Method for enzymatic hydrolysis of proteins immobilised on substrate of scanning probe microscope

FIELD: medicine.

SUBSTANCE: specific enzymatic hydrolysis of proteins immobilised on a substrate surface of a scanning probe microscope is enabled by the fact that proteins are exposed to ultrasound and a site-specific enzyme in a buffer solution of such pH value and temperature to ensure enzyme activity. Before the enzymatic hydrolysis, the substrate of the scanning probe microscope is immersed in 1-5% acetic acid and exposed to high-frequency ultrasound 1-3 MHz and power 1 Wt/cm2 and more at such temperature of the solution to ensure breaking of the protein-surface bonds. Then, the solution containing desorbed proteins is frozen and lyophilised. The substrate of the scanning probe microscope may consist of silicon, glass, mica, sapphire. The substrate surface of the scanning probe microscope whereon protein molecules are activated, is chemically activated by silane.

EFFECT: use of the method allows lowering a detection threshold of protein molecules immobilised on the substrate surface of the scanning probe microscope by the use of a mass spectrometer.

2 ex

 

The invention relates to Biophysics and medical proteomics, in particular the way the site-specific hydrolysis of protein molecules using enzyme immobilized on the surface of the scanning probe microscope. The described method can be used to apply the methods probe microscopy and mass spectrometry for the visualization and identification of protein molecules in proteomic studies. Mass-spectrometric study provides verification of results identification of proteins (protein complexes), obtained by scanning probe microscopy. The invention can be used in medical diagnosis of infectious diseases and cancer in the identification of specific proteins and protein complexes in the studied biological liquids of an organism.

There is a method of hydrolysis of [the patent WO 2004049818]where the protein (protein complex, protein-containing starting material) is exposed to at least one enzyme. However, this method involves the use of arbitrary enzyme that does not provide site-specificity of the hydrolysis process. In the subsequent mass spectroscopic study of the products of this hydrolysis is impossible to identify the original protein, as existing genomic databases is predpolagajut the use of site-specific enzymes, destroying the peptide bond in the presence of the peptide sequence of strictly defined amino acids.

The known method [Deveni So, Gergely J. Amino acids, peptides and proteins. M.: Mir, 1976, 368 S.], where the proposed reaction Sanger, Toppi, - nonspecific partial acid hydrolysis. Its disadvantage is the inability subsequent analysis of mass spectrometric methods.

Known patent [RF 2351932], namely, that uses the hydrolysis of site-specific enzyme - trypsin - specific branch of the parts (namely, peptides) proteins (protein complexes) from the surface. The fact that the hydrolyzable proteins (protein complexes) immobilized on the surface, it is not possible to achieve a high degree of hydrolysis: reaction of interaction of the enzyme and protein (protein complex) is ineffective due to the fact that proteins (protein complexes) are located on the surface, and the enzyme is located in the volume, which leads to poor kinetics of the reaction, which is determined by diffusion to the surface.

The closest in technical essence, adopted for the prototype, is a method of enzymatic hydrolysis, consisting in the use of site-specific enzyme trypsin in conjunction with ultrasonic impact at the stage of conducting the hydrolysis reaction described in the patent [US 20090246813]. Under taccom this way, as in the previous method, is the inefficiency of its application to hydrolysis of proteins immobilized on the surface, due to the complexity of the reaction between a protein (protein complex), located exclusively at the surface, and the enzyme trypsin, which is in volume.

The present invention is to reduce the detection threshold of protein molecules (proteins, protein complexes), immobilized on the surface of the substrate scanning probe microscope using a mass spectrometer to 1011PCs/cm2. This is required to ensure high efficiency of hydrolysis at a relatively low protein concentration, determined by the density of its shrinkage on the surface, which is determined by the possibility of their visualization (registration) using a scanning probe microscope.

Achievable technical result consists in increasing the efficiency of deformirovaniya protein molecules covalently associated with the substrate, and subsequent hydrolysis, which leads to a reduction in the threshold of detection by mass spectrometric analysis. The technical result is achieved by advanced ultrasonic exposure, during which proteins are detached from the surface and pass into the liquid phase, which increases the efficiency of the subsequent hydrolysis. This distinguishes offer aemy way from the prototype, where ultrasonic treatment is used to increase the speed of hydrolysis.

To solve this problem is proposed the method lies in the fact that the protein is exposed to ultrasound and a site-specific enzyme in buffer solution with pH value and the temperature, which provide the activity of the enzyme, and the exposure duration is chosen so to ensure the hydrolysis of the main part of the protein, but to avoid non-specific hydrolysis.

Before the influence of the enzyme substrate scanning probe microscope with an immobilized on the protein extracted from the transport buffer solution, placed in a 1-5% solution of acetic acid and subjected to the influence of high frequency ultrasound with power not less than 1 W/cm2that provides for disconnection of the protein surface. Then the solution containing the desorbed proteins, frozen and subjected to lyophilization.

The method is as follows: initially poured transport buffer solution from the test tube containing the sample (substrate intended to study in a scanning probe microscope, with immobilized her protein molecules with a concentration in the range of 1011-1013PCs/cm2). Next, the sample is poured acetic acid in the concentration range of 1-5% of the AK, to the sample was completely immersed in the solution. The test tube with the sample was placed in an ultrasonic bath with ultrasound frequency of 1-3 MHz and a power of not less than 1 W/cm2at the time, which provides separation of the maximum number of proteins (protein complexes) from the surface, but without destruction of the proteins themselves, at a temperature range which is caused by the diffusion coefficient and the preservation of protein structure (protein complex). Then remove the backing from the tube. Then the tube with the solution containing the desorbed proteins, frozen and subjected to lyophilization. Then produce thawing to room temperature. Add a solution of enzyme and buffer solution, allowing to maintain the pH at a level necessary for the efficient operation of the enzyme. Withstand the resulting solution at a temperature of 37°C. the exposure Duration is chosen so that the largest number of the target protein was obtained and this was to minimize the amount of products Samogitia enzyme. After hydrolysis produce a stop process by changing the pH to slightly acidic environment. Control products Samogitia enzyme is performed by comparison of mass spectra of the reaction products of the analyte obtained at different duration of incubation, and the mass spectrum of the dough the first sample, containing only enzyme used. For control purposes the enzyme also carry out the hydrolysis of known protein under the same conditions as the hydrolysis of the analyte. Then both the solution of the hydrolysis products are transferred into the chromatograph connected to a mass spectrometer or plate MALDI spectrometer, after adding a matrix to the solution.

Example 1

Originally drained transport buffer solution from the tube, in which there was a sample size of 10×20×0.5 mm (substrate scanning probe microscope). Then pour a sample of 2% acetic acid so that the sample was completely immersed in the solution. Next 10 minutes put the test tube with the sample in a thermostatic bath with a capacity of 5 W/cm2at 37°C. After that, take out the substrate and freeze the solution containing the desorbed proteins at -70°C. Then the solution is subjected to freeze-drying for 4 hours, then thawed to room temperature. Add in a test tube with 400 ál of buffer solution NH4HCO3with the concentration of 3.95 µg/µl and 2 µl of trypsin with a concentration of 0.2 µg/µl. Next, the test tube is placed in a thermostat (with constant stirring) at 37°C for 12 hours. Then add MALDI-matrix and applied to the target to obtain a mass spectrum.

Example # 2

P is roncallo drained transport buffer solution from the tube, in which there was a sample size of 10×20×0.5 mm (substrate scanning probe microscope). Then pour a sample of 2% acetic acid so that the sample was completely immersed in the solution. Next 10 minutes put the test tube with the sample in a thermostatic bath with a capacity of 5 W/cm2at 37°C. After that, take out the substrate and freeze the solution containing the desorbed proteins at -70°C. Then the solution is subjected to freeze-drying for 4 hours, then thawed to room temperature. Add in a test tube with 400 ál of buffer solution NH4HCO3with the concentration of 3.95 µg/µl and 1/30 part (with respect to the amount of protein) of crystalline pepsin. Next, the test tube is placed in a thermostat (with constant stirring) at 25°C for 17 hours. Then the sample is analyzed using gas chromatography-mass spectrometer.

The method provides a reduction in the threshold detection of protein molecules (proteins, protein complexes), immobilized on the surface of the substrate scanning probe microscope, using a mass spectrometer to a concentration of 1011PCs/cm2.

1. The method of carrying out specific enzymatic hydrolysis of proteins immobilized on the substrate surface scanning probe microscope, which consists in the fact that proteins podvergautsya ultrasound and site-specific enzyme in buffer solution with pH value and the temperature, which provide the activity of the enzyme, and the exposure duration is chosen so to ensure the hydrolysis of the main part of the protein, but to avoid non-specific hydrolysis, characterized in that before the influence of the enzyme substrate scanning probe microscope with an immobilized on the protein extracted from the transport buffer solution, placed in a 1-5%solution of acetic acid and exposed to ultrasound high frequency of 1-3 MHz and a power of not less than 1 W/cm2providing disconnection of protein-surface, after which the solution containing the desorbed proteins, frozen and subjected to lyophilization.

2. The method according to claim 1, characterized in that the substrate of the scanning probe microscope may consist of silicon, glass, mica, sapphire, and any other rigid material.

3. The method according to claim 1, characterized in that the surface of the substrate scanning probe microscope chemically activated by the silane, which immobilized protein molecules.



 

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