JP2002365177A - Pretreating device of sample for mass spectrometry - Google Patents

Abstract

PROBLEM TO BE SOLVED: To facilitate sample preparation for MALDI-TOF MS. SOLUTION: Gel is imaged by a scanner 2, and a spot is cut off by a cutter of a cutter part 20 and transferred to a well of MTP 10a. The gel is cleaned in the well and dried, and then enzyme liquid is dispensed, to digest a protein into a peptide. Extracted liquid is dispensed, and the peptide is extracted from the gel. Zip chip treatment of the extracted peptide is executed, and the peptide is mixed with a matrix and discharged onto an MS target plate, to thereby prepare the sample.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a MALDI-TOF MS
The present invention relates to an apparatus used for pretreatment of a PMF method (Peptide MS Fingerprinting method) for identifying a protein in a living body using a (matrix-assisted laser desorption / ionization time-of-flight mass spectrometer).

[0002]

2. Description of the Related Art When trying to identify a protein in a living body using MALDI-TOF MS, the following four pretreatment steps are required to prepare a sample for MALDI-TOF MS measurement.

[0003] A step of imaging and analyzing the gel or the like in order to determine a sample spot to be measured on a gel after electrophoresis or on a membrane after transfer. This step is
It is mainly performed using a CCD camera, a computer, etc., but a flatbed scanner may be used for imaging. The step of cutting out the migration spot to be cut out by the cutter, which is determined in the step. A digestion step in which a protein in the cut electrophoretic spot is decomposed into peptides by an enzyme and extracted. A step of preparing a sample for mass spectrometry by placing the extracted peptide on a target plate of a mass spectrometer.

An apparatus having a function of executing each of these four steps independently is used. Further, among these four steps, an apparatus having a function of executing the step and an apparatus having a function of executing the step are also used.

[0005]

For these pretreatments, two or three devices are required, a large installation space is required, and not only the purchase cost of the devices is increased, but also the space between these devices is increased. The movement of the sample required manual labor, which was inconvenient. In addition, an operation for driving is required for each device, which hinders improvement in work efficiency. When the sample is moved between the instruments or in the instrument without the entire cover, the suspended matter (especially keratin) dropped from the air during the operation or at rest of the instrument has an adverse effect on the MALDI-TOF MS measurement due to contamination.

[0006] The present invention provides a sample pretreatment which uses MALDI-TOF MS to facilitate the preparation of a measurement sample for identifying a protein in a living body and which can prevent contamination by airborne suspended matter from the air. It is intended to provide a device.

[0007]

According to the present invention, there is provided a sample pretreatment apparatus for mass spectrometry, which holds an electrophoresed gel or a membrane after transfer, and performs an imaging and analysis of the gel and an optical scanner unit. Holds a container and a container that analyzes the designated electrophoretic spot and extracts the peptide by extracting the spots cut out by the cutter in the container. A digester, a mass loader that holds a target plate of a mass spectrometer, and sets the peptide extracted in the digester on the target plate together with a matrix, and holds enzymes and reagents used in the digester. A reagent portion to be dispensed, the enzyme and the reagent of the reagent portion being dispensed into the digestion portion, and the extracted peptide together with the matrix A pipette mechanism for transferring onto a target plate, a moving mechanism for moving the cutter unit and the pipette mechanism between required positions, and a housing that covers and accommodates these parts and mechanisms. .

In the present invention, by providing four functions in one apparatus, installation space and purchase cost can be reduced, and there is no need to move samples between apparatuses, and the operating environment is unified. Since the entire device was housed in the housing,
Isolation from falling suspended matter becomes possible.

[0009]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS It is preferable that a housing for accommodating each part and each mechanism can be made hermetically closed. This allows better isolation from falling suspended matter. Gels, membranes, containers used for pretreatment, reagents, MS target plates, etc. are installed in the workspace of the device, but there is a moving mechanism in the workspace, which is driven during operation of the device. It is dangerous that an operator manually installs or removes the above members and reagents into and out of the work space during operation of the apparatus.

[0010] In order to avoid such dangers and to allow replacement of gels and reagents even during operation of the apparatus, the gel or the membrane after transfer is held on an optical scanner unit and can be transported inside and outside the casing. It is preferable to provide a tray to be used. It is also preferable to provide a tray that holds the enzyme, the reagent, and the target plate, and that can be transported inside and outside the casing.

[0011]

FIG. 1 is a plan view of an internal main portion of an embodiment. Reference numeral 2 denotes an optical scanner for imaging and analyzing a gel or a membrane, which is a sample placed on the top, and uses a flatbed scanner here. Scanner 2
A sample tray 4 is provided for placing a gel or a membrane thereon, and the tray 4 holds a transparent glass plate above the scanner 2 so that a gel or a membrane can be placed on the glass plate. ing.

A tray 6 for installing reagents and the like is disposed adjacent to the tray 4. A sample section MTP (microtiter plate) as a digestion section for accommodating a reagent section 8 holding enzymes and reagents and a sample spot cut out on the scanner 2 on the tray 6 and extracting the peptide by digestion with the enzyme is stored. ) 10a and MT containing desalting solution
P10b, a pipette chip housing section 12 housing a pipette chip for dispensing enzymes and reagents for digesting proteins and extracting peptides, and a μ-C18 Zip chip (Millipore) for desalting and concentrating the extracted peptides ) And a target plate 16 for MALDI. An MTP with 96 wells is used as the MTP 10a, and an MTP with 384 wells is used as the MTP 10b.
Are arranged at the maximum, and one MTP 10b is arranged. Each member on the tray 6 is detachably mounted on the tray 6.

A cutter section 20 is provided to cut out an electrophoretic spot on the scanner 2. The cutter unit 20 has a hollow pipe-shaped cutter installed in a vertical direction, and presses the tip of the pipe in the thickness direction of the gel or membrane on a scanner to pack and cut the gel or membrane into the pipe. Cutter part 2
No. 0 also has a mechanism for supplying pure water to discharge the gel or membrane cut by the pipe-shaped cutter into the wells of the MTP 10a.

As the pipette mechanism, eight pipettes are arranged, and a probe 22 which is operated simultaneously is arranged. The eight pipettes are driven by respective syringe pumps 24. The pipette tip of the pipette tip housing section 12 or the Zip tip of the Zip tip housing section 14 is detachably attached to the tip of the pipette.

The cutter section 20 and the probe 22 are moved in the Z direction.
(Z perpendicular to the paper surface: perpendicular to the horizontal plane)
It is attached to the moving mechanism. The Z moving mechanism includes an air cylinder driven by the supply of compressed air, and can move the cutter unit 20 and the probe 22 up and down (Z direction). A clutch mechanism is provided between the cutter unit 20 and the probe 22. When the probe 22 is moved up and down, the cutter unit 20 is also moved at the same time. When the cutter unit 20 is moved up and down, the cutter 22 is stopped. The clutch mechanism is operated so that only the part 20 moves up and down.

The Z-direction moving mechanism is attached to an XY moving mechanism that moves on each portion on the trays 4 and 6 in the X direction (horizontal direction in the figure) and the Y direction (vertical direction in the figure) in a horizontal plane. I have. In the XY moving mechanism, a Y guide shaft 26 fixed in the Y-axis direction is fixed to a housing 40, and a Y moving unit 28 that moves along the Y guide shaft 26 is provided on the Y guide shaft 26. ing. The Y moving portion 28 is screwed with a bar screw 27 extending in the Y axis direction, and is moved in the Y direction along the Y guide shaft 26 by rotating the bar screw 27. Reference numeral 29 denotes a Y drive motor for rotating the bar screw 27.

An X guide shaft 30 which extends in the X direction below the Y guide shaft 26 (vertically below) is fixed to the Y moving portion 28. Cutter section 20 and probe 22
Is movably supported by the X guide shaft 30, and the
At the intersection with the guide shaft 26, X
It is attached to the guide shaft 30. The Z drive unit of the cutter unit 20 and the probe 22 is a bar screw 32 extending in the X-axis direction.
And the rod screw 32 is rotated in the X direction along the X guide shaft 30 by rotation. 3
Reference numeral 4 denotes an X drive motor for rotating the rod screw 32.
By this moving mechanism, the cutter unit 20 and the probe 22
Can move freely in the X, Y, and Z directions and can be transferred to any position.

As shown in FIG. 2, the pretreatment device is housed in a housing 40, which covers these parts and is housed in a sealed state. The trays 4 and 6 can be independently transferred inside and outside the housing 40, and exchange gels or membranes, reagents, chips or M during operation of the apparatus.
Exchange and replenishment of TP and the like can be performed.

Next, the operation of this embodiment will be described. (1) Gel imaging and cutting The gel is placed on the tray 4 and placed on the scanner 2, and at least one MTP 10 a is arranged at the position of the sample MTP 10 a on the tray 6. Up to four MTPs 10a can be installed on the tray 6. The MTP 10a has 96 wells, and a maximum of 96 sample spots can be collected for one MTP 10a. 2. An image is taken by the scanner 2. 3. A spot to be cut is selected, and a well of the MTP 10a to which the cut spot is to be transferred is designated. 4. Start cutting the gel. 5. The cutter of the cutter unit 20 moves onto a spot to be cut, and discharges a predetermined amount of water onto the gel. The spot is cut out by the cutter descending onto the spot and pressed in the thickness direction of the gel, and the cut gel is stored in the cutter by suction. The cutter is raised, moved by the moving mechanism onto the well of the designated MTP 10a, and discharged to discharge the cut gel together with water into the well. 6. Repeat step 5 until all necessary spots have been cut out.

(2) Washing and decolorization of cut gel (2-1) Removal of water when discharging gel Since the tip used in the previous operation is mounted on the pipette of the probe 22, the probe 22 is moved to the tip discharge position and the tip is discharged. The probe 22
Is provided with eight pipettes, and the processing is simultaneously performed on these eight pipettes. The same applies to the following steps. 2. The probe 22 is moved to the drain and the pipette is washed by discharging water with a syringe pump 24. 3. The probe 22 moves to the position of the pipette tip section 12 and mounts the tip. 4. The probe 22 moves to the sample MTP 10a, and sucks the solution in the well. 5. The probe 22 moves to the drain, and discharges the sucked solution. 6. Step 4-5 until all wells are drained
repeat.

(2-2) Washing of gel The probe 22 moves onto the washing solution in the reagent section 8 and sucks a predetermined amount of the washing solution. 9. The probe 22 moves onto the sample MTP 10a,
Dispense the washing solution into the wells. 10. Steps 8 and 9 are repeated, and the washing liquid is poured into all the wells of the MTP 10a. 11. Hold at room temperature or 37 ° C. for a certain time. 12. The probe 22 moves to the MTP 10a and sucks the cleaning liquid. 13. The probe 22 moves to the drain and discharges the sucked cleaning liquid. 14． Steps 8-13 are repeated several times until the gel discolors.

(2-3) Drying of gel The probe 22 moves onto the dry solvent of the reagent section 8,
A predetermined amount of the dry solvent is sucked. 22. The probe 22 moves to the MTP 10a, and dispenses the dry solvent. 23. Steps 21 to 22 are repeated, and the dry solvent is dispensed to all the wells of the MTP 10a. 24. Hold at room temperature or 37 ° C. for a predetermined time. 25. The probe 22 moves to the MTP 10a, and sucks the dry solvent in the well. 26. The probe 22 moves to the drain, and discharges the sucked dry solvent. 27. Steps 25-26 are repeated to remove dry solvent from all wells of MTP 10a. (Steps 21-27 here can also be omitted.) The probe 22 moves to the chip discharging position and discharges the chip. 29. The probe 22 moves to the drain and supplies water from the syringe pump 24 to clean the pipette. 30. The MTP 10a is moved to a vacuum dryer to dry the gel spot. Alternatively, it may be dried by holding at 37 ° C. for a predetermined time. The transfer of the MTP 10a in this step 30 is performed manually.

(3) Digestion in gel (3-1) Preparation of chip After the gel has been dehydrated, the MTP 10a is placed on the tray 6 and returned into the device. This step is also performed manually. 2. The probe 22 moves to the chip discharging position and discharges the chip. 3. The probe 22 moves to the drain and cleans the pipette by supplying water from the syringe pump 24. 4. The probe 22 moves to the pipette tip housing section 12 and mounts the tip. 5. A predetermined amount of air is sucked.

(3-2) Dispensing of enzyme solution The probe 22 moves to the position of the enzyme solution in the reagent section 8, and aspirates a predetermined amount of the enzyme solution. 7. The probe 22 moves to the MTP 10a, and dispenses the enzyme solution into the well. 8. Steps 6-7 are repeated, and the enzyme solution is added to all the wells of the MTP 10a.

(3-3) Digestion The probe 22 moves to the chip discharging position and discharges the chip. 10. The probe 22 moves to the drain and supplies water from the syringe pump 24 to wash the pipette. 11. The probe 22 returns to the home position. 12. MTP10a is sealed with a sealing tape and heated at 37 ° C.
For at least 4 hours or at 30 ° C. overnight. This step 12 is performed manually.

(4) Extraction (4-1) Preparation of chip The MTP 10a is placed on the tray 6 and returned into the device. This operation is also performed manually. 2. The probe 22 moves to the chip discharging position and discharges the chip. 3. The probe 22 moves to the drain and supplies water from the syringe pump 24 to wash the pipette. 4. The probe 22 moves to the pipette tip section 12 and attaches the tip.

(4-2) Dispensing of extract solution The probe 22 moves to the position of the extract in the reagent section 8,
A predetermined amount of the extract is aspirated. 6. The probe 22 moves to the MTP 10a, and dispenses the extract into the well. 7. Steps 5-6 are repeated, and the extract is added to all the wells of the MTP 10a.

(4-3) Extraction The MTP 10a is moved to an incubator and kept at 30 ° C. for 30 minutes to remove the remaining organic solvent in the extract.

(5) Zip chip treatment and sample preparation on MS target plate (5-1) Preparation of desalting solution for MTP 10b The MTP 10a is placed on the tray 6 and returned into the apparatus. This step is performed manually. The probe 22 moves to the chip discharging position and discharges the chip. 3. The probe 22 moves to the drain and supplies water from the syringe pump 24 to wash the pipette. 4. The probe 22 moves to the desalting solution of the reagent section 8 and aspirates a predetermined amount of the desalting solution into the syringe. 5. The probe 22 moves to the MTP 10b, and the desalted solution is dispensed to each well. The position of the well of the MTP 10b corresponds to the position on the MS target plate 16. 6. Steps 4-5 are repeated and the desalted solution is added to all designated wells.

(5-2) Equilibration of Zip chip The probe 22 moves to the Zip tip section 14 and
Attach p-tip. 8. The probe 22 moves to the wetting solution in the reagent section 8 and repeats suction and discharge of the wetting solution several times to wet the Zip tip.

(5-3) Washing Zip Chip The probe 22 moves to the equilibrium solution in the reagent section 8, and the suction and discharge of the equilibrium solution are repeated several times to equilibrate the Zip tip.

(5-4) Adsorption of sample The probe 22 moves to the MTP 10a, and the suction and discharge of the sample are repeated several times at the same position.

(5-5) Desalting The probe 22 moves to the MTP 10b, and the MTP 1
A predetermined amount of the desalting solution in the well 0b is sucked.
The probe 22 moves to the drain and discharges the desalted solution. 12. Step 11 is repeated several more times.

(5-6) Aspiration of eluate of matrix mixture After the probe 22 moves to the matrix solution of the reagent section 8 and sucks a predetermined amount of air, the probe 22 descends and the pipette sucks the matrix by a predetermined amount.

(5-7) Discharge to MS target plate The probe 22 moves to the MS target plate 16. The position corresponds to the designated well of the MTP 10a. 15. The probe 22 descends, and the Zip tip contacts the surface of the MS target plate 16. Thereby, the solution is dispensed to the MS target plate 16. 16. The probe 22 moves to the tip discharge position, and the Zip
Discharge chips. 17． Steps 7-16 are repeated until all samples have been processed. 18. When the sample on the MS target plate 16 is completely dried, the MS target plate 16 can be set on a mass spectrometer to acquire a spectrum.

In the embodiment shown in FIG. 1, a flatbed scanner is used as the scanner 2, but the scanner is mounted on the moving mechanism for moving the light receiving element in the Y direction in the embodiment, and the light receiving element is scanned. May be used. Further, the temperature of the MTP 10a may be controlled by a computer so that each constant temperature holding step is performed on the tray 6. Further, by automating the sealing of the MTP 10a, it is possible to completely automate the entire process.

[0037]

The sample pretreatment device for mass spectrometry according to the present invention
Since the four functions required for mass spectrometry sample adjustment can be realized with a single device, installation space and purchase costs can be reduced, and there is no need to move samples between devices, which reduces the operating environment. Unified. In addition, since the entire device is housed in the housing, it is possible to isolate the device from falling suspended matters. If the inside of the housing can be sealed,
Better isolation from falling objects,
Higher precision analysis becomes possible. Gels, membranes,
If containers, reagents, chips, MS target plates, and the like used for pretreatment are set on a tray and the tray can be transferred inside and outside the housing, exchange and setting of those members and reagents can be performed outside the workspace. Can be performed, it is safe for the operator, and the work efficiency can be improved.

[Brief description of the drawings]

FIG. 1 is a plan view of a main part of one embodiment.

FIG. 2 shows two scanners 4, 6 trays 8 reagent parts 10a, 10b microtiter plate 12 pipette chip storage part 14 Zip chip storage part 16 MALDI target plate 20 cutter part 22 probe with pipette 26 Y guide shaft 28 Y movement Part 30 X guide shaft

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court ゛ (Reference) G01N 1/28 G (72) Inventor Chau Hoang Thanh Nguyen Australia 3079 Victoria A Ivanhoe Beatty Street 57 (72) Inventor William Samuel Hunter Australia 3032 Victoria A Scottvale The Parade 28 (72) Inventor Robert John Ramsden Australia 2079 NSW Mount Tocoller Sprig Place 29 F-term (reference) 2G045 BA11 BB60 DA36 FB01 FB05 JA11 2G052 AA28 AB16 AB18 AD17 BA02 BA15 CA03 CA04 CA18 FD03 GA24

Claims (4)

[Claims] 1. An optical scanner unit for holding a gel or a transferred membrane after electrophoresis is completed, and performing imaging and analysis thereof, and cutting out a designated electrophoretic spot analyzed by the optical scanner unit. A cutter unit, holding a container, housing the spot cut out by the cutter unit in the container, digesting the protein of the spot to extract a peptide, and holding a target plate of a mass spectrometer, A mass loader section for placing the peptide extracted in the digestion section together with a matrix on the target plate; a reagent section for holding enzymes and reagents used in the digestion section; and an enzyme and reagent for the reagent section in the digestion section. A pipette mechanism for dispensing the extracted peptide together with the matrix onto the target plate, A pretreatment apparatus for a sample for mass spectrometry, comprising: a moving mechanism for moving the cutter unit and the pipette mechanism between required positions; and a housing for accommodating these units and each mechanism. 2. The pre-processing device according to claim 1, wherein the housing is capable of sealing the inside thereof. 3. The pretreatment device according to claim 1, further comprising a tray that holds the gel or the membrane after the transfer on an optical scanner unit and enables the gel or the membrane to be transferred inside and outside the housing. 4. The pretreatment apparatus according to claim 1, further comprising a tray that holds the enzyme, the reagent, and the target plate and allows the enzyme, the reagent, and the target plate to be transferred inside and outside the casing.