JP7327096B2 - Molecular separation method - Google Patents

Description

The present invention relates to a method for separating molecules.

Molecules such as antibodies that bind to target molecules have been used to separate target molecules in a sample. A B/F (Bound/Free) separation method has been proposed for separating antibody-bound target molecules from antibody-unbound molecules contained in a sample (see Non-Patent Document 1). Patent Literature 1 describes that a sample is subjected to an immunoprecipitation operation using immobilized beads to separate molecules to be analyzed, and then the sample is subjected to mass spectrometry.

Japanese Patent No. 6424757

Ishikawa, "Principles and Problems of Enzyme Immunoassay," Clinical Chemistry, (Japan), Japan Society of Clinical Chemistry, 1978, Vol. 6, No. 2, p.106-115

It is preferable to separate target molecules more quickly and easily.

In a first aspect of the present invention, a sample and a second molecule that binds to the first molecule to be separated are prepared, and the sample is brought into contact with the liberated second molecule in a solution. , forming a complex in which the first molecule and the second molecule bind, purifying the complex based on molecular weight, and separating the first molecule from the purified complex and subjecting a sample for mass spectrometry analysis to mass spectrometry analysis.

According to the present invention, target molecules can be separated more quickly and easily.

FIG. 1 is a conceptual diagram for explaining a molecule separation method according to one embodiment. FIG. 2 is a flow chart showing the flow of the molecule separation method according to one embodiment. FIG. 3 is a conceptual diagram for explaining a molecule separation method according to a modification. FIG. 4 is a mass spectrum of a solution to which verotoxin 1 and verotoxin 2 were added in an example. FIG. 5 is a mass spectrum of a sample trapped on an ultrafiltration membrane in an example. FIG. 6 is a mass spectrum of the ultrafiltration filtrate in the example.

Embodiments for carrying out the present invention will be described below with reference to the drawings.

-Embodiment-
In the method for separating molecules of the present embodiment, a complex formed by binding a target molecule and a molecule that binds to the target molecule in a solution is separated based on the molecular weight. Here, the target molecule is a molecule to be separated. Hereinafter, a molecule that binds to a target molecule is referred to as a target-binding molecule, and a complex formed by binding a target molecule and a target-binding molecule is simply referred to as a complex.

FIG. 1 is a conceptual diagram showing the molecular separation method of this embodiment. The sample contains target molecule T and non-target molecule N1 and is dispersed in solution SN stored in tube TB (upper left of FIG. 1). The non-target molecule N1 is a molecule other than the target molecule in the sample, such as a contaminant. At this time, if the sample is subjected to mass spectrometry, a peak PT corresponding to the target molecule T and a peak PN corresponding to the non-target molecule N1 will be observed in the mass spectrum M1 obtained by the mass spectrometry. Become. Here, the mass spectrum is a graph in which the horizontal axis indicates the m/z of ions detected by mass spectrometry, and the vertical axis indicates the intensity of the detection signal of the ions. Although m/z is used as the mass-to-charge ratio below, it is not particularly limited as long as it represents the ratio of mass to charge.

In the molecular separation method of the present embodiment, the sample is brought into contact with the released target binding molecule TBM in a solution to generate a complex CX in which the target molecule T and the target binding molecule TBM are bound. In the example of FIG. 1, the arrow A1 schematically indicates that the solution containing the target binding molecule TBM is added to the solution SN containing the sample.

Conjugate CX is purified on the basis of molecular weight. In the example of FIG. 1, ultrafiltration is performed using an ultrafiltration membrane F that allows the non-target molecule N1 to permeate but does not allow the complex CX to permeate (arrow A2). In ultrafiltration, after the solution SN containing the complex CX and the like is added to the ultrafiltration membrane F, it is preferable to perform filtration using centrifugal force. The complex CX is captured by the ultrafiltration membrane F and the non-target molecule N1 is contained in the filtrate FT stored in the filtrate receiver R. After the target molecule T is separated from the complex CX captured by the ultrafiltration membrane F, the separated target molecule T can be detected by subjecting the resulting solution to mass spectrometry. In the mass spectrum M2 obtained by this mass spectrometry, the peak corresponding to the non-target molecule N1 does not appear and the peak PT corresponding to the target molecule T is observed.

FIG. 2 is a flow chart showing the flow of the molecule separation method of this embodiment. In step S1001, a sample and target binding molecules are provided. A target-binding molecule is provided that is not bound to a solid support.

(Regarding the sample)
The sample is not particularly limited as long as it is a liquid, solid, or the like that contains or may contain the target molecule to be separated. The target molecule is not particularly limited as long as it can be ionized and the target binding molecule is available. The molecular separation method of the present embodiment is also a mass spectrometry method, and can be applied to a molecular detection method by mass spectrometry. This embodiment can also be used as a method for isolating molecules. From this point of view, the target molecule can be a molecule to be detected, for example a biomarker or a hazardous substance such as a toxin. As an example, the target molecule may be verotoxin, and the method of the present embodiment may be applied to testing for food poisoning.

(Regarding target binding molecules)
The target-binding molecule is not particularly limited as long as it can be purified by utilizing the increase in molecular weight due to binding between the target molecule and the target-binding molecule during the operation of purifying the complex based on molecular weight (step S1005). From this point of view, the molecular weight of the target-binding molecule is preferably 1 kDa or more, more preferably 30 kDa or more.

The target-binding molecule is preferably a molecule capable of binding to the target molecule through antigen-antibody reaction, ligand-receptor reaction or sugar chain-lectin interaction. Thereby, the specificity of these reactions or interactions can be used to more reliably separate the complex.

When using an antigen-antibody reaction, it is preferable that the target molecule is an antigen and the target-binding molecule is an antibody, but the reverse is also possible. In the following embodiments, the term "antibody" includes immunoglobulins such as IgG (Immunoglobulin G), as well as immunoglobulin-like molecules that are not immunoglobulins but that contain variable regions responsible for antigen specificity in immunoglobulins. For example, the target molecule can be verotoxin and the target binding molecule can be an anti-verotoxin antibody.

When using a ligand-receptor reaction, the target molecule is the ligand and the target binding molecule is the receptor, or the target molecule is the receptor and the target binding molecule is the ligand. For example, the target molecule can be verotoxin and the target binding molecule can be globotriaosylceramide. When sugar chain-lectin interaction is used, it is preferable that the target molecule is a sugar chain and the target binding molecule is a lectin, but the reverse is also possible.

A target binding molecule may be a monoclonal antibody or a polyclonal antibody. In addition, in this embodiment, multiple types of molecules may be used as target-binding molecules. Target binding molecules can include multiple types of antibodies with different antigen specificities or structures.

Returning to FIG. 2 , after step S1001 ends, step S1003 is started. In step S1003, an operation is performed to bind the target molecule and the target binding molecule. This operation is hereinafter referred to as a join operation. By the binding operation, when the target molecule is contained in the sample, the target molecule and the target-binding molecule react and bind to form a complex. This reaction is hereinafter referred to as binding reaction.

The binding procedure involves contacting the sample with free target binding molecules in solution. For example, a solution containing a target binding molecule not bound to a solid support is added to a solution containing a sample. In the binding procedure, the sample and free target binding molecules are dispersed in solution. From the viewpoint of rapid binding reaction, it is preferable that the solution containing the sample and the target binding molecule is appropriately mixed by shaking or a vortex mixer.

In this embodiment, the binding reaction is performed such that the released target molecule and the released target-binding molecule bind. As a result, the reaction time can be significantly shortened compared to binding the target molecule to the target-binding molecule bound to the solid-phase carrier. The incubation time for the binding reaction in the binding procedure is preferably less than 6 hours, more preferably less than 3 hours, even more preferably less than 1 hour, and even more preferably 30 minutes or less. In addition, it is not easy to immobilize an antibody, which is a protein, to a solid-phase carrier while maintaining its activity, and it takes time and effort. However, the method of the present embodiment does not require that, and is efficient from this point of view as well. In addition, some of the activity is lost when the antibody is immobilized on the solid-phase carrier, but the method of the present embodiment also reduces such a problem.

After step S1003 ends, step S1005 is started. In step S1005, an operation for purifying the complex is performed. This operation is hereinafter referred to as a refinement operation. When a complex is formed in step S1003 by the purification operation, the complex is purified in the sample. Here, purification of the complex means that the concentration of at least part of the molecules other than the complex in the sample is relatively reduced compared to the concentration of the complex.

The purification operation is not particularly limited as long as the complex is purified based on its molecular weight. In the purification procedure, it is preferable to remove molecules having at least the same molecular weight as the target molecule from the complex-containing fraction of the sample by utilizing the increase in molecular weight due to the binding of the target-binding molecule to the target molecule. As a result, in the mass spectrometry in step S1009, it is possible to prevent the accuracy of detection of the target molecule from deteriorating due to contaminants having m/z similar to that of the target molecule.

Preferably, the purification procedure purifies the conjugate using at least one of ultrafiltration, dialysis, and size exclusion chromatography. In particular, the purification operation is preferably performed by ultrafiltration from the viewpoint of being quick and trouble-free. In ultrafiltration, it is preferable to perform filtration using centrifugal force with a centrifuge from the viewpoint of rapid purification.

In ultrafiltration, macromolecules such as proteins dissolved in water and fine particles can be separated by the pores in the porous ultrafiltration membrane. Because of the variation in pore size and the difficulty of measurement, the molecular weight cutoff is used as an index of membrane separation performance instead of the pore size. Each ultrafiltration membrane manufacturer defines the nominal molecular weight limit (NMWL) according to different standards. Molecules with molecular weights comparable to the molecular weight cutoff may or may not permeate the ultrafiltration membrane. Hereinafter, the molecular weight at which the rejection rate is 90% in the fractionation curve obtained by introducing a plurality of standard substances having different molecular weights into the ultrafiltration membrane is defined as the molecular weight cutoff.

In ultrafiltration, it is preferable to use an ultrafiltration membrane that allows the passage of molecules having a molecular weight similar to that of the target molecule but does not allow the passage of complexes. From this point of view, the molecular weight cutoff of the ultrafiltration membrane is preferably higher than the molecular weight of the target molecule and lower than the molecular weight of the complex. From the viewpoint of ensuring the permeation of molecules with a molecular weight similar to that of the target molecule, the cutoff molecular weight of the ultrafiltration membrane is more preferably higher than twice the molecular weight of the target molecule. In order to more reliably trap the complex on the ultrafiltration membrane, the cutoff molecular weight of the ultrafiltration membrane is more preferably less than 80% of the molecular weight of the complex.

For example, the target molecule is a substance with a molecular weight of 20 kDa or less, which can be measured particularly accurately by time-of-flight mass spectrometry, and the target-binding molecule is antibody IgG. IgG has a molecular weight of approximately 150 kDa. In this case, the cutoff molecular weight of the ultrafiltration membrane is preferably 30 kDa or more, more preferably 60 kDa or more, and even more preferably 90 kDa or more. The higher the molecular weight cutoff, the more reliably permeation and separation of molecules with a molecular weight similar to that of the target molecule can be achieved. In the above case, the cutoff molecular weight of the ultrafiltration membrane is preferably 130 kDa or less, more preferably 110 kDa or less. The lower the molecular weight cut off, the more reliably the complex can be trapped on the ultrafiltration membrane and the loss of the target molecule in the sample can be prevented. As a more specific example, if the target molecule is the B subunit of verotoxin (molecular weight: 7,500 to 8,000 Da) and the target binding molecule is an anti-verotoxin antibody (molecular weight: about 150 kDa), based on the above numerical examples, the limit The molecular weight cut off of the filtration membrane can be set.
The above is just an example, and the cutoff molecular weight of the ultrafiltration membrane is appropriately set according to the molecular weights of the target molecule and the complex. Alternatively, or in addition to ultrafiltration, microfiltration may be performed using microfiltration membranes having comparable properties with respect to the molecular weight of molecules to be permeated. Also in microfiltration, it is preferable from the viewpoint of rapid purification to perform filtration using centrifugal force with a centrifuge.

After step S1005 ends, step S1007 is started. In step S1007, a sample for mass spectrometry containing the target molecule separated from the complex is prepared. A sample for mass spectrometry is prepared from the purified fraction obtained in the purification procedure, which contains the complex when the sample contains the target molecule.

An operation is performed to separate the target molecule from the complex. This operation is hereinafter referred to as a separation operation. When the complex purified in step S1005 is obtained by the separation operation, the target molecule is separated from the complex.

A method for separating the target molecule from the complex is not particularly limited as long as mass spectrometry of the separated target molecule is possible. For example, the target molecule can be separated from the complex by contacting the purified fraction containing the complex with an organic solvent such as acetonitrile or an acid such as sulfuric acid.
If an organic solvent is used in the preparation of the sample for mass spectrometry, the target molecule is separated from the complex during the preparation, so there is no need to perform a separate separation operation for the main purpose of separating the target molecule. . If the mass spectrometry sample containing the target molecule separated from the complex can be obtained in this way, the separation operation can be omitted.

(Preparation of sample for mass spectrometry)
The method for preparing the sample for mass spectrometry is not particularly limited as long as it is performed according to the type of ionization in mass spectrometry (step S1009) described later. In order to reduce the amount of contaminants, an operation such as desalting may be performed as appropriate.

An example of performing matrix-assisted laser desorption/ionization (MALDI) in mass spectrometry will be described below. A solution containing or possibly containing the complex obtained by the purification operation is prepared. When the above-mentioned ultrafiltration is performed, a buffer solution such as PBS (Phosphate Buffered Saline) is added to the components remaining on the ultrafiltration membrane to appropriately desalt them to prepare the solution. This solution is called the purified solution. A solution containing a matrix (hereinafter referred to as matrix solution) is added to the purified solution, dropped onto a MALDI sample plate, and dried to obtain a crystal containing the sample and the matrix. This crystal serves as a sample for mass spectrometry. After placing the purified solution on the MALDI sample plate, the matrix solution may be added. The type of matrix is not particularly limited, and CHCA (α-cyano-4-hydroxycinnamic acid), sinapinic acid, DHB (2,5-dihydroxybenzoic acid), or the like can be used as appropriate. As a solvent for the matrix solution, an aqueous solution containing several tens of volume % of an organic solvent such as acetonitrile, to which 0 to 3 volume % of trifluoroacetic acid (TFA) is added, or the like can be used. In this case, the complexes are dissociated into target molecules by contact with the matrix solution.
In addition, the sample for mass spectrometry may be prepared using an additive for appropriately enhancing the sensitivity and the like.

After step S1007 ends, step S1009 starts. In step S1009, mass spectrometry is performed on the sample for mass spectrometry prepared in step S1007 and containing the target molecule separated from the complex.

The method of mass spectrometry is not particularly limited as long as ions derived from target molecules can be detected by mass separation. Mass spectrometry can be of any type, such as quadrupole, ion trap or time-of-flight. When the target molecule has a high mass of several kDa or more, time-of-flight mass spectrometry is preferable from the viewpoint of accurate mass separation. In the mass spectrometry, one stage of mass separation may be performed, or two or more stages of mass separation may be performed. In the case of single mass spectrometry by one-step mass separation, the target molecule can be detected quickly without much trouble. In two or more stages of mass spectrometry, more detailed information can be obtained from the detected ions, such as confirmation of whether the detected ions are derived from the target molecule, by analyzing the structure of the detected ions.

Mass spectrometry can be performed by a mass spectrometer, including one or more of any mass spectrometer, such as a quadrupole, ion trap, or time-of-flight type. Gas chromatography or liquid chromatography may be performed prior to mass spectrometry.

The ionization method in mass spectrometry is not particularly limited. From the viewpoint of ionizing macromolecules such as peptides or proteins without fragmenting them, it is preferable to perform MALDI or electrospray ionization (ESI). As the ionization method, MALDI is preferable from the viewpoint that monovalent ions are easily generated and data that is easy to analyze can be obtained. In the case of MALDI, a sample for mass spectrometry prepared as described above is irradiated with laser light to ionize the sample.

In mass spectrometry, it is preferable to scan the m/z of ions to be mass-separated to acquire data for obtaining a mass spectrum. The data obtained by mass spectrometry are called mass spectrometry data. The mass spectrometry data is stored in a storage medium that can be referenced from a processing device such as a computer.

After step S1009 ends, step S1011 is started. In step S1011, mass spectrometry data are analyzed. Analysis of the mass spectrometry data is performed by a processing device such as an electronic computer. It is preferred to generate mass spectral data corresponding to the mass spectrum from the mass spectrometry data. For example, in time-of-flight mass spectrometry, the time-of-flight and the intensity of the detection signal of ions detected at each time-of-flight are associated in the mass spectrometry data. The time-of-flight is converted to m/z based on pre-obtained calibration data, and mass spectral data in which m/z and intensity are associated can be obtained.

From the m/z of the ions originating from the target molecule, it is determined whether ions with m/z within an acceptable range based on the accuracy of the mass spectrometry were detected. A target molecule is detected if an ion having an m/z in the acceptable range is detected. As the m/z of the ions derived from the target molecule, a past actual measurement value or a value calculated based on the molecular weight of the target molecule and the ions added during ionization can be used. For example, in the case of MALDI, a value obtained by adding the molecular weight of protons to the molecular weight of the target molecule can be used assuming that monovalent ions to which protons are added to the target molecule are detected. Cations or anions other than protons may be added. The method of analyzing data obtained by mass spectrometry is not particularly limited, and the target molecule can be quantified as appropriate.

When the purpose is to detect the presence or absence of a target molecule in a sample, the presence or absence of the target molecule can be determined from the data obtained by mass spectrometry. When the separation of molecules itself is the objective, after confirming that the target molecules have been separated by mass spectrometry, the sample containing the target molecules separated from the complex may be used for other purposes.

The following modifications are also within the scope of the present invention and can be combined with the above-described embodiments. In the following modified examples, the same reference numerals are used to refer to parts having the same structures and functions as those of the above-described embodiment, and description thereof will be omitted as appropriate.

(Modification 1)
In the above-described embodiment, prior to the operation of contacting the sample with the target-binding molecule (step S1003 in FIG. 2), molecules having a molecular weight equal to or higher than that of the complex or the target-binding molecule are filtered by ultrafiltration or microfiltration. An operation may be performed to remove at least a portion of molecules having a molecular weight greater than that of the binding molecule. Hereinafter, this ultrafiltration or microfiltration is referred to as first filtration, and the filtration in step S1005 is referred to as second filtration. In particular, the first filtration preferably removes from the sample molecules having a molecular weight equal to or higher than that of the complex.

FIG. 3 is a conceptual diagram for explaining the molecule separation method of this modified example. A solution SN containing a sample is stored in the tube TB. In addition to the target molecule T, the solution SN contains a non-target molecule N1 having a molecular weight smaller than that of the complex and a non-target molecule N2 having a molecular weight equal to or greater than that of the complex.

Prior to adding the target binding molecule to solution SN, a first filtration using filtration membrane F1 is performed (arrow A3). The filtrate FT1 that has passed through the filtration membrane F1 is stored in the filtrate reservoir R1. The target molecule T is contained in the filtrate FT1. Each step after step S1003 (FIG. 2) described above is performed using the filtrate FT1 as a sample. Non-target molecules N2 are captured by the filtration membrane F1 and removed from the sample. As a result, after the second filtration is performed in step S1005, molecules other than the complex contained in the filtration membrane of the second filtration can be reduced, and mass spectrometry can be performed with higher accuracy.

The filtration membrane F1 is not particularly limited in molecular weight cut-off or pore size as long as it can allow the target molecule T to permeate through it and can capture non-target molecules N2 and the like. The target molecule T is a substance with a molecular weight of 20 kDa or less, which can be measured with particularly high accuracy by time-of-flight mass spectrometry, and the target binding molecule is IgG with a molecular weight of about 150 kDa. In this case, the molecular weight cutoff of the filtration membrane F1 is preferably 30 kDa or more, more preferably 60 kDa or more, and even more preferably 90 kDa or more. The higher the cut-off molecular weight, the more surely the target molecule T can be permeated, and the loss of the target molecule T can be suppressed. In the above case, the molecular weight cut off of the filtration membrane F1 is preferably 130 kDa or less, more preferably 110 kDa or less. The lower the molecular weight cut off, the more reliably the non-target molecule N2 having a molecular weight equal to or higher than that of the complex can be captured by the filtration membrane F1 and removed from the sample.

(mode)
It will be appreciated by those skilled in the art that the multiple exemplary embodiments described above, or variations thereof, are specific examples of the following aspects.

(Section 1) A method for separating molecules according to one aspect comprises preparing a sample, a second molecule (target binding molecule) that binds to a first molecule (target molecule) to be separated, and contacting the sample with the liberated second molecule to generate a complex in which the first molecule and the second molecule are bound; purifying the complex based on molecular weight; subjecting a sample for mass spectrometry containing the first molecule separated from the purified complex to mass spectrometry. This makes it possible to separate target molecules more quickly and easily.

(Section 2) In a method for separating molecules according to another aspect, in the method for separating molecules according to the aspect of the first aspect, the second molecule is an antibody, a ligand-receptor reaction, or a sugar chain-lectin interaction. is a molecule that binds to the first molecule by Thus, the specificity of these reactions can be used to more reliably separate target molecules.

(Section 3) In a method for separating molecules according to another aspect, in the method for separating molecules according to the aspect of the first or second aspect, the complex is subjected to ultrafiltration, microfiltration, dialysis and size exclusion. Purified using at least one of chromatography. As a result, the target molecules can be separated with high accuracy by utilizing the change in the molecular weight of the target molecules due to the binding of the target-binding molecules.

(Section 4) In a method for separating molecules according to another aspect, in the method for separating molecules according to the aspect of the third aspect, the complex is purified by ultrafiltration or microfiltration, and the ultrafiltration or microfiltration In filtration, centrifugal force is used to filter. This allows rapid filtration.

(Section 5) In the method for separating molecules according to another aspect, in the method for separating molecules according to the aspects of the third or fourth aspect, before the sample and the second molecule are brought into contact, At least a portion of molecules having a molecular weight larger than the molecular weight of the first molecule is removed from the sample by a first filtration that is filtration or microfiltration, and after the sample and the complex are contacted, the complex is removed. The body is purified by a second filtration, ultrafiltration or microfiltration. As a result, contaminants and the like captured in the second filtration can be reduced, and mass spectrometry can be performed with higher accuracy.

(Section 6) A method for separating molecules according to another aspect is the method for separating molecules according to any one of aspects 1 to 5, wherein the mass spectrometry includes a matrix-assisted laser desorption/ionization method. Alternatively, ionization is performed by an electrospray method. Thereby, macromolecules such as peptides or proteins can be ionized without being fragmented.

The present invention is not limited to the contents of the above embodiments. Other aspects conceivable within the scope of the technical idea of the present invention are also included in the scope of the present invention.

Examples according to this embodiment are shown below, but the present invention is not intended to be limited to the following examples.

In this example, verotoxin 1 (Nacalai Tesque, 01770-74) was used as the target molecule, and anti-verotoxin 1 antibody (Nacalai Tesque, 20272-14) was used as the target binding molecule, and these complexes were purified by ultrafiltration. did. A sample for mass spectrometry was prepared using the purified complex and subjected to mass spectrometry. Samples included verotoxin 2 (Nacalai Tesque, 01771-64) as a non-target molecule.

Verotoxin 1 and verotoxin 2 were added to 1 mL of PBS buffer solution containing 2.5 mM n-octyl-β-D-glucoside so that each concentration was 1 μg/mL. 100 μL of the solution containing verotoxin 1 and verotoxin 2 was desalted using a solid-phase extraction chip (Agilent Technologies, Bond Elut OMIX, A57009100). The desalted solution was dropped onto a stainless steel plate, which is a sample plate for MALDI, and mass spectrometry was performed using a mass spectrometer (Shimadzu Corporation, MALDI-8020). For mass spectrometry, ionization was performed by MALDI and time-of-flight mass spectrometry was performed. The same applies to mass spectrometry below.

FIG. 4 is a diagram showing mass spectra of solutions containing verotoxin 1 and verotoxin 2 obtained by the mass spectrometry. In the mass spectrum of FIG. 4, the monovalent ion peak P11 (m/z 7689.7) derived from verotoxin 1, the divalent ion peak P12 derived from verotoxin 1, and the monovalent ion derived from verotoxin 2 An ion peak P21 (m/z 7818.1) and a bivalent ion peak P22 derived from verotoxin 2 were clearly observed. This confirmed that verotoxin 1 and verotoxin 2 were contained at detectable levels using the mass spectrometer.

Next, 150 μL of this sample solution containing Verotoxin 1 and Verotoxin 2 was aliquoted. 1 μg of anti-verotoxin 1 antibody was added to each sample solution obtained by fractionation and incubated for 30 minutes. The total amount of the reaction solution after incubation was applied to an ultrafiltration spin column (NMWL: 100K Da, Merck Millipore, UFC510096) and separated by centrifugation (14,000 G, 5 minutes). 100 μL of PBS buffer solution was added to the residue liquid (about 25 μL) in the filter of the ultrafiltration spin column to collect the residue. The solution containing the residue was desalted with a solid phase extraction tip. The desalted solution was dropped onto a stainless plate, which is a sample plate for MALDI, and subjected to mass spectrometry using a mass spectrometer. The total amount of ultrafiltration filtrate was similarly subjected to desalting and mass spectrometry.

FIG. 5 is a diagram showing a mass spectrum obtained by mass spectrometry of a solution containing ultrafiltration residue. Only the monovalent ion peak P11 (m/z 7690.1) and the divalent ion peak P12 derived from verotoxin 1 were clearly observed. It was shown that it did not pass through the filter of This was probably because verotoxin 1 bound to an anti-verotoxin 1 antibody (molecular weight: about 150K Da).

FIG. 6 is a diagram showing a mass spectrum obtained by mass spectrometry of the ultrafiltration filtrate. Only the monovalent ion peak P21 (m/z 7814.7) and the divalent ion peak P22 from verotoxin 2 were clearly observed, and no affinity for the antibody, i.e. molecules not bound to the antibody, were present in the filtrate. It was confirmed that it penetrated to the side.

From the above, it is possible to separate molecules bound to antibodies from molecules not bound to antibodies by filtration (ultrafiltration) with pores of the same size as the molecules, and to detect each molecule using mass spectrometry. Proven.

CX... complex, F... ultrafiltration membrane, F1... filtration membrane, FT, FT1... filtrate, M1, M2... mass spectrum, N1, N2... non-target molecule, T... target molecule, TB... tube, TBM ... target binding molecule, PN ... non-target molecule peak, PT ... target molecule peak, R, R1 ... filtrate reservoir, SN ... solution.

Claims (4)

providing a sample and a second molecule that binds to the first molecule to be separated;
contacting the sample with the released second molecule in a solution to generate a complex in which the first molecule and the second molecule are bound;
purifying the conjugate based on molecular weight;
subjecting a mass spectrometry sample containing the first molecule separated from the purified complex to mass spectrometry ;
At least a portion of molecules having a molecular weight greater than that of the first molecule are removed from the sample by first filtration, which may be ultrafiltration or microfiltration, prior to contacting the sample with the second molecule. ,
A method of separating molecules, wherein after contacting the sample with the complex, the complex is purified by a second filtration, which is ultrafiltration or microfiltration. In the method for separating molecules according to claim 1,
A method for separating molecules, wherein the second molecule is an antibody or a molecule that binds to the first molecule by ligand-receptor reaction or sugar chain-lectin interaction. In the method for separating molecules according to claim 1 or 2 ,
A molecular separation method, wherein centrifugal force is used in the first filtration and the second filtration. In the method for separating molecules according to any one of claims 1 to 3 ,
A method for separating molecules, wherein in said mass spectrometry ionization is performed by matrix-assisted laser desorption ionization or electrospray.

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