JP2003149204A - Method for separating and identifying hardly soluble protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To develop a proteome technique capable of highly sensitively and simply separating and identifying hardly soluble proteins and proteins of large molecular weight. SOLUTION: By performing separation by chromatography instead of two-dimensional electrophoresis, it is possible to separate and identify the hardly soluble proteins and the proteins of large molecular weight which have been difficult by a conventional method.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to a method for separating and identifying a poorly soluble protein.

2. Description of the Related Art In the post-genome era, it has become more important to separate and identify proteins in the living body. In particular, it is known that there are very sparingly soluble molecules in membrane proteins such as receptors. Therefore, a means for efficiently separating and identifying refractory molecules can be found in drug discovery and medical science. Considered useful for development. Because
This is because, among poorly soluble proteins, membrane proteins have potential as target molecules for drug discovery and as target molecules for diagnosis and treatment of diseases. At the same time, proteome technology has been attracting attention as a method for identifying a large number of proteins. The proteome technique is to decompose and extract a protein from each spot separated by two-dimensional electrophoresis, measure its molecular weight by mass spectrometry, and search the database for a protein that may generate a fragment of that molecular weight, and then search for the protein. Is a method of identifying. Since this method separates proteins in two dimensions, it has excellent protein resolution, but poorly soluble proteins that are likely to be target molecules for drug discovery and proteins with a molecular weight of 120 kDa or more penetrate into the first dimension gel. Therefore, it is known that it is difficult to use for the separation of poorly soluble proteins (Rabilloud T.
et al. Electrophoresis 18: 307-316 (1997), Oh-Ishi
M. et al. Electrophoresis 21: 1653-1669 (200
0)).

[0001]

SUMMARY OF THE INVENTION An object of the present invention is to develop a proteome technique capable of separating and identifying a poorly soluble protein and a protein having a molecular weight of 120 kDa or more with high sensitivity and in a simple manner.

[0002]

The present inventors compared with the first step of two-dimensional electrophoresis, isoelectric focusing,
Separation of poorly soluble proteins is easy and separation of proteins with large molecular weight is also possible by chromatography, especially ion exchange chromatography, and then SD
Separation by S polyacrylamide gel electrophoresis (SDS-PAGE), in-gel digestion and extraction, and mass spectrometry can be performed to separate and identify insoluble proteins that could not be identified by conventional methods using two-dimensional electrophoresis. Found.

That is, the present invention is as follows: The following steps, 1) step of separating proteins by chromatography, 2) step of separating separated proteins by SDS polyacrylamide gel electrophoresis, 3) digestion of separated proteins in gel, extraction and identification by mass spectrometry A method for separating and identifying a protein, which comprises the step of: Here, the protein is not particularly limited, but is preferably a protein having a high solubility or a large molecular weight, which is difficult to separate by ordinary two-dimensional electrophoresis. More preferably, it is desirable to preliminarily separate only the sparingly soluble protein with a neutral surfactant such as Triton-X114. Identification by mass spectrometry means that the molecular weight of an oligopeptide obtained by degrading a protein is measured by mass spectrometry, and a protein that may generate a fragment of that molecular weight is searched from a database to identify the protein. It means to do. 2. The method according to 1, wherein the chromatography is ion exchange chromatography.

[0004]

BEST MODE FOR CARRYING OUT THE INVENTION According to the present invention, a protein is decomposed and extracted from each spot separated by two-dimensional electrophoresis, and its molecular weight is measured by mass spectrometry. The proteome technique of identifying the protein by searching from a database is improved so that it can be applied to a poorly soluble protein and a protein having a large molecular weight. Hereinafter, embodiments of the present invention, in particular, steps before reaching SDS-PAGE, which is different from ordinary proteome technology, will be described.

The protein applied to the present method is not particularly limited, but is preferably a poorly soluble protein having a large molecular weight, which is difficult to separate by ordinary two-dimensional electrophoresis. More preferably, a sparingly soluble protein, for example, a protein strongly bound to the cytoskeleton (postsynaptic thickening protein, etc.) is preliminarily separated and concentrated with a neutral detergent, for example, Triton-X114 etc. It is desirable to set it. Specifically, a poorly soluble protein is a tissue / cell / cell fraction 0.1-5%,
Preferably 1-2% neutral detergent, preferably Triton-
It can be homogenized in a buffer containing X100 or Triton-X114 and centrifuged to collect an insoluble fraction.

The poorly soluble protein collected is subjected to a buffer containing a substance that denatures the protein, such as urea, thiourea, guanidine hydrochloride, potassium isothiocyanate, guanidine isothiocyanate, potassium iodide, and the like, followed by ion exchange chromatography. When used, it is preferably dissolved in a buffer solution containing uncharged urea / thiourea. The dissolved protein is separated by chromatography using a column equilibrated with a buffer containing a substance that denatures the same protein. Chromatography may be any principle of chromatography, but ion exchange chromatography that separates proteins by their electrical properties is preferable.

If necessary, the separated protein is precipitated with TCA (trichloroacetic acid), concentrated, dissolved in a buffer containing SDS, and separated by SDS-PAGE electrophoresis. After separation, in accordance with normal proteome technology, in-gel digestion and
Extraction and mass spectrometry can be performed (Oda Y. et al. Na
t. Biotechnol. 19: 379-382 (2001)).

[0008]

Industrial Applicability According to the present invention, it becomes possible to easily separate and identify a poorly soluble protein and a protein having a large molecular weight, which were difficult by the conventional methods.

[0009]

EXAMPLES The present invention will be described below with reference to specific examples, but the present invention is not limited thereto. [Example 1] Post-synaptic thickening (PSD) fraction of Triton-X114
Isolation and identification of proteins in the insoluble fraction 1) Preparation of Triton-X114 insoluble fraction Taken et al. (Takeuch et al.
i M. et al. J. Biol. Chem. 272: 11943-11951 (1997))
The PSD fraction was purified by the method described in 1. The resulting PSD fraction (total protein mass 19.4 mg) was adjusted to a protein concentration of 1 mg / ml with buffer A (20 mM Tris / Cl pH 7.4, 100 mM NaCl, 2 mM EDT).
A, 1 mM DTT, 2% Triron-X114) was homogenized and then stirred at 4 degrees for 15 minutes. Then 30 at 196,000 xg
Centrifugation was performed, and the resulting precipitate was used as a Triton-X114 insoluble fraction. 2) Separation by ion exchange chromatography Triton-X114 insoluble fraction (total protein mass 8.35 mg) was added to buffer B (20 mM Hepes pH 7.5, 7 M Urea, 2 M Thiourea, 0.
9% CHAPS, 10mM DTT) Suspended in 8 ml, 1 at 10,000 xg
Centrifuge for 0 minutes and add the dilution buffer (20 mM Hepes pH 7.5,
After adding 4 ml of 1 mM DTT), the supernatant was centrifuged again at 10,000 xg for 10 minutes and subjected to ion exchange chromatography.
Equilibration buffer (20 mM Hepes pH 7.5, 6 M Urea, 0.6% C
After applying the sample to a MonoQ 5/5 column (Amersham Pharmacia) equilibrated with HAPS, 1 mM DTT), the sample was eluted with a concentration gradient up to a NaCl concentration of 1 M. A part of the eluted fractions was separated by SDS-PAGE on a 10% polyacrylamide gel and then stained with silver. The image is shown in FIG. 3) Identification of proteins by mass spectrometry of fractions 17 to 24 Of the obtained fractions, the fractions 17 to 24, which contained a large amount of post-synaptic thickening protein in the antibody search, were concentrated by TCA precipitation, Separation by SDS-PAGE on 10% polyacrylamide gel. After negative staining was performed using Zinc stain kit (Bio-Rad), the protein band was divided into 65 bands. The excised gel was prepared by Oda et al. (Oda Y. et al. Nat. Biotechnol. 19: 379-
382 (2001)), the protein was identified by in-gel digestion and mass spectrometry. Table 1 for a list of identified proteins
It was shown to.

[Table 1] 4) Identified proteins We were able to identify cytoskeletal systems, scaffold molecules, and membrane proteins that were already known to exist in PSD. The PSD method that is the conventional method is used as is for SDS-P
Method to separate by AGE and identify by mass spectrometry (Wal
ikonis RS. et al. J. Neurosci. 20: 4069-4080 (200
0)), SAPAP (Hirao
K. et al. Genes Cells 5: 203-210 (2000)) could be identified. In addition, De, which is generally considered to be insoluble,
Membrane proteins such as nsin-180 could also be identified. Based on the above, this method was considered to be a means for identifying poorly soluble molecules that could not be detected up to now.

[0010]

[Brief description of drawings]

FIG. 1: SDS-PAGE of each fraction obtained by separating the post-synaptic thickening fraction by ion exchange chromatography.

Continued front page    (72) Inventor Shoichi Takeuchi             3317 Shalom, Nishi Shichijo Ishiimachi, Shimogyo-ku, Kyoto-shi             No. 33 202 F-term (reference) 2G052 AB18 AD29 AD46 CA03 CA04                       ED04 ED07 GA22 GA24

Claims (2)

[Claims] 1. The following steps, 1) a step of separating proteins by chromatography, 2) a step of separating separated proteins by SDS polyacrylamide gel electrophoresis, 3) digestion of the separated proteins in a gel and extraction. A method for separating and identifying proteins, which comprises the step of identifying by means of mass spectrometry. 2. The method of claim 1, wherein the chromatography is ion exchange chromatography.