U.S. patent application number 14/119639 was filed with the patent office on 2014-08-07 for system and method for retrieval treatment of proteins in formalin-fixed paraffin-embedded tissue section.
This patent application is currently assigned to SHIZUOKA PREFECTURE. The applicant listed for this patent is SHIMADZU CORPORATION, SHIZUOKA PREFECTURE. Invention is credited to Yutaka Aoki, Keiichi Hatakeyama, Isamu Hayashi, Shun-ichiro Ogura, Taka-Aki Sato, Yukari Umino.
Application Number | 20140220624 14/119639 |
Document ID | / |
Family ID | 47217017 |
Filed Date | 2014-08-07 |
United States Patent
Application |
20140220624 |
Kind Code |
A1 |
Aoki; Yutaka ; et
al. |
August 7, 2014 |
SYSTEM AND METHOD FOR RETRIEVAL TREATMENT OF PROTEINS IN
FORMALIN-FIXED PARAFFIN-EMBEDDED TISSUE SECTION
Abstract
A protein retrieval treatment system used for activating
proteins contained in a deparaffinized tissue section obtained by
removing paraffin from a formalin-fixed paraffin-embedded tissue
section. The protein retrieval treatment system includes: a
dispensing unit for dispensing a retrieval treatment solution over
a dispensing area including a measurement area on the
deparaffinized tissue section; and a moist-heat treatment unit for
heating, in a saturated water vapor, the deparaffinized tissue
section with the dispensing area covered with the retrieval
treatment solution.
Inventors: |
Aoki; Yutaka; (Nerima-ku,
JP) ; Umino; Yukari; (Yokohama-shi, JP) ;
Sato; Taka-Aki; (Shinagawa-ku, JP) ; Hayashi;
Isamu; (Meguro-ku, JP) ; Hatakeyama; Keiichi;
(Sunto-gun, JP) ; Ogura; Shun-ichiro;
(Yokohama-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SHIZUOKA PREFECTURE
SHIMADZU CORPORATION |
Shizuoka-city, Shizuoka
Kyoto-shi, Kyoto |
|
JP
JP |
|
|
Assignee: |
SHIZUOKA PREFECTURE
Shizuoka-city, Shizuoka
JP
SHIMADZU CORPORATION
Kyoto-shi, Kyoto
JP
|
Family ID: |
47217017 |
Appl. No.: |
14/119639 |
Filed: |
April 25, 2012 |
PCT Filed: |
April 25, 2012 |
PCT NO: |
PCT/JP2012/061090 |
371 Date: |
March 5, 2014 |
Current U.S.
Class: |
435/40.52 ;
422/521 |
Current CPC
Class: |
G01N 33/6848 20130101;
G01N 33/68 20130101; G01N 1/30 20130101 |
Class at
Publication: |
435/40.52 ;
422/521 |
International
Class: |
G01N 1/30 20060101
G01N001/30; G01N 33/68 20060101 G01N033/68 |
Foreign Application Data
Date |
Code |
Application Number |
May 25, 2011 |
JP |
2011-117334 |
Claims
1. A protein retrieval treatment system for retrieval of protein
molecules in a measurement area on a deparaffinized tissue section
prepared by removing paraffin from a formalin-fixed
paraffin-embedded tissue section obtained by a formalin-fixing
treatment followed by a paraffin-embedding treatment, the retrieval
being performed for a mass spectrometry of the measurement area and
achieved by dissociating a crosslink resulting from formalin
fixation in the protein molecules contained in the measurement
area, and the system comprising: a dispensing unit for dispensing a
retrieval treatment solution over a dispensing area including the
measurement area on the deparaffinized tissue section; and a
moist-heat treatment unit for heating, by using a saturated water
vapor, the deparaffinized tissue section with the retrieval
treatment solution put on the dispensing area.
2. The protein retrieval treatment system according to claim 1,
wherein the dispensing unit dispenses the retrieval treatment
solution at predetermined space intervals so as to form a plurality
of droplets separated from each other within the dispensing
area.
3. The protein retrieval treatment system according to claim 2,
wherein a volume of each of the droplets and the space intervals of
the droplets are set so that the dispensing area is covered with
the plurality of droplets.
4. The protein retrieval treatment system according to claim 1,
wherein the dispensing unit dispenses the retrieval treatment
solution a plurality of times onto a same spot within the
dispensing area.
5. A protein retrieval treatment method for retrieval of protein
molecules in a measurement area on a deparaffinized tissue section
prepared by removing paraffin from a formalin-fixed
paraffin-embedded tissue section obtained by a formalin-fixing
treatment followed by a paraffin-embedding treatment, the retrieval
being performed for a mass spectrometry of the measurement area and
achieved by dissociating a crosslink resulting from formalin
fixation in protein molecules contained in the measurement area,
and the method comprising: a dispensing process in which a
retrieval treatment solution is dispensed over a dispensing area
including the measurement area on the deparaffinized tissue
section; and a moist-heat treatment process in which the
deparaffinized tissue section with the retrieval treatment solution
put on the dispensing area is heated by using a saturated water
vapor.
6. The protein retrieval treatment method according to claim 5,
wherein the retrieval treatment solution is dispensed at
predetermined space intervals so as to form a plurality of droplets
separated from each other within the dispensing area.
7. The protein retrieval treatment method according to claim 6,
wherein a volume of each of the droplets and the space intervals of
the droplets are set so that the dispensing area is covered with
the plurality of droplets.
8. The protein retrieval treatment method according to claim 5,
wherein the retrieval treatment solution is dispensed a plurality
of times onto a same spot within the dispensing area.
9. The protein retrieval treatment method according to claim 5,
wherein a pH of the retrieval treatment solution is equal to or
higher than 9.
Description
TECHNICAL FIELD
[0001] The present invention relates to a system and method for a
retrieval treatment of proteins contained in a formalin-fixed
paraffin-embedded tissue section obtained by a formalin-fixing
treatment followed by a paraffin-embedding treatment (such a tissue
section is hereinafter called the "paraffin-embedded tissue
section"), and more specifically, to a retrieval treatment system
and method for pretreating the aforementioned tissue section in
advance of a mass spectrometry.
BACKGROUND ART
[0002] In recent years, the bio-imaging technique for imaging a
distribution of biological substances (such as proteins or
metabolites) in a section of biological tissue has been used in the
fields of clinical diagnoses or drug discoveries. In particular,
this technique is widely used for an investigation of a
distribution of biological substances, in which a section sample
prepared by an appropriate pretreatment of a paraffin-embedded
tissue section is scanned by a MALDI (matrix-assisted laser
desorption ionization) mass spectrometer, and the thereby measured
masses of biological substances in the section sample are
comprehensively analyzed to determine the distribution of the
biological substances.
[0003] A pretreating method described in Non-Patent Documents 1, 2,
3 and 4 has conventionally been used to make a paraffin-embedded
tissue section available as a section sample for a mass
spectrometry. This conventional pretreatment method is hereinafter
described by means of the flowchart shown in FIG. 2.
[0004] Initially, paraffin is removed from the paraffin-embedded
tissue section, after which a hydration treatment is performed to
obtain a deparaffinized tissue section (S1). Inside the molecules
of the proteins contained in the deparaffinized tissue section thus
obtained, bonding of single amino acid residues or a formation of
methylene crosslink by a hydroxyl group, in particular, a strong
methylene crosslink via a primary amino or phenyl radical, have
occurred. To dissociate such bonds or crosslinks (both are referred
to as "crosslinks" hereinafter), the tissue section is subjected to
a heat treatment in a retrieval treatment solution (S2: this
treatment is hereinafter called the "retrieval treatment").
Subsequently, the retrieval treatment solution which has adhered to
the deparaffinized tissue section during the retrieval treatment is
washed off (S3). After the washing, the deparaffinized tissue
section is treated with a digestive enzyme, such as trypsin, to
hydrolyze proteins contained in the tissue section into peptides
(S4). Lastly, after the enzymatic treatment, a matrix solution is
dropped onto the deparaffinized tissue section to deposit the
matrix on it (S5). The section sample thus prepared is subjected to
the MALDI mass spectrometry.
[0005] The retrieval treatment in the previously described
conventional pretreatment method is not only indispensable for
restoring two-dimensional and three-dimensional structures of the
protein molecules which have been damaged due to the crosslinking
reaction caused by the formalin fixation treatment, but also for
facilitating an approach of the digestive enzyme to protein
molecules in the subsequent enzymatic treatment. In one
conventional retrieval treatment, the deparaffinized tissue section
is immersed in a retrieval treatment solution containing a
surfactant, and a heat treatment (in a hot bath, a microwave oven
or an autoclave) is performed on the immersed sample for a
predetermined period of time.
BACKGROUND ART DOCUMENT
Non-Patent Document
[0006] Non-Patent Document 1: Maurizio Ronci, Elena Bonanno,
Alfredo Colantoni, Luisa Pieroni, Carmine Di Ilio, Luigi Giusto
Spagnoli, Giorgio Federici and Andrea Urbani, "Protein unlocking
procedures of formalin-fixed paraffin-embedded tissues: Application
to MALDI-TOF Imaging MS investigations", Proteomics, vol. 8, 2008,
pp. 3702-3714
[0007] Non-Patent Document 2: M. Reid Groseclose, Pierre P.
Massion, Pierre Chaurand, and Richard M. Caprioli, "High-throughput
proteomic analysis of formalin-fixed paraffin-embedded tissue
microarrays using MALDI imaging mass spectrometry", Proteomics,
Vol. 8, No. 18, 2008, pp. 3715-3724
[0008] Non-Patent Document 3: Marie-Claude Djidja, Emmanuelle
Claude, Marten F. Sne, Peter Scriven, Simona Francese, Vikki
Carolan and Malcolm R. Clench, "MALDI-Ion Mobility Separation-Mass
Spectrometry Imaging of Glucose-Regulated Protein 78 kDa (Grp78) in
Human Formalin-Fixed, Paraffin-Embedded Pancreatic Adenocarcinoma
Tissue Sections", Journal of Proteome Research, Vol. 8, No. 10,
2009, pp. 4876-4884
[0009] Non-Patent Document 4: Johan O. R. Gustafsson, Martin K.
Oehler, Shaun R. McColl and Peter Hoffmann, "Citric Acid Antigen
Retrieval (CAAR) for Tryptic Peptide Imaging Directly on Archived
Formalin-Fixed Paraffin-Embedded Tissue", Journal of Proteome
Research, Vol. 9, No. 9, 2010, pp. 4315-4328
SUMMARY OF THE INVENTION
Problem to be Solved by the Invention
[0010] However, when the deparaffinized tissue section is immersed
in the retrieval treatment solution, a portion of the biological
substances in the deparaffinized tissue section flows into the
retrieval treatment solution and is lost. The loss of biological
substances from the tissue section also occurs in the process of
washing off the retrieval treatment solution adhered to the
deparaffinized tissue section after the retrieval treatment. As a
result, when a section sample prepared through the conventional
retrieval treatment is subjected to a MALDI mass spectrometry, the
intensities of the peaks originating from the biological substances
become low, making the result of a quantitative determination
incorrect or preventing a qualitative analysis of a trace amount of
component. Such a situation leads to the problem that, when an
image mapping is performed based on the result of the MALDI mass
spectrometry, it is difficult to create a mapping image with a
sufficient amount of information for correctly reflecting the true
concentrations of the biological substances.
[0011] Thus, the problem to be solved by the present invention is
to provide a system and method for a retrieval treatment of
proteins in a deparaffinized tissue section in which the loss of
biological substances from the tissue section does not occur in the
process of activating the proteins in the deparaffinized tissue
section, and therefore, the peaks can be detected with adequate
intensities that cause no problems in a quantitative or qualitative
analysis in a MALDI mass spectrometry, so that a larger amount of
information is available for the image mapping.
Means for Solving the Problem
[0012] A system for a retrieval treatment of proteins in a
deparaffinized tissue section according to the present invention
aimed at solving the previously describe problem is a protein
retrieval treatment system for retrieval of protein molecules in a
measurement area on a deparaffinized tissue section prepared by
removing paraffin from a formalin-fixed paraffin-embedded tissue
section obtained by a formalin-fixing treatment followed by a
paraffin-embedding treatment, the retrieval being performed for a
mass spectrometry of the measurement area and achieved by
dissociating a crosslink resulting from formalin fixation in the
protein molecules contained in the measurement area, and the system
including:
[0013] a dispensing unit for dispensing a retrieval treatment
solution over a dispensing area including the measurement area on
the deparaffinized tissue section; and
[0014] a moist-heat treatment unit for heating, by using a
saturated water vapor, the deparaffinized tissue section with the
retrieval treatment solution put on the dispensing area.
[0015] The system for a retrieval treatment of proteins in a
deparaffinized tissue section according to the present invention is
designed so as to exclude, from the retrieval treatment, the
conventional process of immersing the deparaffinized tissue section
in a retrieval treatment solution and washing the tissue section
after the immersing. After a retrieval treatment solution is
dispensed in the dispensing unit onto the deparaffinized tissue
section, the deparaffinized tissue section is subjected to the
moist-heat treatment. To make this treatment as effective as the
process of immersing the deparaffinized tissue section in a
retrieval treatment solution, the retrieval treatment solution
should preferably contain a surfactant. Adding a surfactant to the
retrieval treatment solution improves the affinity between the
retrieval treatment solution and the deparaffinized tissue section,
so that the crosslink resulting from the formalin fixation can be
adequately dissociated when subjected to the moist-heat
treatment.
[0016] In the previously described retrieval treatment system, the
dispensing unit may dispense the retrieval treatment solution at
predetermined space intervals so as to form a plurality of droplets
separated from each other within the dispensing area.
[0017] In this case, the volume of each of the droplets and the
space intervals of the droplets should preferably be set so that
the dispensing area is covered with the plurality of droplets.
[0018] The dispensing unit may be configured so that it dispenses
the retrieval treatment solution a plurality of times onto the same
spot within the dispensing area.
[0019] A method for a retrieval treatment of proteins in a
deparaffinized tissue section according to the present invention
aimed at solving the previously described problem is a protein
retrieval treatment method for retrieval of protein molecules in a
measurement area on a deparaffinized tissue section prepared by
removing paraffin from a formalin-fixed paraffin-embedded tissue
section obtained by a formalin-fixing treatment followed by a
paraffin-embedding treatment, the retrieval being performed for a
mass spectrometry of the measurement area and achieved by
dissociating a crosslink resulting from formalin fixation in
protein molecules contained in the measurement area, and the method
including:
[0020] a dispensing process in which a retrieval treatment solution
is dispensed over a dispensing area including the measurement area
on the deparaffinized tissue section; and
[0021] a moist-heat treatment process in which the deparaffinized
tissue section with the retrieval treatment solution put on the
dispensing area is heated by using a saturated water vapor.
[0022] In the dispensing process of the previously described
invention, the retrieval treatment solution may be dispensed at
predetermined space intervals so as to form a plurality of droplets
separated from each other within the dispensing area.
[0023] In this case, the volume of each of the droplets and the
space intervals of the droplets should preferably be set so that
the dispensing area is covered with the plurality of droplets.
[0024] In the dispensing process, the retrieval treatment solution
may be dispensed a plurality of times onto the same spot within the
dispensing area.
[0025] The pH of the retrieval treatment solution may be equal to
or higher than 9.
Effect of the Invention
[0026] In the system and method for a retrieval treatment of
proteins in a deparaffinized tissue section according to the
present invention, the loss of biological substances from the
deparaffinized tissue section due to the immersing does not occur,
since the deparaffinized tissue section is not immersed in a
retrieval treatment solution as in the conventional case; instead,
the retrieval treatment solution is dispensed on the deparaffinized
tissue section before the moist-heat treatment is performed.
Furthermore, since the washing process which has conventionally
been needed after the retrieval treatment is omitted, the loss of
biological substances from the deparaffinized tissue section due to
the washing does not occur. Accordingly, the actual amounts of
biological substances will be correctly reflected in the peak
intensities, so that the peak intensities obtained by a MALDI mass
spectrometry will be higher than in the conventional case. The
increase in the peak intensities leads to an increased number of
peaks whose chemical structures can be identified by a qualitative
analysis as well as an improved detection limit in a quantitative
analysis. As a result, a more accurate mapping image with a greater
amount of information can be created.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 is a flowchart showing a method of pretreating a
paraffin-embedded tissue section by using a retrieval treatment
system according to the present invention.
[0028] FIG. 2 is a flowchart showing a method of pretreating a
paraffin-embedded tissue section by using a conventional retrieval
treatment method.
[0029] FIG. 3 is a configuration diagram showing the main
components of a retrieval treatment system according to one
embodiment of the present invention.
[0030] FIG. 4A is a model diagram illustrating a retrieval
treatment according to one example of the present invention, and
FIG. 4B is a model diagram illustrating a MALDI mass spectrometry
according to the present example.
[0031] FIG. 5 is a bar graph showing the number of detected peaks
with respect to the dispensing amount of the retrieval treatment
solution.
[0032] FIG. 6 shows mass peak profiles obtained in the present
example and in a comparative example for each of the predetermined
mass ranges.
[0033] FIG. 7 shows enlarged views of major peaks detected in the
present example and in the comparative example, where the thicker
and thinner lines correspond to the present example and the
comparative example, respectively.
[0034] FIG. 8 is a bar graph showing, for each mass range, the
intensity ratios of the peaks detected in the present example to
those of the peaks detected in the comparative example.
MODE FOR CARRYING OUT THE INVENTION
[0035] One embodiment of the system for a retrieval treatment of
proteins in a deparaffinized tissue section according to the
present invention is hereinafter described with reference to the
attached drawings (the system is hereinafter simply called the
"retrieval treatment system"). FIG. 3 is a configuration diagram
showing the main components of the retrieval treatment system 1
according to the present embodiment.
[0036] The retrieval treatment system 1 is composed of a dispensing
unit 10 for dispensing a retrieval treatment solution onto a
deparaffinized tissue section 30 and a moist-heat treatment unit 20
for heating, in a saturated water vapor, the deparaffinized tissue
section 30 with a retrieval treatment solution 31 put on it.
[0037] The dispensing unit 10 includes: a platform 11 on which a
sample plate 34 with the deparaffinized tissue section 30 put
thereon is to be placed, a solution storage container (not shown)
for holding a retrieval treatment solution, and a dispenser 12 for
dispensing the retrieval treatment solution.
[0038] In the moist-heat treatment unit 20, a sample plate 34
carrying the deparaffinized tissue section 30 with the retrieval
treatment solution 31 put thereon is loaded in a pressure-resistant
container, and the deparaffinized tissue section 30 is heated with
a saturated water vapor at a predetermined temperature for a
predetermined period of time.
[0039] As the dispensing unit 10, any dispensing device capable of
dispensing a trace amount of solution onto a specified area can be
used. A particularly suitable example is a chemical printer
CHIP-1000 (manufactured by Shimadzu Corporation), which is capable
of dispensing a picoliter-size droplet. As the moist-heat treatment
unit 20, an autoclave can suitably be used.
[0040] The dispensing unit 10 and the moist-heat treatment unit 20
may be provided separately from each other or integrally as a
single unit. The dispensing unit 10 can not only be used for
dispensing the retrieval treatment solution but also for applying a
predetermined treatment solution to the deparaffinized tissue
section 30 in the subsequent enzymatic treatment and the matrix
deposition treatment.
EXAMPLE
[0041] One example of the method for a retrieval treatment of
proteins in a deparaffinized tissue section, which was performed by
the previously described retrieval treatment system 1, is
hereinafter described (this method is hereinafter simply called the
"retrieval treatment method"). In the present example, the
retrieval treatment was performed under a plurality of dispensing
conditions in order to find an optimum dispensing amount of the
retrieval treatment solution.
[0042] In the present example, the aforementioned chemical printer
CHIP-1000 was used as the dispensing unit 10 of the retrieval
treatment system 1, and the autoclave KS-243 (manufactured by TOMY
SEIKO CO., LTD) was used as the moist-heat treatment unit 20.
[0043] A piece of kidney tissue of a mouse was fixed with formalin,
after which the tissue was embedded in paraffin and sliced to
obtain a formalin-fixed paraffin-embedded kidney tissue section (3
.mu.m thick). Then, the tissue section was laid on an electrically
conductive support and dried. The obtained formalin-fixed
paraffin-embedded kidney tissue section was exposed to xylene at
room temperature for 30 minutes to remove paraffin and obtain a
deparaffinized kidney tissue section 30. Subsequently, as a
hydration treatment, the deparaffinized kidney tissue section 30
was sequentially exposed to 100% ethanol (twice), 90% ethanol, 80%
ethanol and 70% ethanol, 10 seconds in each.
[0044] After the hydration treatment, the deparaffinized kidney
tissue section 30 was put on a sample plate for slide glass and set
in the chemical printer CHIP-1000. As the retrieval treatment
solution, a 0.1% n-octyl-.beta.-D-glucoside solution/10 mM Tris-HCl
(pH 10.0) solution was put in the solution storage container.
Furthermore, as shown in FIG. 4A, the retrieval treatment solution
31 was dispensed at intervals of 200 .mu.m within each of a
plurality of 1000-.mu.m square dispensing areas 32 on the
deparaffinized kidney tissue section 30. That is to say, six spots
were set at intervals of 200 .mu.m on each side of one dispensing
area 32, and the retrieval treatment solution 31 was dispensed onto
a total of 36 spots (6 spots.times.6 spots).
[0045] To find an optimum dispensing amount, the measurement was
performed under 11 conditions with different dispensing amount per
spot: 500 pL, 1000 pL, 2000 pL, 2500 pL, 3000 pL, 3500 pL, 4000 pL,
4500 pL, 5000 pL, 7500 pL and 10000 pL. The volume of the retrieval
treatment solution dispensed onto each spot was adjusted as desired
by dropping an appropriate number of 100-pL droplets according to
the required quantity (e.g., for a spot onto which a total of 2500
pL must be dispensed, a 100-pL droplet was dropped 25 times onto
the tissue).
[0046] After the dispensing process, the deparaffinized kidney
tissue section 30 was put in a stainless box together with wet
paper material and subjected to a moist-heat treatment in the
autoclave at 110 degrees Celsius for 10 minutes.
[0047] After the moist-heat treatment, the deparaffinized kidney
tissue section 30 was once more set in the chemical printer
CHIP-1000. As an enzymatic treatment solution, a 100 .mu.g/mL
trypsin/10 mM ammonium bicarbonate/5% isopropanol solution was put
in the solution storage container. A total of 5000 pL of the
enzymatic treatment solution was dispensed onto every spot where
the retrieval treatment solution 31 had been dispensed, after which
the tissue section was left in a thermostatic chamber at 37 degrees
Celsius for 180 minutes to hydrolyze proteins contained in the
dispensing area 32 of the deparaffinized kidney tissue section 30.
Then, as a matrix solution, a 50 mg/mL 2,5-dihydroxy benzoic
acid/50% acetonitrile/0.1% trifluoroacetic acid solution was put in
the solution storage container and dispensed at intervals of 150
.mu.m with a total of 7500 pL for each dispensing spot so as to
deposit 2,5-dihydroxy benzoic acid on the deparaffinized kidney
tissue section 30. The section sample thus obtained was subjected
to a MALDI mass spectrometry. The conditions of the MALDI mass
spectrometry will be described later.
[0048] The retrieval treatment solution used in the previously
described retrieval treatment was adjusted at pH 9.6. Biasing the
pH of the retrieval treatment solution toward the basic side in
this manner improves the reaction rate of the hydrolysis of
proteins by trypsin in the subsequent enzymatic treatment since
trypsin has an weakly-basic optimum pH (from pH 8 to 9).
COMPARATIVE EXAMPLE
[0049] As a comparative example, a formalin-fixed paraffin-embedded
kidney tissue section of a mouse was treated as follows based on
the conventional method.
[0050] The deparaffinized kidney tissue section 30 which had
undergone the deparaffinization and hydration treatments in the
same manner as in the previous example was immersed in a retrieval
treatment solution having the same composition as in the previous
example and subjected to a moist-heat treatment in the autoclave at
110 degrees Celsius for 10 minutes. After the moist-heat treatment,
the deparaffinized kidney tissue section 30 was taken out from the
retrieval treatment solution and immersed in 70% ethanol for five
minutes to adequately wash the deparaffinized kidney tissue section
30. After the washing, the enzymatic treatment and the matrix
deposition treatment of the deparaffinized kidney tissue section 30
were performed under the same conditions as in the previous
example. The obtained section sample was subjected to a MALDI mass
spectrometry under the same measurement conditions as in the
previous example.
[0051] For each of the section samples in the present example and
the comparative example, a MALDI mass spectrometry was performed
under the following conditions:
[0052] Device: Matrix-assisted laser desorption/ionization
time-of-flight mass spectrometer AXIMA-QIT (manufactured by
Shimadzu Corporation)
[0053] Laser: 5 Hz, 2 shots/point
[0054] Space intervals of irradiation: 50 .mu.m
[0055] Ion polarity: positive
[0056] Mode of measurement mass area: MID MASS
[0057] Mass range to be measured: m/z 700-2500
[0058] Measurement area: 500 .mu.m.times.500 .mu.m
[0059] Measurement points: 11.times.11=121 points
[0060] As schematically shown in FIG. 4B, for each of the 121
points arranged at intervals of 50 .mu.m within each of the
500-.mu.m square measurement areas 33 included in the dispensing
area 32, the MALDI mass spectrometry was performed by irradiating
two shots of laser beam at each point.
[0061] Within the aforementioned mass range to be measured, the
peak intensity at each mass-to-charge ratio was acquired. The
minimum intensity of the 100 peaks on both sides of each detection
peak was regarded as the background for that peak and subtracted
from the peak intensity. After that, each peak intensity was
corrected by being divided by the total of all the peak
intensities. Each peak detected in the present example was compared
with the peak detected at the same mass-to-charge ratio in the
comparative example, and those detection peaks were selected whose
intensities were equal to or higher than two times those of the
comparative example and which were statistically significant
(p<0.05).
[0062] The results of the MALDI mass spectrometry are hereinafter
described.
[0063] FIG. 5 is a graph showing the number of detected peaks with
respect to the dispensing amount of the retrieval treatment
solution in the present example. The number of detected peaks
showed the tendency of increasing with the dispensing amount of the
retrieval treatment solution until the amount reaches 1000 pL. The
number of detected peaks at a dispensing amount of 1000 pL was
approximately two times the value at a dispensing amount of 500 pL.
The number of detected peaks did not significantly increase while
the dispensing amount was within a range from 1000 pL to 2500 pL.
These results demonstrate that 500 pL is not a sufficient volume of
the retrieval treatment solution for activating proteins in the
dispensing area 32, while the proteins in the dispensing area 32
can be adequately activated when the dispensing amount is 1000 pL
or more.
[0064] On the other hand, the number of detected peaks dramatically
decreased after the dispensing amount of the retrieval treatment
solution exceeded 3000 pL. A probable reason is that, when the
dispensing amount of the retrieval treatment solution exceeds a
certain level, the droplets of the retrieval treatment solution
formed on the deparaffinized kidney tissue section 30 become
combined with each other, causing the retrieval treatment solution
to flow to the outside of the dispensing area 32, so that the
biological substances which have been transferred into the
retrieval treatment solution are lost from the dispensing area
32.
[0065] To support this reasoning, the deparaffinized kidney tissue
section 30 after the dispensing process was visually examined in
the two cases where the dispensing amount of the retrieval
treatment solution per spot was 2500 pL and 5000 pL, respectively.
It was confirmed that, in the case of the dispensing amount of 2500
pL, the droplet at each spot retained a good shape, whereas, in the
case of the dispensing amount of 5000 pL, the droplets at the
neighboring spots were combined together, causing the retrieval
treatment solution to flow to the outside of the dispensing area
32.
[0066] From these results, it can be said that the dispensing
amount per spot should be 2500 pL or less so that the retrieval
treatment solution can be dispensed in the form of separate
droplets.
[0067] FIG. 6 shows mass peak profiles obtained in the present
example in which the retrieval treatment solution was dispensed
with a volume of 2500 pL each spot (i.e. by the largest possible
dispensing amount within a range where the droplets of the
retrieval treatment solution can retain a good shape) and those
obtained in the comparative example, with each profile showing one
of the three predetermined mass ranges of m/z 750-1150, m/z
1150-1650 and m/z 1650-2250. The profiles of the present example
generally show a larger number of clear peaks than those of the
comparative example. This tendency is particularly noticeable
within the mass range of m/z 1650-2250.
[0068] FIG. 7 shows enlarged views of representative peaks
(detected at m/z 1274, m/z 1443, m/z 1509, m/z 1529, m/z 1745 and
m/z 1961) extracted from the mass peak profiles of FIG. 6. The
thicker lines show the peaks obtained in the present example in
which the retrieval treatment solution was dispensed with a volume
of 2500 pL each spot, while the thinner lines show the peaks
obtained in the comparative example. At any of those m/z values,
the intensities of the peaks detected in the present example were
equal to or higher than two times the corresponding peaks detected
in the comparative example.
[0069] FIG. 8 specifically shows, for each mass range,
peak-intensity ratios of 461 peaks which were extracted from the
peaks detected in the MALDI mass spectrometry of the section sample
obtained in the present example in which the retrieval treatment
solution was dispensed with a volume of 2500 pL each spot, and each
of which had a peak intensity equal to or higher than two times the
peak detected at the same mass-to-charge ratio in the comparative
example. A peak detected at a higher mass-to-charge ratio had a
tendency to have a higher peak-intensity ratio. In the
aforementioned 461 peaks, 182 peaks (39.5%) had their intensities
improved to 2-3 times the levels in the comparative example, 149
peaks (32.3%) had their intensities improved to 3-5 times, 104
peaks (22.6%) had their intensities improved to 5-10 times, and 26
peaks (5.6%) had their intensities improved to higher than 10
times.
[0070] As described thus far, by using the retrieval treatment
system and retrieval treatment method according to the present
invention, a larger number of peaks will be detected with a higher
detection sensitivity in a MALDI mass spectrometry than in the case
where the retrieval treatment is performed by the conventional
method. This is probably because the conventional process of
immersing the sample in the retrieval treatment solution and
subsequently washing the sample is omitted, and therefore, the loss
of biological substances from the deparaffinized tissue section is
avoided. Furthermore, it is expected that the molecules of the
biological substances and those of the components of the retrieval
treatment solution frequently collide with each other inside the
micro-sized droplets of the retrieval treatment solution during the
moist-heat treatment. This phenomenon probably promotes the
retrieval and thereby improves the detection sensitivity.
[0071] It should be noted that the specific forms of the present
invention described thus far do not limit the present invention.
For example, although the dispensing areas and the measurement
areas shown in the drawings were shaped rectangular, they may have
any other shapes, such as a circular shape. Various other changes
can be made without departing from the spirit of the present
invention.
EXPLANATION OF NUMERALS
[0072] 1 . . . Retrieval Treatment System [0073] 10 . . .
Dispensing Unit [0074] 11 . . . Platform [0075] 12 . . . Dispenser
[0076] 20 . . . Moist-Heat Treatment Unit [0077] 30 . . .
Deparaffinized Tissue Section [0078] 31 . . . Retrieval Treatment
Solution [0079] 32 . . . Dispensing Area [0080] 33 . . .
Measurement Area [0081] 34 . . . Sample Plate
* * * * *