JPH0399264A - Analysis of protein and polypeptide - Google Patents

Abstract

PURPOSE:To achieve a reduction in a analyzing time by dansylation of amino acid AA isolated by Edman degradation conducted several times on a thin porous glass layer. CONSTITUTION:Glass composed of SiO2, 45-70, B2O3 8-30, CaO 8-25, Al2O3 5-15, Na2O 3-8, K2O 1-5, Na2O + K2O 4-13 and MgO 0-8 (all % by weight) undergoes a thermal treatment and a phase splitting is performed based on Ba2O3 and CaO to obtain a thin porous glass layer by removing the phases resulted by dissolution. N spots of protein or polypeptide are applied on the glass thin layer, at (i)th (i=1-N) spotting, 2-mercaptoethanol, phenyl isocyanate and trifluoroacetate are spotted sequentially and Edman's decomposition is conducted (i) times to isolate AA. Then, dansylation of AA is performed by the total spotting and subsequently, a development is performed on the above glass to detect AA as dansyl amino acid. This enables determination of a primary structure of protein or the like simply in a short time.

Description

[Detailed description of the invention] (Industrial application field) The present invention relates to a method for analyzing proteins and polypeptides.

(Prior Art) A protein or polypeptide (abbreviated as protein, etc.) has a structure in which a large number of amino acids (abbreviated as AA) are bonded together through a dehydration reaction.

In order to analyze the primary structure of a protein, etc. (referred to as main analysis), the protein, etc. is decomposed, AA is sequentially released from the terminal end, and separated and quantified by thin layer chromatography (TLC) or the like.

Phenyl isocyanic acid (PITC) is used to degrade proteins, etc.
) and the Edman degradation method using trifluoroacetic acid (TFA) are widely used.

(Problem to be solved by the invention) The conventional technology has the following problems.

Sequencers that automate Edman degradation are commercially available and in widespread use. In this method (referred to as an automatic method), AA residues at the N-terminus of proteins, etc. are sequentially released as phenylthiohydantoin (PTH)-AA by PITC, and free fiPT
H-AA is separated and quantified by gas chromatography or high performance liquid chromatography.

When using an automatic method, it is possible to determine the sequence of a large number of AA from the N terminus, but there are problems in that the sequencer is not only extremely expensive, but also requires skill to operate, which is laborious and time-consuming. there were.

The protein, etc. is manually subjected to Edman degradation in a test tube to release the AA residue at one end of the first N, and then the AA residue that becomes the one end of the second N is dansylated (dansylated) with dansyl chloride (DNS-C1). A method has also been attempted in which DAN-AA is separated and fixed by TLC using polyamide. This method also has the problem that it not only requires skill to operate, is laborious and time consuming, but also usually only determines the arrangement of several AA from one end of N.

The present invention solves the above-mentioned problems of the prior art, and makes it possible to easily analyze the primary structure of proteins, etc., in a short period of time without using expensive equipment or requiring skill. The purpose is to provide a method for analyzing peptides.

(Means for Solving the Problem) In order to achieve the above object, in the present invention, N proteins or polypeptides are spotted on a porous glass thin layer, and the first (i=1 to N) The protein or polypeptide is subjected to Edman degradation i times to decompose the protein or polypeptide to liberate the amino acid, and then the amino acid is dansylated in all the spots, and then developed on the glass mentioned above to detect the amino acid as dansyl amino acid. , to analyze polypeptides.

Edman degradation is also carried out by sequentially spotting 2-mercaptoethanol, phenyl isocyanic acid and trifluoroacetic acid.

Furthermore, as porous glass, 5i0245~70w
t%, 32Q, 8-30 wt%, CaO
8-25 wt%, Al2035-15 wt%, Na,
03-8 wt%, K2O1-5 wt%, Na2O+
K2O4~13 wt%, MgOO~8 wt%, or Sin, 45~70 wt%, B20: +8'''
-30wt%, CaO3~25wt%, Al2035~
B20 is obtained by heat treating a glass having a composition of 15 wt%.
．． , the one obtained by phase-separating a phase mainly composed of CaO and dissolving and removing this phase is used.

Next, the present invention will be explained in more detail.

The inventor has developed a porous glass thin layer (porous glass sheet)
is chemically and mechanically stable, and the porous glass body has T
The surface of the porous glass sheet We conducted experiments based on the belief that if Edman degradation, hydrolysis, dansylation (DNS formation), and TLC of proteins, etc. were carried out, the reaction would proceed with a small amount of sample and reagent, and structure determination would be possible.

The above experiment revealed that hydrolysis occurs simultaneously during Edman degradation, and the AA residue at the N end is liberated.

By converting this AA residue into DNS and using it as a porous glass sheet carrier and performing TLC, AA
It was possible to separate and fix residues. Furthermore, it has been found that 10 residues from one N end can be separated easily and quickly on a single sheet by repeating Edman degradation by repeatedly spotting reagents.

The present invention is a new proposal based on the above knowledge. Next, the configuration of the present invention will be explained in further detail.

The porous glass sheet has a thickness of 0.2 to 2 cm, a size of 2 to 10 cm x 2 to 10 cm, and an average diameter of pores of 1.
．． 000-12,0OOA, preferably 3.000-8
．． 000 A is suitable.

Such sheets have sufficient mechanical strength and do not need to be reinforced with a substrate.

As such porous glass, porous glass obtained using glass having phase separation property, especially Sin, 4
5-70 wt%, B2O38-30 wt%, (:a0
8-25 wt%, Al2035-15 wt%, Na
2O3-8 wt%, K2O1-5 wt%, Na, 0
+K2O4~13wt%, MgO0~8wt%, or 5i0245~70wt%, B20.8~30wt
%, CaO3~25at%, Al2035~15wt%
A porous glass (hereinafter referred to as porous glass A and B, respectively) obtained by heat-treating a glass molded body having a composition (composition) to separate a phase mainly composed of B2O3 and CaO, and dissolving and removing this phase. ) is appropriate.

Note that the porous glass sheet can be suitably manufactured by subjecting a block-shaped glass molded body to phase separation treatment, slicing it, and treating the obtained sheet with an acid.

As the glass having phase separation properties, it is particularly desirable to use composition A or composition glass (hereinafter collectively referred to as the present glass), which has high pressure resistance, high mechanical strength, and high chemical resistance. , pHO in the range of 14 to 14, the pore diameter is uniform, and a thin layer chromatography sheet with high separation accuracy can be obtained.

In addition, the glass of composition A has a small pore diameter of 3,000 to 100
．． It is suitable for obtaining a porous glass with a relatively large pore diameter in the range of 000 A, and the glass composition is suitable for obtaining a porous glass with a relatively small pore diameter in the range of several hundred to 20,00 A. Suitable for obtaining glass.

The present glass and the method for producing a sheet using the present glass will be described in more detail below.

Among the components of this glass, SiO2 is the basic component for forming the skeleton of the porous glass obtained by the phase separation and removal process, and A1□03 is an auxiliary component that reduces the brittleness of the porous glass obtained. have

B20. On the one hand, CaO functions as an auxiliary component to form the framework of the porous glass, but on the other hand, in cooperation with CaO, it has the effect of producing minute phase separation by heat treatment. Then, porous glass is formed by dissolving and removing the thus generated separated phase mainly composed of CaO1B203.

As can be seen from the above explanation, B2O3 is an important factor that determines the size of the pores.
It has been found that the amount of 2O3 has a close relationship with the uniformity of the pore diameter.

The blended raw materials having the above composition are melted and formed into a molded body of a desired shape. There are no particular limitations on the method for producing the molded body, and conventional methods can be used.

Note that since a part of 8203 evaporates and escapes during melting of the batch, it is necessary to determine the amount of B2O3 in the hatch in consideration of this amount of evaporation.

A molded body made of glass having composition A or B (hereinafter referred to as the present glass) is heat-treated to phase-separate a phase mainly composed of CaO1B, 0.3 (hereinafter referred to as CaO1B21t layer). The higher the heat treatment temperature and the longer the heat treatment time, the Ca
The O1B2O3 phase tends to become larger, and the pore diameter can be set to a desired value by selecting heat treatment conditions.

When using this glass, pores of uniform size can be formed, and the diameter can be set in the range of 50 to 100,000 A by selecting the processing conditions and composition. It has been found that especially favorable results can be obtained when the amount of the 1,000 to 12,000 is 1,000 to 12,000. The sheet is obtained by slicing the heat-treated molded body of the glass into a sheet, and then immersing the sheet in an acid such as HCI H2SO, HNO, etc. to dissolve and remove the CaO1B203 phase. I can do it. Note that prior to acid treatment, it is desirable to etching the surface for a short time with an HF solution.

As mentioned above, the diameter of the pores in the resulting porous glass can be controlled by the conditions of heat treatment, but the diameter of the pores changes depending on the amount of B2O3 remaining in the porous glass, and this B2O3 It has been found that the amount of is influenced by the conditions of heat treatment and acid treatment. It has been found that particularly favorable results can be obtained when porous glass is used as a sheet for thin layer chromatography by setting these conditions such that B2O3 remains preferably at least 0.5 wt%.

Desirable processing conditions are as follows.

Heating temperature: 600 to 850°C Heating time: 2 to 48 hr, preferably 12 to 48 hr,
Type of acid HC statement, HzSO<, concentration of NH3 acid
0.01-2. ON, preferably 0.1 to 1. ON Processing time: 2-20 hours, preferably 4-16 hours
The temperature is 50 to 95°C, preferably 80 to 90°C.The sheet obtained as above is used for analysis of various proteins as described below.

(a) Along one side of such a porous glass sheet,
Spot the sample at N locations. 30 samples per location
About 1 pmol is sufficient. After spotting, dry the sample.

You can also dry it by air drying using a dryer, but
It is preferable to cover the sheet with a bag made of polyethylene or the like and dry it anaerobically by sending gas or the like into the bag, which has the effect of sharpening the spots obtained by TLC.

(b) Then spot the volatile reducing agent on all N points. 2-mercaptoethanol is suitable as a volatile reducing agent.

Although the effect of 2-mercaptoethanol is not clear, it has been found to have the effect of promoting the decomposition of proteins, etc.

An amount of 2-mercaptoethanol of about 900 mM is sufficient.

Next, PITC and TFA are spotted to decompose proteins and the like.

PITC is suitably used as a pyridine solution of about 1.8 mM.

After spotting PITC, TFA is spotted to decompose proteins and the like.

The next reaction liberates AA at one N end of the protein, etc., and generates phenylthiohydantoin (PTH)-AA.

PITC protein, etc.-→PTH-AA Then, AA at one end of the first N is liberated by the next reaction.

PTH-AA+TFA→AA at one end of the first N (in the presence of 2-mercaptoethanol) 2-mercaptoethanol is extremely effective in carrying out Edman decomposition on a glass sheet in a short time. Furthermore, it is necessary to spot PITC and TFA in this order; if the order is reversed, the reaction will not proceed.

(c) After completing the operation in (b) above, perform the same operation as in (b) for the remaining N-1 spots excluding the first spot. By this operation, AA at one end of the second N is released in N-1 spots. At the same time, AA at one end of the first N reacts with PITC, etc., and no longer contributes to the spot during the development described below.

(d) Perform the same operation as in (b) for <N-2 spots excluding the first and second spots.

Thereafter, the number of spots is decreased one by one and the operation (b) is repeated, and for the first spot, the operation (b) is repeated i times. As a result, at the i-th spot, the i-th
AA, which becomes one end of N, is released.

(e) Next, a solution of DNS-CJI is spotted, and DAN-AA is generated by the following reaction.

AA+DNS-C1-→DAN-AA DNS-C1 is preferably used as an acetone solution of about 90 pmollo and 1 g.

At this time, it is preferable to spot with a buffer, preferably an acetone solution of trimethylamine, so that a clearer spot can be drawn.

In the i-th spot, a DAN compound (dansyl amino acid) of AA at the i-th N end is generated.

(f) DAN-AA produced in the reaction of (e) is developed with chloroform or the like, and irradiated with a wavelength of 2.537 nm using a UV lamp to detect and fix AA by fluorescence.

At the i-th spot, AA, which is the one end of the i-th N, is D
Detected as AN compound. The N-end AA with a number smaller than i reacts as described in (c) and does not contribute to the spot, and only the i-th N-end AA is detected in the i-th spot. .

(Function) Eight proteins or polypeptides are spotted on a thin porous glass layer, and Edman degradation is performed i times on the first spot (i = 1 to N) to degrade the proteins or polypeptides. The amino acids are liberated, and then the amino acids are dansylated in all spots, and then developed on the above glass to detect the amino acids as dansylated chloride.
On N spots, the 1st to Nth N-end AA are simultaneously separated and fixed with high accuracy, and the primary structure of proteins, etc. is analyzed.

By performing Edman degradation by sequentially spotting 2-mercaptoethanol, phenyl isocyanic acid and trifluoroacetic acid, AA can be liberated efficiently in a short time.

As porous glass, SiO245-70wt%, B
2O3a ~ 30 wt%, CaO3 ~ 25 wt%, A
l2035~15 wt%, Na=03~8 wt%,
K2O1~5wt%, Na, 0 + N204~13
wt%, MgO0-8wt%, or SiO245-7
0 wt%, B2O33~30wt%, CaO3~2
A glass having a composition of 5wt% and A12035 to 15wt% is heat-treated to obtain B20. By using a product obtained by phase-separating a phase mainly composed of CraO and dissolving and removing this phase, the dry chemistry reaction can be carried out smoothly.

(Example) Sin245-70wt%, B20:l 8-30
wt%, CaO3~25wt%, Al2O+ 5~
15 wt%, Na2O3~8wt%, N201
A glass having a composition of ~5 wt% and MgO0~8 wt% was molded into a block shape and heated at 600 to 800°C for 20
It was heated for an hour and cut into 5 cm x 5 cm x 5 mm sheets. Soak the sheet in INHC: sentence, 80-90
It was heated at <0>C for 4-16 hours to make it porous. The pore diameter was measured by the mercury pressure method using Shimadzu Bore Sizer 9305 (Shimadzu Corporation, Kyoto) and was 4.400 A.

Using the above sheet, the following experiment was conducted using lysozyme as a polypeptide with a known structure.

0.3 mM aqueous solution of lysozyme (30 poa+I/
0.1 JLl) was prepared. 1 of this liquid on a glass sheet
Spotted 0 pieces. , IIL capillary (Drum
ond) to 1/10, and attach a 3crs rubber tube to the other end.
Then, the above solution was aspirated and discharged, and 0.141 pieces of the above solution were spotted.

Note that the subsequent spots are set to 0. I did it one by one.

The sheet was wrapped in a polyethylene bag and dried anaerobically by supplying N2 gas.

After drying, 888mM 2-mercaptoethanol was spotted. Next, 1.8 mM P I TC in pyridine, 150 mM triethylamine (1,5 nmol
A solution of 0.1 pi) TFA was spotted and Edman degradation was performed.

The above Edman decomposition was repeated, and Edman decomposition was performed i times on the first spot.

Next, add the above trimethylamine solution and 0.9 mM DNS.
-CI acetone solution (90 piol/0.1 island 1)
was spotted. This was developed with chloroform. (Time required: 10 minutes) DNS-AA uses a UV lamp (Min.
eralight, San Gabriel, GA
) at a wavelength of 253.7 nm and detected by fluorescence.

FIG. 1 is a chromatogram obtained in this manner, and a clear spot completely matching the known structure of lysozyme was obtained.

In addition, in the figure, Lys stands for Lysine and Val stands for Vali.
ne, Phe, Phenylanine, Gu
y stands for Glycine, Arg stands for Argine, Cy
s stands for Cystine, Glu stands for Gulaminic
Ac1d, Leu for Leucine, Ala for Al
Indicates anine.

The total time required for the analysis was 70 minutes.

(Effects of the Invention) The primary structure of proteins, etc. can be easily determined in a short time without requiring expensive equipment or skill.

Further, the amount of sample required is small, and about 30 pmol is sufficient.

[Brief explanation of drawings]

FIG. 1 is a chromatogram obtained by analyzing lysozyme by the method of the present invention.

Claims (3)

[Claims] (1) Spot N proteins or polypeptides on a thin porous glass layer, perform Edman degradation i times on the i-th spot (i = 1 to N), and decompose the proteins or polypeptides. A protein that releases amino acids, then dansylates the amino acids in all spots, and then develops the amino acids on the glass and detects the amino acids as dansyl amino acids;
Polypeptide analysis method. (2) The analytical method according to claim 1, wherein the Edman degradation is carried out by sequentially spotting 2-mercaptoethanol, phenyl isocyanic acid and trifluoroacetic acid. (3) Porous glass is SiO_245-70wt%, B
_2O_38-30wt%, CaO8-25wt%, A
l_2O_35-15wt%, Na_2O3-8wt%
, K_2O1~5wt%, Na_2O+K_2O4~1
3wt%, MgO0~8wt%, or SiO_245~
70wt%, B_2O_38~30wt%, CaO8~
The protein according to claim 1 or 2, which is obtained by heat-treating a glass having a composition of 25 wt%, Al_2O_35 to 15 wt% to separate a phase mainly composed of B_2O_3 and CaO, and dissolving and removing this phase. ,
Polypeptide analysis method.