Patent ID: 12235196

DESCRIPTION OF EMBODIMENTS

The present invention provides a method for pretreatment of a biological sample in order to measure a protein contained in the biological sample by liquid chromatography/mass spectrometry. The method includes the step of adding an acetic acid aqueous solution to the biological sample, so as to prepare a pretreatment sample containing an acetic acid at a concentration within a predetermined range. The pretreatment sample preferably contains the acetic acid at a concentration ranging from 20 to 50 weight percent, more preferably at a concentration ranging from 35 to 45 weight percent.

With the method according to the present invention, the organic solvent, the surfactant, or the peptide, each added to the biological sample in a conventional method to prevent the protein or the peptide from being adsorbed to the sample container, is no longer required to be added or may be reduced in amount. Further, a surface treatment of the sample container, which has been required in the conventional method to prevent the protein or the peptide from being adsorbed to the sample container, is no longer required.

FIG.1is a diagram showing a schematic flow of the method for pretreatment of the biological sample, the method according to the present invention. In an example described here, the biological sample corresponds to plasma, and the method for pretreatment of the plasma uses solid phase extraction.

<Sample Preparation>

Blood is collected from a subject. A blood collection tube is used to collect the blood from the subject, the blood collection tube where protease inhibitor cocktail is previously contained. After a stable isotope-labeled peptide or the like, as an internal standard substance, is added to the blood collected, the blood is separated into the plasma and a blood cell component by a centrifugal separator. 500 μL of the plasma is collected to be put in the sample container.

Next, 10 μL of a 40% acetic acid aqueous solution is added to the sample container to be stirred therein at room temperature for 5 to 10 minutes. As a result, each of an interaction between the proteins in the plasma and an interaction between the peptides in the plasma is released.

Subsequently, 500 μL of a 5% ammonium hydrate is added to the sample container and stirred by vortex mixer to obtain 1000 μL of the pretreatment sample.

<Solid Phase Extraction (SPE)>

Next, a solid-phase extraction column is used for pretreatment for removing an unwanted component contained in the pretreatment sample obtained above. The solid-phase extraction column may be a solid-phase extraction column using a non-polar filler of, for example, C18, or a solid-phase extraction column using an anion or cation exchange filler.

First, the filler of the solid-phase extraction column is wetted with an organic solvent (e.g., methanol) to activate the filler (conditioning).

Next, the pretreatment sample above (1000 μL) is flowed through the filler of the solid-phase extraction column at a predetermined flow rate (sample loading).

Subsequently, a cleaning solution is appropriately matched to polarity of the target protein and a polarity of the unwanted component, where both the protein and the unwanted component are contained in the pretreatment sample, and the cleaning solution is flowed through the filler (cleaning). As a result, the unwanted component that has been adsorbed into the filler is discharged from the solid-phase extraction column. The cleaning solution may be, for example, the 5% ammonium hydrate or a 10% acetonitrile (ACN).

Finally, an eluent is flowed through the filler to elute the protein as the target component that has been retained in the filler. The eluent may be, for example, a solution prepared by mixing the ACN, water, and the acetic acid in a ratio of 45/25/30. The acetic acid may be, for example, a 30% acetic acid.

A 60% acetic acid aqueous solution is added to an eluate obtained above, so as to prepare an analysis sample which contains the acetic acid at concentration of 40%. Then, the analysis sample is introduced into the liquid chromatograph mass spectrometer (LC/MS), so that the liquid chromatography/mass spectrometry (LC/MS/MS) is executed on the sample.

As has been described above, with the method for pretreatment of the sample, the method according to the present invention, the acetic acid aqueous solution is used as the solvent to prepare the pretreatment sample or the analysis sample. Apart from the acetic acid aqueous solution, the pretreatment may be carried out using the same solvents and instruments as in the conventional methods.

In the description above, the method for the pretreatment uses the solid phase extraction (SPE), but the method may also include the centrifugal separation performed in the step of preparing the sample. In other words, the method for pretreatment of the sample according to the present invention is applicable to a plurality of means of pretreatment, such that the protein contained in the biological sample is measured using the LC/MS or the LC/MS/MS.

In the description above, a biological sample collected from the subject is used as an example, but a peptide sample commercially available may also be the biological sample. In this case, the acetic acid aqueous solution may be used as a solvent for dissolving the peptide.

Example

Next, the present invention will be described specifically with reference to examples below, but the present invention is not limited to these examples. The following description assumes that “%” represents weight percent.

First Example

[Preparation of sample (pretreatment according to the present invention)]A glucagon sample commercially available (product name: “Glucagon Human”; manufacturer: Fujifilm Wako Pure Chemical Corporation) was put in a sample container made of polypropylene (product name: “Protein LoBind tubes”; manufacturer: Eppendorf Ltd), into which a sample preparation solvent was added. The sample preparation solvent was diluted by a diluent solvent step by step, so that five types of glucagon solution containing the glucagon at molar concentrations of 0.01 nM, 0.1 nM, 1 nM, 10 nM, and 100 nM or four types of glucagon solution containing the glucagon at molar concentrations of 0.01 nM, 0.1 nM, 1 nM, and 10 nM were prepared.

As the sample preparation solvent, any one of the 40% acetic acid aqueous solution, a 0.1% formic acid, a physiological salt solution, and a phosphate buffer solution was used; and as the diluent solvent, the 40% acetic acid aqueous solution or the 0.1% formic acid was used. Then, in each of seven different combinations of the sample preparation solvent and the diluent solvent, a set of the glucagon solutions containing the glucagon at the molar concentrations above was prepared.

[Liquid Chromatography/Mass Spectrometry (LC/MS/MS)]

Each of the seven sets of the glucagon solutions obtained above was introduced into the liquid chromatograph mass spectrometer (ultra-fast triple quadrupole LC/MS/MS system: LCMS-8060; Shimadzu Corporation), so that the LC/MS/MS was performed on the corresponding set of the glucagon solutions. The LC/MS/MS was performed three times on each of the seven sets.

As a result of the LC/MS/MS, a product spectrum of a precursor ion (m/z 940.10) derived from the glucagon was obtained, and based on the product spectrum, an intensity of a product ion (m/z 697.15) was acquired.FIG.2shows a result of each of the seven sets, each subjected to the LC/MS/MS three times. As seen fromFIG.2, when each of the sample preparation solvent and the diluent solvent corresponded to the 40% acetic acid aqueous solution, a detection intensity (area value) in the LC/MS/MS is significantly increased, as compared with the other combinations of the sample preparation solvent and the diluent solvent.

Second Example

With regard to an insulin sample commercially available (product name: “Human Insulin”; Sigma-Aldrich), as in the first example, the sample preparation solvent and the diluent solvent were used, so that five types of insulin solution containing the insulin at molar concentrations of 0.01 nM, 0.1 nM, 1 nM, 10 nM, and 100 nM or four types of insulin solution containing the insulin at molar concentrations of 0.01 nM, 0.1 nM, 1 nM, and 10 nM were prepared.

As a result, each of seven sets of the insulin solutions was obtained, and as in the first example, each of the seven sets was introduced into the liquid chromatograph mass spectrometer, so that the LC/MS/MS was performed on the corresponding set. As a result of the LC/MS/MS, a product spectrum of a precursor ion (m/z 1162.50) derived from the insulin was obtained, and based on the product spectrum, an intensity of a product ion (m/z [0]345.15) was acquired.FIG.3shows a result of each of the seven sets, each subjected to the LC/MS/MS three times. As seen fromFIG.3, in the second example too, when each of the sample preparation solvent and the diluent solvent corresponded to the 40% acetic acid aqueous solution, a detection intensity (area value) in the LC/MS/MS is significantly increased, as compared with the other combinations of the sample preparation solvent and the diluent solvent.

Third Example

As each of the sample preparation solvent and the diluent solvent, the acetic acid aqueous solution was prepared at concentration of 10%, 20%, 30%, 40%, and 50% (hereinafter, referred to as the 10% acetic acid aqueous solution, the 20% acetic acid aqueous solution, 30% acetic acid aqueous solution, the 40% acetic acid aqueous solution, and the 50% acetic acid aqueous solution). Then, in the same procedures as in the first example, the glucagon sample commercially available was prepared and diluted in a sample container made of polypropylene (PP) (product name: “TORAST-H Bio Vial”; Shimadzu GLC Ltd.), so that the glucagon solution containing the glucagon at molar concentration of 100 nM was obtained.

Additionally, as each of the sample preparation solvent and the diluent solvent, the 10% acetic acid aqueous solution, the 30% acetic acid aqueous solution, and the 50% acetic acid aqueous solution were prepared. Then, in the same procedures as in the first example, the glucagon sample commercially available was prepared and diluted in a sample container made of glass (product name: “1.5 mL Vial”; Shimadzu GLC Ltd.), so that the glucagon solution containing the glucagon at molar concentration of 100 nM was obtained.

As in the first example, each of the glucagon solutions obtained above was introduced into the liquid chromatograph mass spectrometer where the LC/MS/MS was performed on the corresponding glucagon solution. As a result, an intensity of the product ion was acquired. Each of the glucagon solutions was subjected to the LC/MS/MS 10 times.FIG.4shows the results. As seen fromFIG.4, in any one of the sample container of PP and the sample container of glass, when each of the sample preparation solvent and the diluent solvent corresponded to the 10% acetic acid aqueous solution, the detection intensity was lowest. Additionally, when each of the sample preparation solvent and the diluent solvent corresponded to the 20% to 50% acetic acid aqueous solutions, the detection intensity was higher in the sample container of PP than in the sample container of glass; and particularly, when each of the sample preparation solvent and the diluent solvent corresponded to the 30% to 50% acetic acid aqueous solutions, the detection intensity was significantly higher in the sample container of PP.

As has been described above, when the sample container of PP was used, each of the 20% to 50% acetic acid aqueous solutions as the sample preparation solvent and the diluent solvent was highly effective to suppress an adsorption of the glucagon. Alternatively, when the sample container of glass was used, each of the 30% to 50% acetic acid aqueous solutions as the sample preparation solvent and the diluent solvent was also highly effective, although not as effective as with the sample container of PP, to suppress the adsorption of the glucagon.

Fourth Example

Here, as each of the sample preparation solvent and the diluent solvent, the 40% acetic acid aqueous solution was prepared, and six of the glucagon solutions, each containing the glucagon at molar concentration of 100 nM, were prepared. Then, out of the six, three of the glucagon solutions were immediately introduced into the liquid chromatograph mass spectrometer where the LC/MS/MS was performed; and the other three were left at a temperature of 4° C. for 19 hours, before being introduced into the liquid chromatograph mass spectrometer where the LC/MS/MS was performed.FIG.5shows the results. Note that, each of the glucagon sample, the sample container, and the liquid chromatograph mass spectrometer used in this example was the same as in the first example.

As seen fromFIG.5, the results show no significant difference between the LC/MS/MS performed immediately after the preparation of the glucagon solutions and the LC/MS/MS performed 19 hours after the preparation. The results above presume that the glucagon solution, prepared by using the 40% acetic acid aqueous solution as the sample preparation solvent and the diluent solvent, was already effective to suppress the adsorption of the glucagon when prepared in the sample container.