JP5119053B2 - Biological sample separation method, biological sample detection method, biological sample separation system, and biological sample separation / detection system - Google Patents

Description

  The present invention relates to a biological sample separation method, a biological sample detection method, a biological sample separation system, or a biological sample separation / detection system.

  In recent years, researches for searching for new biomarkers from components present in minute amounts in biological samples have been performed all over the world, and their application to clinical tests is expected. Small molecules in blood such as peptides are attracting particular attention as effective markers.

  A biological sample is a complex mixture of many components. Therefore, in order to analyze this, first, components are separated by high performance liquid chromatography (HPLC) or the like to reduce the complexity of the sample, and then unknown components are analyzed by mass spectrometry (MS) capable of highly sensitive detection. The method is widely used. And, in order to detect a marker present in a trace amount in a biological sample by MS, for example, about 20 μL (1 mg in protein amount) is necessary for serum.

  In HPLC, a column having a larger protein capacity is generally required to have a higher flow rate for separation. For example, when separating 1 mg of the protein, a large-capacity column with a flow rate of about 1 mL / min is required. However, in this case, since the volume of the eluted sample increases, it is necessary to concentrate the sample once separated and sorted before the MS measurement. For example, it takes about 3 h to concentrate a 100 μL sample, which causes a reduction in analysis throughput.

  For example, in the case of serum, 90% or more of the components are occupied by some major proteins including albumin (see, for example, Non-Patent Document 1). Therefore, by removing these in advance, it becomes possible to introduce a necessary 20 μL serum sample into a low-volume column with a small flow rate, and the time required for subsequent concentration can be reduced.

  As a major protein removal method, a removal kit using an antibody immobilized on a support is already commercially available. However, it has been found that when these are used, the major protein-binding small molecules are also removed. Among them, there are many effective biomarker candidates, which is a problem in using this method.

  As a technique for reducing the concentration of the organic solvent in the sample eluted from the reverse phase column, a technique for removing the organic solvent by passing the sample containing the organic solvent through a heated capillary nebulizer has been reported (for example, Patent Document 1). reference.). In this method, the organic solvent is evaporated and removed by heating. When an unstable biomolecule such as protein is contained in the sample, the sample may be denatured by heating, and the use of this method is not suitable.

  In addition, a method has been reported in which a biological sample containing protein is introduced into a normal phase column, the protein is passed through and removed, the low molecule adsorbed on the normal phase column is eluted, introduced into the second column, and separated. (For example, refer to Patent Document 2). The technique removes the protein through the first column. In this method, the sample is diluted by adding an organic solvent to dissociate the protein and the low molecule before introducing the sample into the first column.

JP-T-2006-504098 JP 2003-149216 A Mol Cell Proteomics. 2002, 1, 845-67.

  One object of the present invention is to separate and sort biological samples in a stable and small volume.

  In the present invention, a biological sample is adsorbed on a first reversed-phase column, eluted from the first reversed-phase column using an organic solvent, the eluted sample is diluted to reduce the concentration of the organic solvent, and the diluted sample is A biological sample separation method for separating and sorting a biological sample by introducing it into a second reversed-phase column that is thinner than the first column and eluting the sample at a lower flow rate than when it is eluted from the first column. With one feature.

  According to one embodiment of the present invention, it is possible to provide a biological sample separation method that can reduce the volume per fraction and omit or reduce the concentration step.

The embodiment of the present invention has, for example, the following features. A biological sample is adsorbed on the first reversed-phase column, and only low molecules such as peptides are eluted with an organic solvent (acetonitrile, etc., <50%) at a concentration that does not elute the main protein. When the sample is diluted to an adsorbable organic solvent concentration (<25%), and the diluted sample is adsorbed on the second reversed-phase column that is thinner than the first reversed-phase column and eluted from the first reversed-phase column Separate and sort samples at a lower flow rate. As the biological sample, biological samples such as blood (serum, plasma), urine, cerebral spinal fluid, saliva, etc., in which the presence of major proteins hinders marker search are assumed. In addition, low molecular weight compounds such as proteins, peptides, and metabolites are assumed to be analyzed. As a support of the reverse phase column, a low polarity octadecyl group (C18), octyl group (C8), butyl group (C4), trimethyl group (C3), a support such as silica gel, polymer, silica gel and carbon hybrid particles, Those obtained by chemically bonding a phenyl group (Ph) or the like can be used. As the organic solvent, acetonitrile, methanol or the like is mixed with water to prepare an appropriate concentration. In order to adjust the pH, an acid such as trifluoroacetic acid (TFA) is appropriately added. For elution of the sample from the first reversed-phase column, constant concentration of eluent containing a certain concentration of organic solvent, stepwise delivery of multiple types of eluent with different organic solvent concentrations, and gradient of organic solvent concentration Gradient liquid change etc. which were changed automatically are used. A similar technique can be used to elute the sample from the second reverse phase column. For the purpose of separating mixture samples with the second reversed-phase column, stepwise pumping of multiple types of eluents with different organic solvent concentrations, and gradient pumping with organic solvent concentrations changed in gradients are particularly recommended. The In addition, after elution from the first column, dilution with water is performed to promote adsorption of the sample to the second column. Dilution reduces the organic solvent concentration. In addition, by providing a dilution unit between the first column and the second column, liquid feeding to both columns can be performed with a single pump. The present invention can also be used as a technique for adsorbing and removing a protein that is one of biological samples. It is desirable to dilute with water after elution from the first column to promote adsorption of the sample to the second column. On the other hand, in the present invention, by providing a dilution unit between the first column and the second column, it is possible to carry out liquid feeding to both columns with a single pump.

  Hereinafter, an example explains.

  A first embodiment of the present invention will be described with reference to FIGS. In this example, (1) the biological sample is adsorbed on the first reverse phase column, (2) only the peptide is eluted with an organic solvent at a concentration at which the main protein is not eluted, and (3) the eluted peptide is the reverse phase column. Dilute the sample to an organic solvent concentration that can be adsorbed to (4), adsorb the diluted sample to the second reversed-phase column that is thinner than the first reversed-phase column, and (5) elute from the first reversed-phase column The work is proceeded in the order of separating and sorting the sample at a lower flow rate than when it was done. However, the present invention is not limited to this embodiment, and various modifications can be made within the scope of the technical idea.

  As shown in FIG. 1, in this embodiment, a pump 101, an autosampler 102, a UV detector 103, a fraction collector 104, a first flow path switching valve 105a, and a second flow path switching valve 105b are used as an HPLC system. Fig. 1 shows the state of the piping of the HPLC system, and Fig. 2 shows the sequence of liquid feeding.

  Here, the experiment was conducted with the first reversed phase column 106 and the second reversed phase column 107 offline. Off-line is an experimental technique in which a column is not connected in a single flow path, but the sample is once collected after being separated in the first column and then introduced again into the second column. The pipe connects the first reverse-phase column 106 and the second reverse-phase column 107 in parallel from the pump 101 via the first flow path switching valve 105a, and then both columns are connected to the second flow path switching valve 105b. Then, it is introduced into the UV detector 103 and sorted by the fraction collector 104. When the valve positions of the first flow path switching valve 105a and the second flow path switching valve 105b in FIG. 1 are set to a valve position 1 represented by a solid line in the drawing, the first flow path switching valve is supplied from the autosampler 102. The pipes are connected to 105a, the first reverse phase column 106, the second flow path switching valve 105b, and the UV detector 103. When the valve position 2 is represented by a broken line in the figure, the first flow path switching valve 105a, the second reverse phase column 107, the second flow path switching valve 105b, and the UV detector are provided from the autosampler 102. A pipe is connected to 103. As eluent A, 5% acetonitrile containing 0.1% TFA was prepared, and as eluent B, 95% acetonitrile containing 0.1% TFA was prepared and used.

Hereafter, each process of HPLC is demonstrated in detail with reference to FIG.
(1) Step of adsorbing biological sample to first reversed phase column 106: Using autosampler 102, 20 μL of serum is introduced into first reversed phase column 106 (4.6 mm ID × 100 mm, particle diameter 3 μm). . A reverse phase column is a column for sending a liquid mobile phase to a hydrophobic stationary phase and separating various compounds based on the difference in hydrophobic interaction between the two phases.
(2) Step of eluting the peptide from the first reverse phase column 106: The peptide is eluted with a linear gradient having a B solution concentration of 0 to 50% in 5 minutes after the introduction of serum. Considering the length of the pipe from the pump 101 to the UV detector 103, the eluate for 4.5 minutes from the 1 minute after the start of the gradient to just before the albumin is detected (5.5 minutes) (4.5 ml) ).
(3) Step of diluting the eluted peptide to an organic solvent concentration that can be adsorbed on the second reversed-phase column: 4.5 mL of water is added to the recovered 4.5 mL of the eluate, and the final acetonitrile The concentration is about 12%.
(4) Step of adsorbing the diluted sample on the second reverse phase column: 9 mL of the diluted sample solution is applied to the second reverse phase column 107 (2 mm ID x 150 mm, particle diameter 5 μm) using the autosampler 102. Introduce. The second reversed phase column has the same separation characteristics as the first reversed phase column but is thinner than the first reversed phase column.
(5) Separating and separating the sample from the second reversed-phase column: The peptide is eluted with a linear gradient with a B solution concentration of 12 to 50% in 10 minutes after the introduction of the sample. Considering the length of the pipe from the pump 101 to the UV detector 103, the eluate for 10 minutes from the start of the gradient to 1 to 11 minutes (11 to 21 minutes in the entire sequence) is collected every 18 seconds ( 60 μL each).

(Measurement of separated sample by mass spectrometry)
The separated sample is measured using a mass spectrometer 110 (mass spectrometer), for example, a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (MALDI-TOF-MS), and the number of peaks obtained in each fraction is measured. To do. As a result, a total of 3109 peptide peaks were detected during 10 minutes of elution.

  In this example, an example is shown in which all steps are performed online by providing a dilution unit between the first reverse phase column and the second reverse phase column.

  As shown in FIG. 3, in this embodiment, a pump 101, an autosampler 102, a UV detector 103, a fraction collector 104, a first flow path switching valve 105a, and a second flow path switching valve 105b are used as an HPLC system. FIG. 3 shows the state of the piping of the HPLC system, and FIG. 4 shows the liquid feeding sequence. The first reverse phase column 106 is connected from the pump 101 via the first flow path switching valve 105a, then the dilution unit 301 is connected, and then the second reverse phase column via the second flow path switching valve 105b. 107 is connected, and finally introduced into the UV detector 103 and sorted by the fraction collector 104. In this case, in the step of separating and separating the sample from the second reverse phase column, the first reverse phase column 106 and the dilution unit 301 become dead volumes, which may cause deterioration of the separation accuracy in the second reverse phase column. . Therefore, by separately providing a flow path that directly connects the first flow path switching valve 105a and the second flow path switching valve 105b, the pump 101 does not go through the first reverse phase column 106 and the dilution unit 301, and the The liquid is fed to the second reverse phase column 107. When the valve positions of the first flow path switching valve 105a and the second flow path switching valve 105b in FIG. 3 are set to the valve position 1 represented by a solid line in the drawing, the first flow path switching valve is supplied from the autosampler 102. A pipe is connected to 105 a, the first reverse phase column 106, the dilution unit 301, the second flow path switching valve 105 b, the second reverse phase column 107, and the UV detector 103. Further, when the valve position is represented by a broken line in the figure, the first flow path switching valve 105a, the second flow path switching valve 105b, the second reverse phase column 107, and the UV detector are supplied from the autosampler 102. A pipe is connected to 103.

FIG. 5 shows a structural example of the dilution unit. The present dilution unit has a structure in which a hollow portion 503 projects at the lower part of the pipe 502. The volume of the cavity 503 is 9 mL, and a diluent injection port 501 for injecting a diluent is attached to the bottom surface of the cavity 503. Into the cavity 503, 4.5 mL of diluent is introduced in advance via the diluent injection port 501.

  When the sample eluted from the first reverse phase column 106 flows into the dilution unit, the sample is first accumulated in the cavity 503 by gravity. When the amount of the sample that has flowed in exceeds 4.5 mL (9 mL (that is, the volume of the cavity 503) when combined with the previously introduced diluent), the sample diluted with the diluent is added from the cavity 503. It flows out to the second reverse phase column 107. Prior to the sample outflow, the air present in the cavity 503 is pushed out first, so that 4.5 mL corresponding to the volume of the air passes through the second unit between the dilution unit 301 and the second reverse phase column 107. By switching the flow path switching valve 105b, the sample is discharged to the drain, and only the sample that flows out thereafter is introduced into the second reverse phase column 107. That is, the air in the cavity 503 pushed out before the sample outflow is discharged to the drain by switching a valve interposed between the dilution unit and the second reverse-phase column 107, and only the sample that flows out after that is discharged into the second. The reverse phase column 107 is introduced.

Hereinafter, each process of HPLC is demonstrated in detail with reference to FIG.
(1) Step of adsorbing a biological sample to the first reverse phase column: 20 μL of serum is introduced into the first reverse phase column 106 using the autosampler 102.
(2) Step of eluting the peptide from the first reverse phase column: The peptide is eluted with a linear gradient with a B solution concentration of 0 to 50% within 5 minutes after the introduction of serum.
(3) Step of diluting the eluted peptide to an organic solvent concentration that can be adsorbed on the second reverse phase column: The sample eluted from the first reverse phase column 106 Are introduced into a dilution unit 301 provided between the reverse phase column 107 and the reverse phase column 107.
(4) Adsorbing the diluted sample to the second reverse phase column: Prior to the sample outflow from the dilution unit 301, air existing in the cavity inside the dilution unit is pushed out, which corresponds to the volume of air. The 4.5 mL portion is discharged to the drain by switching the valve 150b between the dilution unit 301 and the second reverse phase column 107, and only the sample that flows out thereafter is introduced into the second reverse phase column 107.
(5) Step of separating and separating the sample from the second reverse phase column: 10 minutes after completion of sample introduction into the second reverse phase column 107 (19.5 to 29.5 minutes in the entire sequence), B The peptide is eluted with a linear gradient with a liquid concentration of 12-50%. Considering the length of the pipe from the pump 101 to the UV detector 103, the eluate for 10 minutes from 1 to 11 minutes (20.5 to 30.5 minutes in the whole sequence) after the start of the gradient is added every 18 seconds. Aliquot (60 μL each).

  As described in this example, excess protein such as albumin is removed from the biological sample by the first reversed-phase column, and the total protein concentration of the sample is reduced, so that the remaining components are removed from the first reversed-phase column. A thin second reversed phase column provides a technique for separation at a low flow rate. Specifically, when the method of performing separation at a high flow rate using only a single reverse phase column is used as a comparative example, the comparative example has a sample volume of 300 μL per fraction, When 9 hours are required for the concentration step before MS analysis, the volume per fraction can be reduced to 60 μL by using the method of this example, and the concentration step can be omitted or reduced to 1/5 or less. Therefore, the analysis throughput can be improved by 5 times or more. This technique is particularly effective in high-throughput biological sample analysis using mass spectrometry.

It is explanatory drawing which showed the HPLC system piping example 1. FIG. It is explanatory drawing which showed the liquid feeding sequence in the piping example. It is explanatory drawing which showed the HPLC system piping example 2. FIG. It is explanatory drawing which showed the liquid feeding sequence in the example 2 of piping. It is explanatory drawing which showed the dilution unit structure.

Explanation of symbols

101 Pump 102 Autosampler 103 UV detector 104 Fraction collector 105a First flow path switching valve 105b Second flow path switching valve 106 First reverse phase column 107 Second reverse phase column 301 Dilution unit 501 Diluent injection port

Claims (12)

The sample is introduced into the first reversed-phase column to remove unnecessary components in the sample, and the components of the sample adsorbed on the first reversed-phase column are first eluted by gradient elution using an organic solvent. The sample eluted from the column and diluted from the first reverse phase column is diluted to reduce the organic solvent concentration, and the diluted sample is introduced into the second reverse phase column that is thinner than the first column. than when eluted from a reversed phase column by gradient elution with a low flow rate, specimen separation method the components of the sample you preparative separation · min. According to claim 1, before Ki試 fee includes a protein, the first components other than the main protein from the reverse phase column and eluted in, specimen separating how to characterized in that the eluted sample is diluted with water. In claim 1,
The sample eluted from the first reversed phase column once collected, specimen separating how to introduce the second backhaul column diluted. In claim 1,
The dilution unit is provided between the first reversed phase column and a second reverse phase column, a second specimen you introduced into the backhaul column samples eluted from the first column through the dilution unit separation Method. In claim 4 ,
The dilution unit has a structure in which a cavity protrudes at the lower part of the pipe, and dilution water is introduced into the cavity in advance, so that the sample eluted from the first column flows into the unit by gravity. samples are accumulated in the cavity, when the inflow exceeds the volume of the cavity, specimen separation method you characterized in that sample diluted flows out into the second backhaul column from the cavity. In claim 4,
The air in the cavity that is pushed out prior to the sample outflow is discharged to the drain by switching the valve between the dilution unit and the second column, and then only the sample that flows out is introduced into the second column. specimen separation method it said. In any one of Claims 1 thru | or 5,
Feeding the liquid for the separation in the first reverse phase column, specimen separating how to characterized in that the liquid feed for the separation in the second reverse phase column, carried out by the same pump. Specimen separation detecting how to characterized in that the sample separated by the method of the biological sample separation according to any one of claims 1 to 7, detected by mass spectrometry. A pump, an autosampler, a detector, a first reverse phase column and a second reverse phase column;
The sample introduced from the autosampler is introduced into a first reverse phase column to remove unnecessary components in the sample, and the sample adsorbed on the first reverse phase column is subjected to gradient elution using an organic solvent. The sample eluted from the first reversed-phase column is diluted with the sample eluted from the first reversed-phase column to reduce the concentration of the organic solvent, and the diluted sample is put into a second reversed-phase column thinner than the first column. A sample separation system that separates and separates the components of the sample by introducing gradient elution at a lower flow rate than when elution from the first reversed-phase column . Specimen separation and detection system that, comprising a specimen separation system; and a mass analyzer for detecting components of the fractionated sample to claim 9. In claim 9,
A sample separation system in which a dilution unit is provided between a first reverse phase column and a second reverse phase column, and a sample eluted from the first column is introduced into the second reverse feed column via the dilution unit. In claim 11,
The dilution unit has a structure in which a cavity protrudes at the lower part of the pipe, and dilution water is introduced into the cavity in advance, so that the sample eluted from the first column flows into the unit by gravity. A sample separation system, wherein the sample is accumulated in the cavity and the diluted sample flows out from the cavity to the second reverse feed column when the amount of inflow exceeds the volume of the cavity.

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