Patent Description:
An amino acid sequence analyzer (protein sequencer) utilizing Edman degradation includes an Edman degradation part which configures a device for pretreatment of a sample. In the protein sequencer, Edman degradation in the Edman degradation part causes amino acids to be eliminated from protein (including peptide) which is a target sample for an amino acid sequence analysis such that amino acid residues are eliminated one by one as derivatives of amino acids. Each amino acid eliminated from the protein sample due to Edman degradation is dissolved in a reagent within a vessel called a conversion flask, and then is introduced in a high-performance liquid chromatograph. In the high-performance liquid chromatograph, each amino acid in the reagent is separated while passing through a column and is sequentially detected by a detector, and thus, the amino-acid sequence is analyzed (for example, see Patent Document <NUM> listed below).

Examples of a reagent used for dissolving a derivatized amino acid (amino acid sample) include an organic solvent such as acetonitrile, and a mixed solution of the organic solvent and water. In the above device for pretreatment of a sample, usually, concentration of the organic solvent is set to a predetermined value. Therefore, depending of the type of the column used in the high-performance liquid chromatograph connected to the device for pretreatment of a sample, concentration of the organic solvent may not be suitable for separation of an amino acid sample. For example, in a case where concentration of the organic solvent is high, an amino acid sample cannot be favorably separated, which may have an adverse effect on the analysis result.

In a case where concentration of an organic solvent in a reagent for dissolving an amino acid sample eliminated by Edman degradation is high as described above, the amino acid sample may be dried by using a centrifugal concentrator, the dried amino acid sample may be dissolved again into another solvent suitable for separation, and measurement may be performed again.

However, when the amino acid sample is completely dried in a vessel, the amino acid is precipitated and is likely to adhere to an inner surface of the vessel. Therefore, there is a problem that when the dried amino acid sample is to be dissolved again in the solvent, some acids are not dissolved enough and sample loss occurs.

In addition, work of drying an amino acid sample and dissolving the sample again is usually manually performed by an operator. Therefore, the work takes time and labor, and the work of drying and dissolving again takes time. As a result, there is a problem that an analysis takes a long time.

<CIT> discloses an apparatus for use as an independent unit along with a main sequencer in the overall Edman degradation process.

The present invention is made in view of the above circumstances, and an object of the present invention is to provide a device for pretreatment of a sample which can prevent occurrence of sample loss and can easily and efficiently lower concentration of an organic solvent in a reagent, as defined in claim <NUM>, an analyzer equipped therewith, as defined in claim <NUM>, and a method for pretreatment of a sample, as defined in claim <NUM>.

A device for pretreatment of a sample according to the present invention as defined in claim <NUM>, includes a vessel, a reagent introduction part, a reagent discharge part, a first gas supply part, and a second gas supply part. In the vessel, a reagent containing an organic solvent is introduced and a sample is dissolved in the reagent. The reagent introduction part introduces the reagent into the vessel. The reagent discharge part discharges the reagent in which the sample is dissolved in the vessel to an outside of the vessel. The first gas supply part supplies a gas into the vessel and thus pressurizes the interior of the vessel. The second gas supply part supplies a gas into the reagent in the vessel and thus forms gas bubbles in the reagent.

According to such a configuration, a gas is supplied from the second gas supply part to the reagent in the vessel, and thus gas bubbles are formed in the reagent and volatilization of the organic solvent in the reagent is promoted by the gas bubbles. Therefore, concentration of the organic solvent in the reagent can be easily and efficiently lowered. In addition, differing from the configuration of completely drying a sample, since a sample does not adhere to an inner surface of the vessel, occurrence of sample loss can be prevented.

In a case of forming gas bubbles in the reagent as described above, the area of the gas-liquid interface in the reagent increases. Therefore, it is considered that volatilization of the organic solvent is promoted via the interface.

The second gas supply part supplies a gas into the reagent in the vessel via the reagent discharge part.

According to such a configuration, by supplying a gas into the reagent in the vessel via the reagent discharge part for discharging the reagent in which the sample is dissolved to the outside of the vessel, gas bubbles can be formed in the reagent. Since the reagent discharge part is usually configured to discharge a reagent from a bottom portion inside the vessel, gas bubbles can be formed from lower part of the reagent if a gas is supplied into the vessel via the reagent discharge part.

Since gas bubbles can be favorably formed in the reagent in this manner, concentration of the organic solvent in the reagent can be efficiently lowered. In addition, since gas bubbles can be formed in the reagent without adding a new configuration, concentration of the organic solvent in the reagent can be easily lowered without incurring a cost increase.

The first gas supply part supplies a gas into the vessel via the reagent introduction part.

According to such a configuration, by supplying a gas into the vessel via the reagent introduction part for introducing a reagent into the vessel, the interior of the vessel can be pressurized. Since the reagent introduction part is usually configured to introduce a reagent into the vessel by pressure of a gas, the interior of the vessel can be pressurized without newly adding a configuration if a gas is supplied into the vessel via the reagent introduction part. The first gas supply part may supply a gas from a supply source (for example, a gas cylinder) shared by the second gas supply part, or may supply a gas from a supply source different from the supply source of the second gas supply part. In a case where the gas supplied from the first gas supply part is of the same kind as the gas supplied from the second gas supply part, a common supply source may be used. In a case where the gases are of different kinds, different supply sources may be used.

The device for pretreatment of a sample may further include a setting reception processing unit, and a gas supply control unit. The setting reception processing unit receives setting of a time period for supplying a gas from the second gas supply part. The gas supply control unit controls gas supply from the second gas supply part according to the set time period received by the setting reception processing unit.

According to such a configuration, since the time period for supplying a gas from the second gas supply part can be arbitrarily set, the time period for forming gas bubbles in the reagent can be adjusted by adjusting the set time period, and thus concentration of the organic solvent in the reagent can be arbitrarily adjusted. The gas supply control unit may be configured to control gas supply from the first gas supply part in addition to gas supply from the second gas supply part. In this case, the gas supply control unit may control gas supply from the first gas supply part according to the set time received by the setting reception processing unit.

The device for pretreatment of a sample further includes a sample supply part where a protein sample is subjected to Edman degradation. In this case, an amino acid obtained by Edman degradation in the sample supply part is introduced as a sample into the vessel.

According to such a configuration, in the device for pretreatment of a sample applied to a protein sequencer, occurrence of sample loss can be prevented, and concentration of the organic solvent in the reagent can be easily and efficiently lowered.

An analyzer according to the present invention, as defined in claim <NUM>, includes the device for pretreatment of a sample, and a detector which detects a sample in a reagent discharged via the reagent discharge part.

A method for pretreatment of a sample according to the present invention is defined in claim <NUM>.

According to the present invention, volatilization of the organic solvent in the reagent is promoted by gas bubbles formed in the reagent. Therefore, concentration of the organic solvent in the reagent can be easily and efficiently lowered. In addition, since the sample does not adhere to the inner surface of the vessel, occurrence of sample loss can be prevented.

<FIG> is a block diagram schematically illustrating a configuration example of an analyzer according to an embodiment of the present invention. In <FIG>, the flow of a liquid and a gas is indicated by solid arrows, and the flow of electric signals is indicated by broken line arrows.

As an analyzer in which an embodiment of a device for pretreatment of a sample according to the present invention is adopted, the analyzer according to the present embodiment is an amino acid sequence analyzer (protein sequencer) which can utilize Edman degradation to eliminate amino acids from protein (including peptide) that is a target sample for a sequence analysis, and can analyze the amino acid sequence of the target protein (including peptide, the same applies hereinafter). A description will be given below based on a protein sequencer. The protein sequencer includes a device <NUM> for pretreatment of a sample, a high-performance liquid chromatograph (HPLC) <NUM>, and a control device <NUM>.

The device <NUM> for pretreatment of a sample includes a conversion vessel <NUM>, a sample supply part <NUM>, a reagent supply part <NUM>, a first gas supply part <NUM>, a second gas supply part <NUM>, and the like. In the device <NUM> for pretreatment of a sample, the following process is performed. An amino acid is eliminated from a protein sample by Edman degradation in the sample supply part <NUM> and the eliminated amino acid sample is dissolved in a reagent in the conversion vessel <NUM>.

The conversion vessel <NUM> is a vessel made of, for example, glass. A sample (amino acid sample) is supplied to the conversion vessel <NUM> from the sample supply part <NUM>, and a reagent is supplied to the conversion vessel <NUM> from the reagent supply part <NUM>. The first gas supply part <NUM> supplies an inert gas such as nitrogen gas into the reagent supply part <NUM>, and the pressure of the inert gas causes the reagent to be supplied from the reagent supply part <NUM>. Note that the gas supplied from the first gas supply part <NUM> is not limited to nitrogen gas as long as the gas is an inert gas, and may be another gas such as helium gas or argon gas.

In the sample supply part <NUM>, a protein sample held by a sample holder (not illustrated) such as a glass fiber filter or a PVDF (polyvinylidene fluoride) membrane is set, and a reagent is supplied from the reagent supply part <NUM> to the protein sample. The reagent supplied from the reagent supply part <NUM> to the sample supply part <NUM> is a reagent necessary for Edman degradation. Examples of the reagent include ethyl acetate, n-butyl chloride, trimethylamine, a PITC n-heptane solution, and trifluoroacetic acid; however, the reagent is not limited to the above.

In the sample supply part <NUM>, the reagent supplied from the reagent supply part <NUM> is used to eliminate an amino acid from the protein sample by Edman degradation. Specifically, PTC-protein is generated by a coupling reaction and then the N-terminal amino acid of the PTC-protein is cleaved. Thus, the N-terminal amino acid of the protein sample is eliminated as an ATZ-amino acid. The eliminated ATZ-amino acid is supplied to the conversion vessel <NUM> via a sample introduction tube <NUM>. The sample introduction tube <NUM> configures a sample introduction part for introducing the amino acid sample (ATZ-amino acid) into the conversion vessel <NUM>.

A reagent is introduced from the reagent supply part <NUM> via a reagent introduction tube <NUM> in the conversion vessel <NUM> into which the amino acid sample (ATZ-amino acid) is supplied. The reagent directly introduced into the conversion vessel <NUM> from the reagent supply part <NUM> is of a kind different from the reagent introduced to the sample supply part <NUM>. Examples of the reagent necessary for a conversion reaction from an ATZ-amino acid to a PTH-amino acid include an organic solvent such as acetonitrile, and a mixed solution of the organic solvent and water. The reagent introduction tube <NUM> configures a reagent introduction part for introducing the reagent containing the organic solvent into the conversion vessel <NUM>.

In the conversion vessel <NUM>, the amino acid sample (ATZ-amino acid) introduced from the sample introduction tube <NUM> is stabilized, and then is dissolved in the reagent introduced from the reagent introduction tube <NUM>, and is discharged to the HPLC <NUM> together with the reagent via a reagent discharge tube <NUM>. Specifically, the ATZ-amino acid is converted into a stable PTH-amino acid, and the PTH-amino acid is dissolved in the organic solvent or the mixed solution of the organic solvent and water. The reagent discharge tube <NUM> configures a reagent discharge part which discharges the reagent in which the amino acid sample (PTH-amino acid) is dissolved to the outside the conversion vessel <NUM>.

The HPLC <NUM> includes a column <NUM> and a detector <NUM>. The reagent in which the amino acid sample (PTH-amino acid) is dissolved in the conversion vessel <NUM> is supplied from the device <NUM> for pretreatment of a sample to the column <NUM> via the reagent discharge tube <NUM>, and a sample component is separated while the reagent passes through the column <NUM>. Each sample component separated in the column <NUM> is detected by the detector <NUM>. Examples of the detector <NUM> include an ultraviolet-visible detector (UV/VIS detector); however, the detector <NUM> is not limited to this, and may be another detector such as a photodiode array detector.

In the present embodiment, a gas can be supplied from the first gas supply part <NUM> to the reagent supply part <NUM> and the reagent can be supplied from the reagent supply part <NUM>. In addition, in the present embodiment, a gas can be directly supplied from the first gas supply part <NUM> to the reagent introduction tube <NUM> not through the reagent supply part <NUM>. In this case, not the reagent but the gas is supplied through the reagent introduction tube <NUM> into the conversion vessel <NUM>, and thus, the interior of the conversion vessel <NUM> can be pressurized. Such switchover of a gas supply manner can be performed, for example, by operating a flow channel switchover mechanism including a three-way valve.

The second gas supply part <NUM> can supply a gas into the conversion vessel <NUM> via the reagent discharge tube <NUM>. The gas supplied from the second gas supply part <NUM> is an inert gas such as nitrogen gas. Note that the gas supplied from the second gas supply part <NUM> is not limited to nitrogen gas as long as the gas is an inert gas, and may be another gas such as helium gas or argon gas.

The gas supplied from the second gas supply part <NUM> may be of the same kind as the gas supplied from the first gas supply part <NUM>, or may be of a different kind. The pressure of the gas supplied from the first gas supply part <NUM> and the pressure of the gas supplied from the second gas supply part <NUM> are preferably set to values different from each other.

<FIG> is a schematic cross-sectional view illustrating a configuration example around the conversion vessel <NUM>. The conversion vessel <NUM> is accommodated in a state of being sealed in a heat block <NUM>, and is heated to a set temperature (for example, about <NUM>) by the heat block <NUM>. Note that the heat block <NUM> can be omitted.

Ends of the sample introduction tube <NUM>, the reagent introduction tube <NUM>, and the reagent discharge tube <NUM> are inserted into the conversion vessel <NUM>. The ends of the sample introduction tube <NUM> and the reagent introduction tube <NUM> are located at an upper portion inside the conversion vessel <NUM>, and are located higher than the liquid surface of the reagent <NUM> supplied into the conversion vessel <NUM>. In contrast, the end of the reagent discharge tube <NUM> is located at a bottom portion inside the conversion vessel <NUM>, and is located lower than the liquid surface of the reagent <NUM> supplied into the conversion vessel <NUM>. Note that even though not illustrated in <FIG>, an end of a vent tube for exhausting the gas in the conversion vessel <NUM> into atmosphere may be inserted into the conversion vessel <NUM>.

The amino acid sample (ATZ-amino acid) is introduced from the sample introduction tube <NUM> into the conversion vessel <NUM>. In addition, the reagent <NUM> is introduced from the reagent introduction tube <NUM> into the conversion vessel <NUM>, and therefore the amino acid sample is stabilized and the stabilized amino acid sample (PTH-amino acid) is dissolved in the reagent <NUM> at the bottom portion inside the conversion vessel <NUM>. Then, a gas is supplied from the first gas supply part <NUM> into the conversion vessel <NUM> via the reagent introduction tube <NUM>, and thus air in space <NUM> above the reagent <NUM> in the conversion vessel <NUM> is pressurized.

In this state, a gas is supplied from the second gas supply part <NUM> into the conversion vessel <NUM> via the reagent discharge tube <NUM>. Thus, the gas is supplied into the reagent <NUM> from the end of the reagent discharge tube <NUM> located at the bottom portion inside the conversion vessel <NUM>. As a result, gas bubbles <NUM> are formed in the reagent <NUM>.

After such a process of forming gas bubbles <NUM> in the reagent <NUM> has been performed for only a predetermined time period, the reagent is discharged to the outside of the conversion vessel <NUM> from the reagent discharge tube <NUM>. At that time, the reagent is discharged from the end of the reagent discharge tube <NUM> located at the bottom portion inside the conversion vessel <NUM>, and therefore all of the reagent in the conversion vessel <NUM> can be discharged to the outside.

In the present embodiment, a gas is supplied into the reagent <NUM> in the conversion vessel <NUM> from the second gas supply part <NUM>, and thus gas bubbles <NUM> are formed in the reagent <NUM> and the gas bubbles <NUM> promote volatilization of the organic solvent in the reagent <NUM>. Thus, the organic solvent is volatilized in the space <NUM> above the reagent <NUM> in the conversion vessel <NUM>, and concentration of the organic solvent in the reagent <NUM> can be easily and efficiently lowered. In addition, differing from the configuration of completely drying an amino acid sample (PTH-amino acid) and then adding a solvent for a HPLC, which is a conventional method, since the amino acid sample does not adhere to an inner surface of the conversion vessel <NUM>, occurrence of sample loss can be prevented.

It is considered that since the area of the gas-liquid interface in the reagent <NUM> increases in a case of forming gas bubbles in the reagent <NUM> as described above, volatilization of the organic solvent is promoted via the interface.

In addition, in the present embodiment, a gas is supplied into the reagent <NUM> in the conversion vessel <NUM> via the reagent discharge tube <NUM> for discharging the reagent <NUM> in which the amino acid sample (PTH-amino acid) is dissolved to the outside of the conversion vessel <NUM>, and therefore gas bubbles <NUM> can be formed in the reagent <NUM>. Since the reagent discharge tube <NUM> is usually configured to discharge the reagent <NUM> from the bottom portion inside the conversion vessel <NUM> in the same manner as in the present embodiment, gas bubbles <NUM> can be formed from lower part of the reagent <NUM> if a gas is supplied into the conversion vessel <NUM> via the reagent discharge tube <NUM>.

Therefore, since gas bubbles <NUM> can be favorably formed in the reagent <NUM>, concentration of the organic solvent in the reagent <NUM> can be efficiently lowered. In addition, since gas bubbles <NUM> can be formed in the reagent <NUM> without adding a new configuration, concentration of the organic solvent in the reagent <NUM> can be easily lowered without incurring a cost increase.

Furthermore, in the present embodiment, a gas is supplied into the conversion vessel <NUM> via the reagent introduction tube <NUM> for introducing the reagent <NUM> into the conversion vessel <NUM>, and thus the interior of the conversion vessel <NUM> can be pressurized. Since the reagent introduction tube <NUM> is usually configured to introduce a reagent into the conversion vessel <NUM> by pressure of a gas in the same manner as in the present embodiment, the interior of the conversion vessel <NUM> can be pressurized without newly adding a configuration if a gas is supplied into the conversion vessel <NUM> via the reagent introduction tube <NUM>.

Referring again to <FIG>, the control device <NUM> is configured of a computer, for example, and includes a control unit <NUM>, an operation unit <NUM>, a display unit <NUM>, and the like. The control unit <NUM> is configured to include a CPU (Central Processing Unit), for example. The CPU executes a program, which causes the control unit <NUM> to function as a gas supply control unit <NUM>, a setting reception processing unit <NUM>, an analysis processing unit <NUM>, or the like.

The operation unit <NUM> is configured of, for example, a keyboard and a mouse, and an operator can perform various setting operations by using the operation unit <NUM>. The display unit <NUM> is configured of, for example, a liquid crystal display, and the operator can confirm the operation settings and the operation status of the analyzer by viewing display of the display unit <NUM>.

The gas supply control unit <NUM> controls gas supply from the first gas supply part <NUM> and from the second gas supply part <NUM>. Specifically, by controlling the opening or closing state of a valve (not illustrated) provided to each of the first gas supply part <NUM> and the second gas supply part <NUM>, the gas supply state from each of the first gas supply part <NUM> and the second gas supply part <NUM> is switched over.

The setting reception processing unit <NUM> receives settings regarding gas supply from the first gas supply part <NUM> and from the second gas supply part <NUM>. In the present embodiment, the operator can set a time period for supplying a gas from each of the first gas supply part <NUM> and the second gas supply part <NUM> by operating the operation unit <NUM>. The gas supply control unit <NUM> controls gas supply from each of the first gas supply part <NUM> and the second gas supply part <NUM> according to the set time period received by the setting reception processing unit <NUM> such that a gas is supplied only for the set time period.

As described, in the present embodiment, since the time period for supplying a gas from the second gas supply part <NUM> can be arbitrarily set, the time period for forming gas bubbles <NUM> in the reagent <NUM> can be adjusted by adjusting the set time period, and concentration of the organic solvent in the reagent <NUM> can be arbitrarily adjusted. At that time, since the time period for supplying a gas from the first gas supply part <NUM> can also be arbitrarily set, the interior of the conversion vessel <NUM> can be favorably pressurized in accordance with the time period for forming gas bubbles <NUM>.

The analysis processing unit <NUM> identifies each sample component separated while passing through the column <NUM> according to the detection signal from the detector <NUM>, and analyzes the amino acid sequence of the target protein sample. The analysis result obtained by the analysis processing unit <NUM> is displayed on the display unit <NUM>, and thus the operator is notified of the result. Note that the analysis result obtained by the analysis processing unit <NUM> is not limited to the configuration in which the result is displayed on the display unit <NUM>, and may be, for example, a configuration in which the result is output in another manner such as printing.

<FIG> is a flowchart illustrating a flow of processes performed when a protein sample is analyzed by the analyzer illustrated in <FIG>. When an analysis is started, first, the sample holder holding a protein sample is set in the sample supply part <NUM> (step S101), and then a coupling reaction is performed (step S102).

In the coupling reaction, for example, trimethylamine is supplied from the reagent supply part <NUM> to the sample supply part <NUM>, a reaction chamber of the sample supply part <NUM> is filled with trimethylamine (gas), and then, a PITC n-heptane solution is supplied from the reagent supply part <NUM> into the reaction chamber, the solution is reacted with an N-terminal amino group of protein, and thus PTC-protein is generated. Then, ethyl acetate is supplied from the reagent supply part <NUM> into the reaction chamber to wash out excess reagent and a by-product.

Thereafter, trifluoroacetic acid is supplied from the reagent supply part <NUM> into the reaction chamber, and thus the N-terminal peptide bond of PTC-protein is cleaved, and an ATZ-amino acid is generated (step S103: cleavage reaction). The eliminated ATZ-amino acid is extracted by supplying n-butyl chloride into the reaction chamber from the reagent supply part <NUM>, and the extracted amino acid sample (ATZ-amino acid) is introduced into the conversion vessel <NUM> from the sample supply part <NUM> (step S104).

Next, a conversion reaction is performed in the conversion vessel <NUM> (step S105). In the conversion reaction, for example, a trifluoroacetic acid solution is introduced from the reagent supply part <NUM> into the conversion vessel <NUM>, and thus the ATZ-amino acid is converted into a stable PTH-amino acid. Then, a mixed solution of acetonitrile and water is supplied from the reagent supply part <NUM> into the conversion vessel <NUM>, and thus the PTH-amino acid is dissolved in the mixed solution (step S106). This step S106 constitutes a dissolution step of dissolving the sample (amino acid sample) into the reagent <NUM> containing the organic solvent.

A bubbling process as described with reference to <FIG> is performed for the reagent <NUM> in the conversion vessel <NUM> (step S107). Thereafter, the reagent <NUM> in the conversion vessel <NUM> is discharged from the reagent discharge tube <NUM> to the HPLC <NUM>, and is injected into the column <NUM> of the HPLC <NUM> (step S108). The processes in steps S102 to S108 are repeated until the number of cycles set in advance is reached (Yes in step S109), and the amino acid sample (PTH-amino acid) separated in the column <NUM> is detected by the detector <NUM> (step S110). Then, the detection signal from the detector <NUM> is analyzed by the analysis processing unit <NUM>, and thus data display and record, identification, quantification, yield calculation, or the like of the PTH-amino acid are performed (step Sill).

<FIG> is a flowchart illustrating an example of the bubbling process. In the bubbling process illustrated in step S107 in <FIG>, first, a gas is supplied from the first gas supply part <NUM> into the conversion vessel <NUM> via the reagent introduction tube <NUM>, and thus the interior of the conversion vessel <NUM> is pressurized (step S171: first gas supply step). In addition, a gas is supplied from the second gas supply part <NUM> into the conversion vessel <NUM> via the reagent discharge tube <NUM>, and thus gas bubbles <NUM> are formed in the reagent <NUM> in the conversion vessel <NUM> (step S172: second gas supply step). This state is maintained, and thus concentration of the organic solvent in the reagent <NUM> gradually lowers.

Then, when a time period set in advance as the time period for supplying a gas from each of the first gas supply part <NUM> and the second gas supply part <NUM> has passed (Yes in step S173), gas supply from each of the first gas supply part <NUM> and the second gas supply part <NUM> is stopped (step S174: gas supply stop step), and the reagent <NUM> is discharged from the reagent discharge tube <NUM> to the HPLC <NUM> (reagent discharge step). The time period for supplying a gas from the first gas supply part <NUM> and the time period for supplying a gas from the second gas supply part <NUM> may be identical or different from each other. In addition, the time period for supplying a gas from the first gas supply part <NUM> and the time period for supplying a gas from the second gas supply part <NUM> may be set separately. Alternatively, one of the time periods may be set first and then the other may be set with reference to the time period set first.

Hereinafter, the results of the experiments conducted in order to confirm the effects of the present invention will be described. In the experiments, <NUM>µL of a reagent with concentration of <NUM> pmol/µL obtained by using acetonitrile as an organic solvent and mixing and diluting acetonitrile with water was supplied to an amino acid sample (PTH-amino acid) in the conversion vessel <NUM>.

<FIG> is a diagram illustrating the detection result in a case where an analysis of a protein sample was performed by using a conventional analyzer. <FIG> is a diagram illustrating the detection result in a case where an analysis of the protein sample was performed by using the analyzer according to the present invention. In the experiment illustrated in <FIG>, in a state where the interior of the conversion vessel <NUM> was pressurized by supplying a gas into the conversion vessel <NUM> from the first gas supply part <NUM>, a gas was supplied for <NUM> seconds from the second gas supply part <NUM> into the conversion vessel <NUM> via the reagent discharge tube <NUM>, thereby forming gas bubbles <NUM> in the reagent <NUM> in the conversion vessel <NUM> during that period.

In a case where the conventional analyzer configured to add a solvent for a HPLC after completely drying an amino acid sample (PTH-amino acid) was used, as illustrated in <FIG>, peaks which cannot be favorably separated exist in the former part. This is considered to be due to high concentration of the organic solvent. In contrast, in a case where the analyzer according to the present invention was used, as illustrated in <FIG>, since there is no peak which cannot be favorably separated, it can be seen that concentration of the organic solvent favorably lowered, and the analysis result was improved.

In the above embodiment, the sample introduction part which introduces an amino acid sample (ATZ-amino acid) into the conversion vessel <NUM> is configured of the sample introduction tube <NUM> inserted into the upper portion inside the conversion vessel <NUM>; however, the sample introduction part is not limited to this. For example, an amino acid sample may be introduced by using a member other than a tube. In addition, the reagent introduction part which introduces the reagent <NUM> into the conversion vessel <NUM> is not limited to the reagent introduction tube <NUM> inserted into the upper portion inside the conversion vessel <NUM>. For example, the reagent <NUM> may be introduced by using a member other than a tube. Furthermore, the reagent discharge part which discharges the reagent <NUM> from the conversion vessel <NUM> is not limited to the reagent discharge tube <NUM> inserted into the bottom portion inside the conversion vessel <NUM>. For example, the reagent <NUM> may be discharged by using a member other than a tube.

In addition, in the above embodiment, the configuration where a gas is supplied from the first gas supply part <NUM> into the conversion vessel <NUM> via the reagent introduction tube <NUM> has been described.

Claim 1:
A device (<NUM>) for pretreatment of a sample for a protein sequencer comprising a high-performance liquid chromatograph, the device comprising:
a vessel (<NUM>);
a sample supply part (<NUM>) configured to subject a protein sample to Edman degradation, and to introduce an amino acid obtained by Edman degradation in the sample supply part as a sample into the vessel;
a reagent introduction part (<NUM>) which is configured to introduce a reagent containing an organic solvent into the vessel so that the sample is dissolved in the reagent;
a reagent discharge part (<NUM>) which is configured to discharge the reagent in which a sample is dissolved in the vessel to an outside of the vessel;
a first gas supply part (<NUM>) which is configured to supply a gas into the vessel and thus pressurize an interior of the vessel;
a second gas supply part (<NUM>) which is different from the first gas supply part and which is configured to supply a gas into the reagent in the vessel and thus forms a gas bubble in the reagent; and
a control unit (<NUM>);
wherein the control unit performs;
a first gas supply step of controlling gas supply from the first gas supply part, supplying a gas into the vessel and thus pressurizing an interior of the vessel; and
a second gas supply step of controlling gas supply from the second gas supply part, supplying a gas into the reagent in the vessel and thus forming a gas bubble in the reagent so that the gas bubble volatilizes the organic solvent in the reagent;
wherein the reagent discharge part is configured to discharge the reagent in the vessel from a reagent discharge tube to the high-performance liquid chromatograph after the first gas supply step and the second gas supply step;
wherein the first gas supply part is configured to supply a gas into the vessel via the reagent introduction part, and the second gas supply part is configured to supply a gas into the reagent in the vessel via the reagent discharge part.