. 4 shows the sequence coverage of the proteomes identified in the PC-MudPIT and MudPIT experiments, respectively. In the two-pass model (PC-MudPIT), the median protein sequence coverage of the three replicates averaged around 10%, and in the classical model (MudPIT) the value averaged around 12%.
FIG. 5 shows the repetition rate (A) of the protein and the repetition rate (B) of the peptide fragment in three replicates of the classical model (MudPIT).
FIG. 6 shows the protein (A) and peptide (B) repeats of a triple repeat experiment in a two-pass model (PC-MudPIT).
FIG. 7 shows the total number of peptide fragments identified in the SCX fractionation gradient in the classical MudPIT and PC-MudPIT modes, respectively.
FIG. 8 shows the isoelectric point properties of peptide fragments identified in SCX fractionation gradients in classical MudPIT.
FIG. 9 shows the isoelectric point properties of the peptide fragments identified in the SCX fractionation gradient in PC-MudPIT.
Detailed Description
Example 1 earlier stage of samplePreparation of
1. Cell culture: and (3) taking out the frozen A549 cells from the liquid nitrogen, carrying out water bath oscillation thawing at 37 ℃, centrifuging to remove the supernatant, adding 1ml of fresh culture medium, blowing, uniformly mixing, transferring into a culture bottle, supplementing 9ml of fresh culture medium, uniformly mixing, putting into an incubator at 37 ℃, and culturing for 48 hours until the cells grow over the bottom bottle wall.
2. Cell lysis: after the cells overgrow the bottom bottle wall, the cells are taken out of the incubator, the culture solution is poured out, the cells are washed twice by PBS, 2mL of pancreatin is added into each bottle of cells for enzymolysis for 2 minutes, cell lysate is collected into a 15mL centrifuge tube, the supernatant is removed by centrifugation, and the cells are washed three times by PBS.
3. Protein extraction: 1% protease inhibitor (v: v) was added to RIPA lysate per 10% of the lysate61ml of RIPA lysate was added to each cell, and the cells were lysed on an ice shaker for 30 minutes and blown up with a pipette at intervals of 10 minutes. After centrifugation at 14000rpm for 30 minutes, the supernatant was collected, the protein concentration was measured by BCA method, and the remaining supernatant was transferred to a 1.5mL Eppendorf tube, 1/3 TCA-precipitated protein (TCA: supernatant, v/v) was added thereto, the tube was left at 4 ℃ for 4 hours, and then the tube was removed by centrifugation at 14000rpm for 30 minutes, leaving the precipitate and removing the supernatant. Each tube was resuspended by adding 500mL of-20 ℃ glacial acetone each time, and centrifuged at 14000rpm for 30 minutes in triplicate. The mixture was dried in a vacuum concentrator to remove the residual acetone.
4. Reduction, alkylation and enzymolysis of protein: the dried precipitate was taken out, added with 8M urea solution (solvent formulation: 24g urea + 100. mu. LTris-HCl buffer (500mM, pH8.5) + 220. mu.L double distilled water), and dissolved with ultrasound acceleration. After the precipitate was completely dissolved, TCEP was added to give a final concentration of 5mM in the solution, and the solution was placed in a heated vibration dry bath at 650rpm and vibrated at room temperature for 20 minutes. Iodoacetamide was added to give a final concentration of 10mM in the solution, protected from light, and placed in a heated vibration dry bath at 650rpm for 20 minutes at room temperature. Diluting with 100mM Tris-HCl buffer (pH 8.5) to make the concentration of urea in the solution dilute to 2M, adding pancreatin at a ratio of 1: 50 (pancreatin: protein substrate, w/w) for enzymolysis, placing in a heated vibration dry bath, vibrating at 650rpm and 37 ℃ for 16-20 hours. Finally, a certain volume of 100% pure FA is added to make the concentration of the FA in the solution reach 5%, and the solution is acidified to stop the enzymolysis reaction.
5. Desalting of the sample: monospin C18 desalting column is selected for desalting. The eluate containing the sample was dried in a vacuum concentrator and stored in the form of powder according to the instructions.
Example 2 preparation and Loading of a chromatography column
1. Tip-containing capillary preparation: taking a quartz capillary tube with the length of about 55cm, burning off the polyimide outer layer at the middle part of the capillary tube by using the outer flame of an alcohol lamp, wiping the burnt part by using wiping paper dipped with methanol, and placing the burnt part in a laser micropipette puller to prepare the capillary tube containing the tip for later use.
2. Preparation of capillary tube containing Kasil plug: selecting 15-20 cm long quartz capillary tube with diameter of 250 μm and no deactivationMixing with ammonium formate solution at ratio of 3: 1(v/v), mixing, allowing the mixed solution to enter one end of quartz capillary by capillary phenomenon, and oven standing at 100 deg.C for 4 hr to allow the mixed solution to form porous polymer. Before use, the plugs are cut into 0.5-1mm length by ceramic slices and used for filling the subsequent SCX column.
3. Packing of chromatographic column: the corresponding solid phase filler (Aqua 5 mu m C18; Aqua 3 mu m C18; 5 mu mSCX) is selected and filled in a 1.5mL Eppendorf tube, a proper volume of methanol is added, the mixture is uniformly mixed by vortex, the 1.5mL Eppendorf tube is placed in an air pump, the air pump is closed, the capillary tube needing the filler is placed at the corresponding position of the air pump, after the capillary tube is screwed down, a valve of the air pump connected with a nitrogen tank is opened, and the methanol mixed with the filler is pressed into the prepared capillary tube by the air pressure of about 100 bar. The packing of the analytical column is Aqua 5 mu m C18, and the length of the packing is 15 cm; the packing particle size of the SCX column part was 5 μm, and the packing length was 5 cm. After the capillary tubes were packed with packing, the column was equilibrated with phase a at a pressure greater than 100bar to compact the packing. In addition, the column was activated with a volume of acetonitrile before use, and the SCX column was activated with a volume of 500mM ammonium acetate.
4. Sample loading: before loading, a certain volume of phase A is used to balance the SCX column. The sample was reconstituted with water containing 0.1% (v: v) formic acid and the sample from example 1 was loaded onto an SCX column in the same manner as the packing of a chromatography column. The SCX column was then equilibrated with at least 5 column volumes of phase a.
Example 3 LC-MS/MS analysis
3.1 the apparatus set-up of the classical MudPIT and its liquid phase set-up are as follows, respectively.
(1) Device setup for classical MudPIT: the classical mdpit setup is shown in fig. 1: one end of the PEEK miniature tee joint is connected with a liquid chromatographic pump, the other end of the PEEK miniature tee joint is connected with an SCX chromatographic column, and one end of the vertical flow path is connected with a liquid electric device of a mass spectrum. The liquid electric device is a device for charging an analyte by applying a voltage to a mobile phase at the initial end of a chromatographic column through a metal electrode. The SCX column was then connected to the analytical column via a PEEK mini two-way.
(2) Liquid phase set-up of classical MudPIT: the liquid phase used in this experiment was Thermo EASY-nLCTM1200 liquid phase device, which is used for realizing the fractionation of SCX by placing ammonium acetate with different concentrations at corresponding positions of a sample tray and sucking ammonium acetate with different concentrations by a certain volume in a loading mode, and eluting peptide fragments bound on SCX to a C18 analytical column connected at the back. The parameters for ammonium acetate loading were set as follows: sample pickup, 6 μ L; sample loading, 15 μ L. Furthermore the column was equilibrated to 4. mu.L before the gradient was started. Since the sample is a complex cell lysate, an SCX10 fractionated elution procedure was used, eluting ammonium acetate concentrations of 50mM, 100mM, 150mM, 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, 500mM in that order. The gradient was set as follows: gradient started in 3% B phase; over 100 minutes, from 3% phase B to 35% phase B; from 35% phase B to 50% phase B over 10 minutes; from 50% phase B to 100% phase B over 5 minutes; the 100% B phase eluted the residual peptide fragment from the analytical column for 5 minutes, setting the flow rate at 300 nl/min.
3.2 PC-MudPIT set-up and liquid phase set-up are as follows, respectively.
(1) Device settings for PC-MudPIT: the apparatus of the improved PC-MudPIT (Parallel Channels-MudPIT) is shown in fig. 2A, relative to the existing MudPIT: one end of the SCX column is connected with the liquid phase pump, and the other end of the SCX column is connected with one inlet of the six-way valve; while another liquid phase pump (for example, an RP liquid phase pump in the present embodiment) is connected to the other inlet of the six-way valve. The two outlets of the six-way valve are respectively connected with one RP analytical column through PEEK miniature tees (therefore, two PEEK miniature tees and two RP analytical columns are used in the embodiment), one end of each PEEK miniature tee is connected with the six-way valve, the other end of each PEEK miniature tee is connected with the RP chromatographic column, and one end of the vertical flow path is connected with a mass spectrum hydraulic electric device. The two RP analytical columns can each be switched in two states, either aligned or not aligned with the ion source inlet of the Mass Spectrometer (MS), but not simultaneously aligned with the mass spectrometer inlet. By using PC-MudPIT with this structure, ammonium acetate eluted by SCX can be removed from the pathway before RP analysis is performed, so that ammonium acetate does not enter the mass analyser with the peptide as the RP eluted peptide enters the mass spectrum.
As shown in fig. 2B and 2C, the PC-MudPIT operates in the following mode: (1) as shown in fig. 2B, in this step, RP analysis column 1 is connected and misaligned with the ion source inlet of the mass spectrum through the six-way valve and the SCX analysis column and the SCX liquid phase pump, while RP analysis column 2 is connected and aligned with the ion source inlet of the mass spectrum through the six-way valve and the RP liquid phase pump; after the start of the operation of the device, after the peptide fragment is eluted from the SCX column onto RP analytical column 1(C18 analytical column 1) by ammonium acetate, analytical column 1 with the bound eluted protein is washed with a volume of phase a; since ammonium acetate and C18 packing are not bound, ammonium acetate can be eluted from the analytical column as phase a flows; (2) as shown in fig. 2C, after the washing work of ammonium acetate from the RP analysis column 1 is completed, the position of the RP analysis column 1 is switched to a position aligned with the inlet of the mass spectrometry ion source and the position of the RP analysis column 2 is switched to a position misaligned with the inlet of the mass spectrometry ion source; meanwhile, the state of the six-way valve is switched, so that the RP analytical column 1 is connected with the passage of an RP liquid phase device (liquid phase pump), and the RP analytical column 2 is connected with the SCX column and the SCX liquid phase pump; by means of the change of state of the six-way valve and the position switching of the RP analytical columns 1&2, the peptide fragment bound to the RP analytical column 1 can be eluted by the device with a gradient elution of acetonitrile into the mass spectrum and the bound peptide fragment on SCX is made to elute from the next concentration gradient of ammonium acetate onto the RP analytical column 2 misaligned to the mass spectrum (C18 analytical column 2) and the ammonium acetate on analytical column 2 is removed by washing with phase a. (3) After the RP gradient elution of the RP analytical column 1 and the washing removal of ammonium acetate on the RP analytical column 2 are completed, the positions of the two analytical columns are exchanged again and the state of the six-way valve is switched, so that the PC-MudPIT returns to the state of step (1) (i.e., the state shown in fig. 2B) to continue working, and the step (2) is repeated after the washing work on the RP analytical column 1 is completed; and repeating the steps until the analysis work is finished. In the above process, the switching of the analytical column position and the state of the six-way valve is controlled by a computer program.
In the above-mentioned device, the SCX liquid phase device flow path and SCX column are connected, then connected to the six-way valve by 50 μm 365 μm capillary, the RP liquid phase device flow path is connected to the six-way valve by 50 μm 365 μm capillary, then connected to the two analytical columns by PEEK miniature three-way valve by 50 μm 365 μm capillary from the six-way valve. The states of the six-way valves correspond to the positions of the analytical columns one by one, and the positions of the analytical columns are aligned to the positions of the mass spectrum ion sources only when the analytical columns are connected to a flow path of the RP liquid phase device.
(2) Liquid phase set-up of PC-MudPIT: in contrast to classical MudPIT, since in this mode at least three mobile phase flow paths need to be operated simultaneously, two nano-LC two-path liquid phase devices are required: RP liquid phase apparatus for elution of analytical column RP gradient using Thermo UltiMateTM3000 Series; the SCX liquid phase device is used for the elution of the SCX column and the subsequent washing of ammonium acetate, and Thermo EASY-nLC is usedTM1200. Ammonium acetate at different concentrations in the SCX fractionation was fed to the flow path as a sample, and the fractional concentration of ammonium acetate was the same as in classical MudPIT. The residual ammonium acetate on the analytical column was washed with phase A in a volume of 25. mu.L. The subsequent RP gradient setup is the same as the classical setup of MudPIT, and after the RP gradient of the analytical column is completed, the RP liquid flow path and the connected analytical column are equilibrated with phase A, and the residue of phase B100% of the final RP gradient in the flow path is removed to prevent affecting the separation of the next analytical column, and the phase A thereofThe equilibrium volume should depend on the specific flow path dead volume.
3.3. And (4) related setting of mass spectra. Voltage is applied to the chromatographic column with a tip, and the flow junction, the peptide fragments separated by the analytical column can be directly charged into the mass spectrum by means of ESI. The voltage at the ion source was set to 2.2kV, the mass range (mass range) of MS1 was set to 300-1800m/z, the resolution 60000, and the maximum ion implantation time (mass implantation time) was set to 50 MS; in the data-dependent analysis mode, fragment ions were generated by the precursor ions entering the fragmentation cell at the top 20 of the signal intensity, the MS2 resolution was set to 15000, the normalized collision energy (normalized collision energy) was set to 27%, the maximum ion implantation time (maximum implantation time) was set to 60MS, and the Dynamic exclusion (Dynamic exclusion) duration was set to 30 MS.
3.4 data analysis methods. Spectra from the mass spectrometer were acquired by RawExtract software, pFind analysis software (Chi et al, 2018) was used for proteomic analysis, and the reviewed (Swiss-Prot) human database downloaded from the UniProt database was selected as the a549 cell line is a human non-small cell lung cancer cell line. The library searching parameters are set as follows: enzyme for enzymatic hydrolysis: trysin KR-C, full specificity site, 2 at most of the missed cutting sites, the mass deviation range of the parent ion is set as 10ppm, the mass deviation range of the daughter ion is set as 0.02Da, the urea methyl introduced by IAA alkylation process is used as a fixed modification site, and the oxidation on methionine and the acetylation of the N end of the protein are used as variable modification sites. The false positive rate (FDR) of the identification result is calculated by using reversed protein sequences, and the final result is limited to FDR > 0.1% on the peptide fragment level.
3.5 analysis of samples Using a MudPIT and PC-MudPIT apparatus
In this example, proteomes in the SCX column loaded in example 2 were analyzed using the above-described apparatus for MudPIT and PC-MudPIT. Wherein the analysis of the MudPIT is carried out according to the methods described above in the prior art. The procedure for analyzing the sample using the PC-MudPIT apparatus is described in detail below.
The analytical column of the present invention has both functions of an analytical column and a "trap column". The specific working modes are as follows: two columns were used, column 1 at position 1, column 2 at position 2, and position 2 aligned with the mass spectrometry ion source inlet. First, column 1 was connected to the SCX liquid flow path, and bound to the peptide fraction eluted from the first step gradient of SCX, and ammonium acetate was not bound to the C18 packing in column 1 and was washed out from the tip of the column. Since the position of the column 1 is now misaligned with the mass spectrometry ion source inlet, ammonium acetate does not enter the mass spectrum. The remaining ammonium acetate was then removed using a 25. mu.l phase A equilibration flow path. After completion of the equilibration, the positions of the analytical columns 1 and 2 were switched while switching the six-way valve so that the analytical column 1 was connected to the RP liquid phase flow path, followed by 120 minutes of RP gradient analysis. At this point, the analytical column 2 was connected to the SCX liquid phase flow path, and the peptide fraction eluted by the second fractional gradient of SCX was combined, and after completion of elution, the flow path was equilibrated with 25. mu. l A phase. After the analytical column 1 completes the RP gradient analysis, the A phase equilibrium flow path is used to remove the residual B phase in the RP liquid phase flow path so as not to affect the RP gradient elution of the analytical column 2. In the channel design, in order to reduce the dead volume and not cause the channel blockage, a capillary tube with the diameter of 50 μm is used for connecting the chromatographic column, the liquid phase flow path and the six-way valve; also considering the higher salt loading in the flow path due to the elution of SCX, an analytical column with an internal diameter of 100 μm was chosen, the packing was C185 μm, an SCX column with an internal diameter of 250 μm was chosen, and the packing was SCX/5 μm. To match the inner diameter of the analytical column and the size of the packing, the liquid phase flow rate of the RP gradient was chosen to be 300 nl/min.
When the gradient elution of the analytical column RP is finished, a phase A is used for balancing and connecting a flow path of an RP liquid phase. The specific volume of phase a was determined as follows: the dead volume from the liquid phase outlet to the analytical column junction was theoretically 2. mu.l, and the time it took to flow through one flow path volume was 6.7 minutes at a theoretical flow rate of 300 nl/min. In the initial experiment, the equilibration time was 10 minutes and the flow rate was 300 nl/minute, equilibrating approximately one flow path volume, and no sample peak appeared in the original spectrum at a loading of 50 μ g. To further look for the cause, the amount of samples is first increased. Until the sample amount is increased to 200. mu.g, a sample protein peak appears in the original spectrogram, but the spectrogram peak is wide, the separation effect of the analytical column is influenced by the oversize sample amount, and the oversize sample amount of 200. mu.g is excessive for the scale of the chromatographic column used. With increasing loading, a sample peak appears in the original spectrum, demonstrating to some extent that there is greater sample loss during the elution of the peptide fragment from SCX, to the RP analytical column, until the next RP analytical gradient begins. After the on-line experiment ensures that the two processes of SCX elution and RP binding are not problematic, the analytical column combined with the peptide segment only by aqueous phase equilibrium before the RP analytical column is started is observed, and the appearance of a sample peak is found in an original spectrogram, thereby indicating that a certain organic phase exists in a flow path so that the peptide segment is eluted before the next RP gradient is not started. Thus, the equilibrium time of phase A after the RP gradient was completed was adjusted to 30 minutes, the flow rate was adjusted to 300 nl/minute, and the equilibrium flow path was approximated to three flow path volumes. Under this equilibrium condition, 50. mu.g of the sample was loaded, and the following experimental results were obtained.
Example 4 data analysis and discussion of results
4.1 separation Effect detection for Dual-channel mode MudPIT
According to the characteristic that the MudPIT design has special advantages in analyzing complex proteome, an A549 whole cell lysate is selected as a sample for detecting the separation effect of a double-channel MudPIT mode, and a classical MudPIT mode is adopted as a reference standard. In the experiment, the same SCX and RP liquid phase separation gradient, the same mass spectrum method and the same data analysis mode are adopted in the two modes. The whole process of each mode is repeated three times continuously, and the pFind software is used for carrying out qualitative analysis on the spectrogram result of each time. In the same mode, the spectrograms of the same SCX grading gradient in the three repeated experiments are combined, and the same pFind qualitative analysis method is used for comparing the separation effect of the two modes.
In the two-pass model, MudPIT, 6203 proteomes were identified in three replicates, and 5136 proteomes were identified in each experiment on average; in the classical model of the MudPIT experiment, 7479 proteomes were identified in three replicates, and 6153 proteomes were identified in each experiment (see FIG. 3A). On the peptide fragment level, 25570 peptide fragments are identified in three repeated experiments of the double-channel mode MudPIT; 38354 peptidic messages were identified in classical model MudPIT (see FIG. 3B).
Further analysis of the sequence coverage of the identified proteomes revealed that in the two-pass mode, the mean value of the protein sequence coverage of the three replicates averaged around 10%, and in the classical mode around 12% (see fig. 4).
In order to detect the stability of the dual-channel mode MudPIT system, Wein graph analysis is carried out on proteomes and peptide fragments identified by three experiments respectively carried out on the classical mode MudPIT (figure 5) and the PC-MudPIT (figure 6). As can be seen from fig. 5 and 6, at the protein level, a total of 4195 proteomes were identified in three experiments in the PC-MudPIT model, accounting for 67.6% of the total proteomes; in the MudPIT mode, the number of proteomes identified by three repeated experiments is 4968, which accounts for 66.4% of the total proteome. At the peptide level, the number of peptides identified in the three experiments in the PC-MudPIT model was 11802, accounting for 28.2% of the total, while the PC-MudPIT model was 18491, accounting for 29.4% of the total. In both modes, the duplicate determinations at both proteome and peptidyl levels in the three replicates were similar, indicating that the PC-MudPIT system has comparable stability to the classical mode of MudPIT. Since the data-dependent data collection mode applied in mass spectrometry is to select the parent ion according to the signal intensity to make a secondary spectrum, the parent ion selection mode can result in a low ratio of the number of peptide fragments identified in repeated experiments to the total number.
Compared with the classical mode of MudPIT, the connection mode of the SCX and the RP of the MudPIT (PC-MudPIT) in the double-channel mode is greatly changed. In a double-channel mode, a six-way valve for switching a flow path is added between the SCX and the RP, so that the peptide fragments eluted from the SCX enter the analytical column through the six-way valve and a middle connecting channel; in the classical mode, since the SCX moiety is directly linked to the RP moiety, the peptide fragments eluted from SCX can be directly passed to the analytical column. In the two-pass mode, the peptide fragments eluted from SCX need to be equilibrated by phase a to remove residual ammonium acetate from the flow path when bound to the analytical column. Compared with the classical mode, the main modification of the two-pass mode for the purpose of removing ammonium acetate from the system is focused on the portion of SCX, especially the process of binding peptide fragments eluted from SCX to the analytical column. Therefore, in order to detect whether the change of the double channel can influence the effect of SCX separation, qualitative analysis is carried out on peptide fragments identified by different SCX grading gradients in two modes.
As shown in fig. 7, the bar graph represents the total number of peptide fragments identified in each SCX fractionation gradient, and the dots in the line graph represent the number of newly identified peptide fragments in each SCX fractionation gradient compared to the previous fractionation gradient. In the sample preparation stage, the desalted and dried peptide fragment is redissolved with double distilled water containing 0.1% formic acid, and the pH value of the solution is about 2. Under this pH condition, most of the peptide fragments carry 2-3 positive charges, and the maximum charge is not more than 5. Due to the heterogeneity of the charges carried by the peptide fragments in the sample, most of the peptide fragments are concentrated on the second, third and fourth step gradients to be eluted, and the identified number of the peptide fragments in the subsequent gradients is small. The isoelectric point of the peptide fragment is the pH value of the solution when the peptide fragment is charged to 0. In an acidic solution, the higher the isoelectric point of the peptide fragment, the more tightly it binds to the SCX column, the higher the concentration of ions required for elution, the lower the isoelectric point, the looser it binds to the SCX column, the lower the concentration of ions required for elution. As can be seen from the trend common to both modes, the peptide fragments eluted from the first fractionation gradient, i.e., 50mM ammonium acetate concentration, are less, reflecting the poor analysis effect of peptide fragments with lower isoelectric points in the MudPIT system.
In order to better evaluate the separation effect of SCX in the two-channel mode, the isoelectric point properties of the peptide fragments identified in different SCX fractionation gradients were further analyzed. As shown in FIG. 8 (classical mode of MudPIT) and FIG. 9(PC-MudPIT), comparing the isoelectric point analysis of the eluted peptide fragments with different SCX fractionation gradients in the dual channel mode and the classical mode, it can be seen that most of the peptide fragments have isoelectric points around 5-9, and the more the fractionation gradient is, the higher the mean value of the isoelectric points in the eluted peptide fragments is. Both modes exhibit similar trends. So in the dual channel mode design, although the connection of the SCX part is changed, the SCX separation achieves the same effect as in the classical mode.
Experimental results show that under a dual-channel mode, the separation stability and SCX dimension separation show results similar to those of classical MudPIT, meanwhile, due to the dual-channel design, salt in liquid chromatography is basically completely eluted, then protein enters a mass spectrometer, and the design avoids the salt of a mobile phase in the liquid chromatography process from entering the mass spectrometer, so that the risk of salt pollution of the mass spectrometer is reduced, and the service life of the mass spectrometer is prolonged.
It should be noted that the above-mentioned embodiments illustrate rather than limit the scope of the invention, and that those skilled in the art will be able to make various changes and modifications to the invention without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A multi-dimensional dual channel combined liquid chromatography-mass spectrometry apparatus, the components of the liquid chromatography comprising: the device comprises a first liquid phase pump, a second liquid phase pump, a six-way valve, a primary analytical column, a first secondary analytical column, a second secondary analytical column and an analytical column position conversion device;
wherein the first secondary analytical column and the second secondary analytical column are identical;
wherein the connection state of the component is one selected from state a and state B, and the state a and the state B are switched by the six-way valve;
the state a is composed of a first path and a second path, wherein the first path includes the first liquid phase pump, the primary analytical column, the six-way valve, and the first secondary analytical column in this order, and the second path includes the second liquid phase pump, the six-way valve, the second secondary analytical column in this order;
the state B is composed of a third path and a fourth path, wherein the third path sequentially includes the first liquid phase pump, the primary analytical column, the six-way valve, and the second secondary analytical column, and the fourth path sequentially includes the second liquid phase pump, the six-way valve, and the first secondary analytical column; and is
Wherein the analytical column position switching device is configured to switch the alignment of the outlet of the first or second secondary analytical column with the ion source inlet of the mass spectrometer such that the outlet of the first secondary analytical column is aligned with the ion source inlet of the mass spectrometer and the outlet of the second secondary analytical column is not aligned with the ion source inlet of the mass spectrometer or such that the outlet of the second secondary analytical column is aligned with the ion source inlet of the mass spectrometer and the outlet of the first secondary analytical column is not aligned with the ion source inlet of the mass spectrometer.
2. The LC-MS/MS combination of claim 1, wherein the ion source is an electrospray ionization source.
3. The combined liquid chromatography-mass spectrometry device according to claim 1 or 2, wherein the primary analysis column is a cation exchange chromatography (SCX) column and the first and second analysis columns are both reverse phase chromatography (RP) columns.
4. The LC-MS/MS apparatus of claim 1 or 2, wherein the six-way valve manually or automatically switches the connection state of the component between state A or state B; and the analysis column position conversion device switches the alignment state of the outlet of the first secondary analysis column or the outlet of the second secondary analysis column with the ion source inlet of the mass spectrometer in a manual or automatic manner.
5. The LC-MS/MS combination as claimed in claim 1 or 2, wherein an electrohydraulic means is provided between the first secondary analytical column and the six-way valve, and an electrohydraulic means is provided between the second secondary analytical column and the six-way valve; and the hydraulic pressure device is a PEEK miniature tee joint, a first end or a second end of the PEEK miniature tee joint is respectively connected with the six-way valve and the first secondary analysis column or the second secondary analysis column, and a third end of the PEEK miniature tee joint is connected with the hydraulic pressure device.
6. A method of analyzing a proteome, wherein the method is performed using the liquid chromatography-mass spectrometry combination device of claim 1, and the method comprises the steps of:
(1) bringing the LC-MS apparatus of claim 1 in said state A through said six-way valve and aligning said first secondary analytical column with an ion source inlet of said mass spectrometer and said second secondary analytical column with an ion source inlet of said mass spectrometer through said analytical column position switching device;
(2) injecting a pretreated proteome sample by the liquid chromatography;
(3) passing a sample through the primary analytical column, the six-way valve, and into a first secondary analytical column for separation in sequence by the first liquid phase pump and using a first mobile phase;
(4) switching the six-way valve from the state a to the state B, flushing the sample in the first secondary analytical column by the second liquid phase pump and using a second mobile phase;
(5) aligning the first secondary analytical column with an ion source inlet of the mass spectrometer and the second secondary analytical column with no ion source inlet of the mass spectrometer by the analytical column position translation device such that proteins in the first analytical column enter the mass spectrometer through the ion source inlet of the mass spectrometer for mass spectrometric detection and, at the same time, passing a sample through the primary analytical column, the six-way valve, and into the second secondary analytical column for separation by the first liquid phase pump and using the first mobile phase;
(6) switching the six-way valve from the state B to the state a, flushing the sample in the second secondary analytical column by the second liquid phase pump and using the second mobile phase;
(7) aligning the second secondary analytical column with an ion source inlet of the mass spectrometer and the first secondary analytical column with no ion source inlet of the mass spectrometer by the analytical column position translation device such that proteins in the second analytical column enter the mass spectrometer for detection through the ion source inlet of the mass spectrometer and, at the same time, passing a sample through the primary analytical column, the six-way valve, and into the first secondary analytical column for separation by the first liquid phase pump and using the first mobile phase;
(8) and (5) circularly repeating the step (4) to the step (7) until the analysis of the proteome is finished.
7. The method of claim 6, wherein the time taken for the step (4) and the step (6) is the same, and the time taken for the step (5) and the step (7) is the same; and is
Wherein the time for washing the sample in the first secondary analytical column with the second mobile phase in step (4) is required to be such that the components in the first mobile phase do not affect the mass spectrometric detection in step (5); and, the time for washing the sample in the second secondary analytical column with the second mobile phase in step (6) needs to be such that the components in the first mobile phase do not affect the mass spectrometric detection in step (7).
8. The method of claim 6, wherein the binding of the protein in the sample to the stationary phase in a first secondary analytical column is stronger in the first mobile phase than the binding of the protein in the sample to the stationary phase in a first secondary analytical column in the second mobile phase, and the binding of the protein in the sample to the stationary phase in a second secondary analytical column in the first mobile phase is stronger than the binding of the protein in the sample to the stationary phase in a second secondary analytical column in the second mobile phase.
9. The method of claim 6, wherein the first mobile phase contains ammonium acetate and the second mobile phase does not contain ammonium acetate.
10. Use of a combined liquid chromatography-mass spectrometry device according to any of claims 1-5 in proteomics studies comprising qualitative or quantitative proteomics.
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