Analytical method and apparatus using fingerprints on the basis of types in expression levels of express trace proteins and/or peptides contained in living tissue and/or biological fluid

Abstract

Provided are an analytical method and apparatus using fingerprints on the basis of the types and expression levels of expressed trace proteins and/or peptides contained in living tissue and/or biological fluid. The present invention relates to a method and apparatus for analyzing the status of living tissue and/or biological fluid by binding a specific fluorogenic reagent (such as DAABD-Cl) to expressed trace proteins and/or peptides contained in living tissue and/or biological fluid without degradation treatment followed by precisely detecting and separating by nano-liquid chromatography, and subsequently continuously and quantitatively measuring the separated and purified fluorescent labelled proteins and/or peptides using a fluorescence detector. The use of a nano-liquid chromatograph having a fluorescence detector makes it possible to obtain profiles of expressed trace proteins and/or peptides contained in living tissue and/or biological fluid.

Claims

1. An analytical method using fingerprints on the basis of the types of an expression levels of expressed trace proteins and/or peptides contained in living tissue and/or biological fluid for obtaining a profile on the basis of the types and expression levels of proteins and/or peptides expressed in the living body by detecting and separating proteins and/or peptides using the proteins and/or peptides having a fluorescent marker bound to a target sample followed by continuously quantitatively measuring separated fluorescent labelled proteins and/or peptides by a liquid transfer system capable of transferring liquid at any gradient by combining at least two pumps and a liquid chromatography column provided with a separation column having an inner diameter of 30 to 300 m and a length of 25 cm or more and containing an adsorbent, the separation column including an adsorption layer with a thickness of 1 m or less, the adsorption layer containing a silica base material, and a fluorescence detector.

2. The analytical method using fingerprints on the basis of the types and expression levels of expressed trace proteins and/or peptides contained in living tissue and/or biological fluid according to claim 1, wherein the liquid transfer flow rate of the pumps is 0.1 to 10 L/min.

3. The analytical method using fingerprints on the basis of the types and expression levels of expressed trace proteins and/or peptides contained in living tissue and/or biological fluid according to claim 1, wherein the separation column is a monolithic silica capillary column containing a monolithic silica gel or a monolithic polymer gel.

4. The analytical method using fingerprints on the basis of the types and expression levels of expressed trace proteins and/or peptides contained in living tissue and/or biological fluid according to claim 1, wherein the separation column contains a silica particle or a synthetic polymer particle having a porous surface layer.

5. The analytical method using fingerprints on the basis of the types and expression levels of expressed trace proteins and/or peptides contained in living tissue and/or biological fluid according to claim 1, wherein the number of carbon atoms of an adsorbent ligand for a fluorescent labelled protein bound to the surface of the separation column is 1 to 18.

6. The analytical method using fingerprints on the basis of the types and expression levels of expressed trace proteins and/or peptides contained in living tissue and/or biological fluid according to claim 1, wherein the proteins are proteins extracted from a human, animal, plant, insect, or microorganism, and the total weight of the proteins is 5 g or less.

7. The analytical method using fingerprints on the basis of the types and expression levels of expressed trace proteins and/or peptides contained in living tissue and/or biological fluid according to claim 1, wherein the fluorescent marker is bound to a thiol group or amino group present in the proteins and/or peptides.

8. The analytical method using fingerprints on the basis of the types and expression levels of expressed trace proteins and/or peptides contained in living tissue and/or biological fluid according to claim 1, wherein multiple protein profiles targeted for analysis are selected, and multiple liquid transfer units including at least two liquid transfer pumps are operated in parallel.

9. An apparatus that uses the analytical method using fingerprints, wherein the apparatus comprises as constituents thereof a liquid transfer system capable of transferring liquid at any gradient with at least two pumps and a liquid chromatography column provided with a separation column having an inner diameter of 30 to 300 m and a length of 25 cm or more and containing an adsorbent, the separation column including an adsorption layer with a thickness of 1 m or less, the adsorption layer containing a silica base material, a fluorescence detector, wherein the apparatus is configured to carry out analysis using fingerprints on the basis of the types and expression levels of expressed trace proteins and/or peptides in living tissue and/or biological fluid by supplying as test sample the expressed trace proteins and/or peptides contained labeled with a fluorogenic reagent to the liquid chromatography column provided with a fluorescence detector.

10. The apparatus according to claim 9, which is configured to be operated in parallel by installing in parallel multiple liquid transfer units containing at least two liquid transfer pumps.

11. The apparatus according to claim 9, wherein the separation column is a monolithic silica capillary column containing a monolithic silica gel or a monolithic polymer gel.

12. The apparatus according to any one of claim 9, wherein the adsorbent contains a silica particle or a synthetic polymer particle having a porous surface layer.

13. The apparatus according to claim 9, wherein the adsorbent is subjected to surface modification treatment of the silica base material with an adsorbent ligand or phenyl-form silylating agent having 1 to 18 carbon atoms.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0079] FIG. 1 indicates a schematic diagram (apparatus overview) of an apparatus configuration according to an embodiment of the present invention;

[0080] FIG. 2 indicates a schematic diagram (parallel apparatus overview) of apparatuses arranged in parallel according to an embodiment of the present invention;

[0081] FIG. 3 indicates a highly precise separation diagram on the basis of the results of batch separation of human proteins with a monolithic silica capillary column used in an embodiment of the present invention;

[0082] FIG. 4 indicates a graph representing the results of a separation profile of fluorogenic-labeled proteins derived from yeast obtained in Example 1 (analysis example);

[0083] FIGS. 5A and 5B indicate graphs representing the results of separation profiles of fluorogenic-labeled proteins derived from yeast obtained in Example 2 (effect of length);

[0084] FIGS. 6A to 6C indicate graphs representing the results of separation profiles of fluorogenically-labeled proteins derived from yeast obtained in Example 3 (optimization of gradient time);

[0085] FIGS. 7A to 7C indicate graphs representing the results of separation profiles of fluorogenically-labeled proteins derived from yeast obtained in Example 4 (parallel system operation); and

[0086] FIG. 8 indicates fluorogenic reagents for protein analysis, DAABD-Cl and SBD-F.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0087] Although the following provides an explanation of embodiments of the present invention referring to figures (apparatus configuration examples, test examples or examples and the like), the present invention is not limited in any way by the following configuration examples, test examples or examples and the like.

Apparatus Configuration Example 1

[0088] An example of the configuration of an apparatus using the analytical method according to the present invention that uses fingerprints on the basis of the types and expression levels of proteins and/or peptides is explained in detail on the basis of FIG. 1.

[0089] An analytical apparatus used in the present analytical method was constructed by coupling a liquid transfer unit capable of transferring liquid at any intended gradient over a long period of time by combining at least two liquid transfer pumps A and B of a mobile phase A and a mobile phase B, a separation column having an inner diameter of 30 to 300 m and length of 25 cm or more and containing an adsorbent for which the thickness of the adsorption layer containing a silica base material is 1 m or less, and a nano-liquid chromatography column provided with a fluorescence detector.

[0090] The above-mentioned separation column provided with a fluorescence detector was coupled to the above-mentioned liquid transfer unit via a sample injection switching valve and a thermostatic chamber capable of controlling temperature was provided in the separation column. The above-mentioned liquid transfer unit, sample injection switching valve, separation column and fluorescence detector were coupled to a personal computer for system control to construct a basic configuration for acquiring data, carrying out analyses and the like.

[0091] Each of the constituents containing a high-performance liquid chromatograph provided with a fluorescence detector (FD-LC) is as indicated below.

[0092] LC system: UltiMate 3000 Nano (Thermo Fisher Scientific), detector: FP-2020 Plus with 30 m I.D. capillary cell (JASCO, Ex: 395 nm, Em: 505 nm), columns (monolithic silica capillary column: 0.1 mm I.D.250 mm, length: 700 mm; Ultron C4 and Phenyl Column (Shinwa Chemical))

Apparatus Configuration Example 2

[0093] An example of the configuration of parallel apparatuses using the analytical method according to the present invention that uses fingerprints on the basis of the types and expression levels of proteins and/or peptides is explained in detail on the basis of FIG. 2.

[0094] An analytical apparatus used in the present analytical method was constructed by coupling four liquid transfer units capable of transferring liquid at any intended gradient over a long period of time by combining at least two liquid transfer pumps, separation columns having an inner diameter of 30 to 300 m and length of 25 cm or more and containing an adsorbent for which the thickness of the adsorption layer containing a silica base material is 1 m or more, and nano-liquid chromatography columns provided with fluorescence detectors via sample injection switching valves.

[0095] The mobile phase of a sample was allowed to be injected automatically by coupling an automatic sample injection apparatus (auto sampler) to the above-mentioned liquid transfer units via the above-mentioned sample injection switching valves. Apparatuses for acquiring data and carrying out analyses and the like were provided in parallel by coupling each of the above-mentioned fluorescence detectors to a personal computer for system control to construct a basic configuration. Furthermore, each of the constituents containing a high-performance liquid chromatograph provided with a fluorescence detector (FD-LC) was the same as in Apparatus Configuration Example 1.

Test Example 1

[0096] A fluorogenic reagent (fluorogenic derivatization reagent) was synthesized in the present test example.

Synthesis of DAABD-Cl

[0097] 4-chlorosulfonyl-7-chloro-2,1,3-benzoadiazole (CBD-Cl, 126.53 mg) was dissolved in CH.sub.3CN followed by dropping in N,N-dimethylethylenediamine and adding triethylamine. After stirring for about 10 minutes at room temperature, the reaction liquid was dried under reduced pressure followed by purifying with a silica gel column (CH.sub.2Cl.sub.2) to obtain 4-(dimethylaminoethyl aminosulfonyl)-7-chloro-2,1,3-benzoxadiazole (DAABD-Cl, 20.2 mg, 87.4%).

[0098] Confirmation data of the resulting compound is indicated below.

[0099] .sup.1H-NMR (CD.sub.3OD): 7.94 (1H, d, J=7.5), 7.65 (1H, d, J=7.5), 3.06 (2H, t, J=6.7), 2.30 (2H, t, J=6.7), 2.02 (6H, s); ESI-MS: m/z 305 (M+H).sup.+

Test Example 2

[0100] Sensitivities during MS of fluorogenic reagents (fluorogenic derivatization reagents) were compared in the present test example.

[0101] The sample prepared in the above-mentioned synthesis example detected by LC-MS and not labeled with a fluorogenic derivatization reagent along with that derivatized with SBD-F (FIG. 8) were compared on the basis of relative intensity.

[0102] Relative intensities on the basis of a value of 1 for the respective heights of cysteine, homocysteine, and GHS not labeled with fluorogenic derivatization reagent were as shown in Table 1 below. Furthermore, although a reaction time of 120 minutes was required when carrying out derivatization at 40 C. in the case of SBD-F, in the case of DAABD-Cl, the reaction was completed in 10 to 20 minutes. Thus, a reaction time of 20 minutes was preferable in the case of DAABD-Cl. On the basis of the above, DAABD-Cl was determined to demonstrate high sensitivity in MS. In addition, since the mobile phase is acidic, DAABD-derivatized derivatives were thought to be positively charged and water soluble.

TABLE-US-00001 TABLE 1 SBD-F DAABD-Cl cysteine 23 3.0 10.sup.3 homocysteine 4.0 2.3 10.sup.2 GHS 1.6 2.1 10.sup.2

[0103] Detection Limit of DAABD-Derivatized Peptides/Proteins

[0104] 50 L each of a 10 M mixture of the ten types of peptide/protein standards listed in the following Table 2, 17.5 mM DAABD-Cl, 10 mM EDTA and 50 mM CHAPS were mixed followed by reacting for 30 minutes at pH 9.0 and 40 C. Furthermore, each reagent was dissolved in 0.10 M borate buffer (pH 9.0) containing 6.0 M guanidine hydrochloride. The DAABD-derivatized peptides/proteins that formed were measured using HPLC and detection limits of fluorescence detection were compared with SBD-F.

TABLE-US-00002 TABLE 2 Peptides and Molecular Number of Detection limit (fmol) proteins weight (Da) cystenyl residues DAABD-Cl SBD-F vasopressin 1084 2 7.0 5.0 oxytocin 1007 2 4.5 1.3 somatostatin 1638 2 20 1.8 calcitonin 3418 2 5.0 6.0 amylin (rat) 3920 2 4.5 1.2 insulin 5808 6 2.2 0.7 -acid 21547 4 8.5 1.3 glycoprotein -lactalbumin 16228 8 3.5 0.5 albumin (BSA) 66385 35 0.5 0.2 leptin 16014 2 30 3.0

[0105] Next, a detailed explanation of the present invention is provided on the basis of examples thereof.

Example 1

[0106] A system was constructed using the following apparatus configuration and analysis conditions in order to prepare a profile using proteins extracted from yeast.

[0107] System Configuration [0108] Liquid transfer pump, auto sampler, column oven: UltiMate 3000 Nano (Thermo Fisher Scientific) [0109] Fluorescence detector: FP-2020 (JASCO) [0110] Detection cell: Capillary cell (inner diameter: 30 m, cell length: 1 cm) [0111] Column: C4 modified monolithic silica capillary column (inner diameter: 100 m, length: 70 cm)

[0112] Chromatographic Separation Conditions [0113] Mobile phase A: 0.1% TFA-ultrapure water [0114] Mobile phase B: 0.1% TFA-acetonitrile [0115] Liquid transfer flow rate: 0.5 L/min [0116] Gradient conditions: 20% to 35% B (360 min) [0117] Column temperature: 50 C. [0118] Fluorescence wavelengths: Excitation wavelength (Ex) 395 nm, detection wavelength (Em) 505 nm, gain 1000 [0119] Sample volume: 0.5 L [0120] Sample weight: 0.5 g

[0121] The analysis sample was prepared under the following conditions according to the patent literature.

Preparation of Analysis Sample

[0122] Protein extracted from Saccharomyces cerevisiae (trade name: MS Compatible Yeast Protein Extract, Intact, Promega) was used for the sample. The yeast is composed of 12 million bases and roughly 6,000 genes are involved in protein expression.

[0123] After adding 6 M guanidine solution (600 L), 100 mM EDTA-6 M guanidine solution (200 L), 10 mM TCEP-6 M guanidine solution (50 L) and 140 mM DAABD-C-acetonitrile solution (50 L) to 1 mg of the sample and allowing to react for 10 minutes at 40 C., 10% TFA (30 L) was added while cooling with ice, resulting in the obtaining of 1 mg/mL solution of fluorogenic-labeled protein.

[0124] The results of analyzing over the course of six hours are shown in FIG. 4. The vertical axis of the graph indicates detected values (mV) as determined by fluorescence emission. As a result, in addition to obtaining about 350 peaks according to the type of protein, a profile was obtained that is derived from proteins expressed by the yeast of the sample (genome size: 12.1 million bases, number of genes: 6,275). The amount of solvent used in this measurement was about 0.2 mL.

Example 2

[0125] Columns having lengths of 25 cm and 70 cm were used and analyses were carried out for analysis times suitable for each length in order to confirm the effect of separation column length on profile. The analysis conditions consisted of changing only the gradient conditions, using a gradient of 10% to 55% B (60 min) for the column having a length of 25 cm and a gradient of 20% to 55% B (240 min) for the column having a length of 70 cm. The results are shown in FIGS. 5A and 5B. The vertical axis of the graph indicates detected values (mV) as determined by fluorescence emission.

[0126] FIG. 5A is the profile obtained for the 25 cm column while FIG. 5B is the profile obtained for the 70 cm column. The profile obtained with the 25 cm column of FIG. 5A is not considered to exhibit favorable separation in comparison with the profile obtained with the 70 cm column of FIG. 5B. On the other hand, both profiles can be seen to exhibit similar shapes.

[0127] In the analytical method using fingerprints on the basis of the types and expression levels of expressed trace proteins and/or peptides contained in living tissue and/or biological fluid, extremely high-precision separation is not necessarily required. In addition, since species having a small number of genes have a small number of protein types, a column length suitable for that species can be selected. The results of Example 2 indicate that the present invention enables the selection of column length that is suitable for the particular species.

Example 3

[0128] The relationship between separation time and the number of protein and/or peptide peaks detected was verified in an attempt to obtain more precise protein and/or peptide information. The results are shown in FIGS. 6A to 6C. The vertical axis of the graph indicates detected values (mV) as determined by fluorescence emission. The analysis conditions consisted of using respective gradient of 25% to 45% for 240 minutes, 360 minutes and 600 minutes. As a result, the numbers of peaks detected were 301, 392 and 706, respectively.

[0129] Since a greater number of protein peaks were detected as the gradient time increased, longer and gentler gradient conditions were indicated to be suitable for obtaining a more precise protein profile. Thus, the present invention was demonstrated to be useful with respect to being able to be stably operated for a longer period of time in the case of, for example, searching for disease markers targeted at humans having a larger number of proteins.

Example 4

[0130] It was presumed from Example 3 that gradient analysis for several tens of hours is required for a more precise fingerprint analysis of species predicted to have a large number of genes and express a large number of proteins and/or peptides such as in the case of humans.

[0131] On the other hand, since disease-specific profiles and disease-related proteins and/or peptides are expected to be discovered by comparing the profiles of multiple specimens, the above-mentioned analytical method according to the present invention is required to analyze multiple specimens in a single day. The operation of multiple apparatuses in parallel was verified in order to realize highly precise analyses in a short period of time by the above-mentioned analytical method and apparatus using fingerprints according to the present invention.

[0132] FIG. 7A is a profile obtained by applying gradient conditions consisting of 20% to 45% B for an analysis time of 600 minutes. In contrast, a profile obtained by applying gradient conditions consisting of 20% to 35% B for the same analysis time is shown in FIG. 7B, while a profile obtained by applying gradient conditions consisting of 30% to 45% B for the same analysis time is shown in FIG. 7C.

[0133] Gradient conditions consisting of 20% to 35% B of FIG. 7B resulted in more precise separation corresponding to the 0 to 360 minutes of FIG. 7A while retaining the same profile. In addition, gradient conditions consisting of 30% to 45% B of FIG. 7C achieved more precise separation corresponding to the 240 to 600 minutes of FIG. 7A while retaining the same profile.

[0134] In this manner, since proteins and/or peptides are eluted and detected corresponding to the concentration of the eluent in accordance with the principle of liquid chromatography, profiles corresponding to the concentration of the eluent were determined to be obtained even through gradient conditions differed. In addition, more precise separation was achieved since the gradient conditions were gentler.

[0135] Thus, as FIG. 2, this system was verified to make it possible to obtain highly precise protein and/or peptide profiles obtained by analyses equivalent to 100 hours in an analysis time of just 10 hours, or obtain analysis results obtained in analyses equivalent to 10 hours in just 1 hour, by operating multiple apparatuses in parallel, such as by using 10 apparatuses, and shifting their respective gradient conditions.

[0136] Since the present invention uses a micro column that precisely separates proteins and/or peptides and greatly reduces the consumption of solvent required for analyses, it is realistic to operate this type of system in parallel, and is expected to be widely used in, for example, the diagnosis of diseases in which proteins and/or peptides are involved or prediction of the efficacy of personalized treatment.

[0137] As was previously described in detail, the present invention relates to an analytical method and apparatus using fingerprints on the basis of the types and expression levels of expressed trace proteins and/or peptides contained in living tissue and/or biological fluid, and together with providing a novel analytical method using fingerprints on the basis of the types and expression levels of expressed trace proteins and/or peptides expressed in living tissue and/or biological fluid, the present invention provides an analytical method and an analytical apparatus using fingerprints on the basis of the types and expression levels of expressed trace proteins and/or peptides of living tissue and/or biological fluid obtained through continuous analyses of multiple specimens by using nano-liquid chromatography technology. The present invention is useful not only in the fields of medicine and pharmacology, but also in numerous industries ranging from agriculture to food science, such as through improvement of culturing technology using microorganisms or improving microbial gene modification technology towards increased efficiency thereof, by using the above-mentioned analytical method and apparatus using fingerprints.