An automated mechanical cutting method for adhesive strip samples

Abstract

This invention provides an automated mechanical cutting method for adhesive strip samples. The method involves loading the adhesive strip into centrifuge tubes containing three stainless steel beads of different sizes and 75% ethanol. Each centrifuge tube is then placed in a 48-channel high-throughput tissue homogenizer for high-frequency oscillation and homogenization. The shear force generated by the stainless steel beads cuts the adhesive strip into relatively uniform fragments. By optimizing the size of the steel beads, the oscillation time, and the concentration and protective solution components of the adhesive strip, highly efficient automated cutting is achieved before subsequent experiments. Mass spectrometry analysis of the automatically cut samples shows significantly higher peptide and protein identification rates compared to traditional manual cutting methods.

Description

Technical Field

[0001] This invention belongs to the field of protein identification technology and relates to an automated mechanical cutting method for gel strip samples. Background Technology

[0002] The principle of gel-strip mass spectrometry identification is to enzymatically digest protein bands into a peptide mixture within the gel, extract the peptide mixture, and then identify it using LC-MS / MS (liquid chromatography-mass spectrometry). Search software compares the secondary mass spectrometry information with relevant databases, combining matching scores and false positive control to obtain the exact sequence of the peptides. This allows for the assembly of individual protein sequences, thus enabling protein identification.

[0003] Two-dimensional gel electrophoresis (2-DE) and SDS-PAGE (polyacrylamide gel electrophoresis) are commonly used in biological research to separate protein mixtures, collect target protein spots and strips, and identify proteins using mass spectrometry. This method is particularly effective for tasks such as identifying differentially interesting bands after gel electrophoresis (e.g., immunoprecipitation), removing high-abundance proteins from plasma samples, and identifying mass spectrometry-incompatible substances in solution samples that are difficult to remove.

[0004] The standard procedure for in-gel digestion is: gel cutting – decolorization – disulfide bond reduction – cysteine ​​alkylation – enzymatic digestion – peptide extraction – solution displacement – ​​desalting. The gel cutting step is typically done manually. However, manual gel cutting is prone to introducing contaminants due to the numerous steps involved. Furthermore, gel samples are highly susceptible to keratin contamination, especially silver-stained samples, requiring careful pretreatment. When cutting gel blocks from different samples, care must be taken to avoid cross-contamination from unclean tools and workbenches. In addition to these issues, manual gel cutting also suffers from low efficiency (only one gel block can be processed at a time, requiring 5 minutes per cycle) and poor parallelism of manual operation. Therefore, a more efficient and reproducible automated gel cutting method is urgently needed. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to provide an automated mechanical cutting method for adhesive strip samples. This method involves loading the adhesive strip into centrifuge tubes containing three stainless steel beads of different sizes, and then placing each centrifuge tube in a 48-channel high-throughput tissue homogenizer for high-frequency oscillation and homogenization. The shear force generated by the stainless steel beads cuts the adhesive strip into relatively uniform fragments, thus achieving highly efficient automated cutting for subsequent experiments. Mass spectrometry analysis of the automatically cut samples shows significantly higher peptide and protein identification rates compared to traditional manual cutting methods.

[0006] On the one hand, the present invention provides an automated mechanical cutting method for adhesive strip samples, which mainly involves loading the adhesive strip into a container with abrasive media and crushing it by vibration on an instrument capable of vibration. The abrasive media can generate shear force on the adhesive strip during the vibration process; the container is also filled with a protective liquid.

[0007] This invention presents a mechanical gel cutting method that can replace traditional manual gel cutting, achieving automated mechanical gel cutting. The method primarily involves adding grinding media to a centrifuge tube and using external force to induce mechanical oscillation. The shear force generated by the grinding media breaks down the gel strip. Studies have shown that mass spectrometry analysis of automatically cut gel samples using this invention significantly outperforms traditional manual gel cutting in terms of both peptide and protein identification numbers.

[0008] Since the oscillation process may cause some damage to the gel strip sample, which may affect the accuracy of peptide and protein identification, a protective solution needs to be added during the oscillation and disruption process. The protective solution can help decolorize the colloid and also protect the gel strip sample to maintain its normal state and preserve the integrity of peptides and proteins in the gel strip.

[0009] The protective solution can be water, ethanol, liquid nitrogen, etc., as long as it can provide protection for the peptide, with ethanol being the most preferred.

[0010] Furthermore, the instrument capable of oscillation is any one of a tissue grinder, mechanical grinder, oscillating grinder, or mechanical oscillator; the protective solution is 75% ethanol.

[0011] Any instrument that can provide external force for repeated oscillation can generate shear force on the rubber strip through the grinding medium, thereby breaking the rubber strip, and can be used for automatic rubber cutting.

[0012] 75% ethanol can serve as an excellent decolorizing and protective solution. Automatic gel cutting can shorten the decolorization time to within 3 hours by using 75% ethanol. Moreover, 75% ethanol can effectively decolorize while protecting the integrity of peptides and proteins in the gel particles, thereby resulting in higher identification results and higher recovery rates for peptides and proteins.

[0013] Furthermore, the grinding medium is a spherical, blocky, or granular material made of metal, glass, or stone.

[0014] Grinding media are materials with a certain degree of rigidity, such as metal, glass, or stone. From a usage perspective, disregarding cost, the differences between grinding media made of different materials are: 1) the mass of the media. The greater the mass, the greater the shear force generated by oscillation, and the shorter the time required; 2) the mechanical strength of the media itself. Mechanical strength determines the service life of the media; 3) the purity of the media. The media must have good chemical resistance and not contain biological components, and will not introduce other interfering substances.

[0015] In some methods, glass beads are prone to breakage, so if glass beads are used, the mechanical strength needs to be reduced and the oscillation time needs to be extended.

[0016] The grinding media can be of any shape, such as flakes, layers, spheres, blocks, granules, or other regular or irregular shapes.

[0017] Furthermore, the container is a plastic tube with a specification of 1 to 10 mL, the side length of the adhesive strip does not exceed 2 cm, and each plastic tube contains at least one adhesive strip.

[0018] The size of the plastic tube can be selected according to the specifications of the vibrating instrument. Since the colloid size is generally small, a plastic tube of 1 to 10 mL can be selected.

[0019] Furthermore, the plastic tube is a 1.5mL centrifuge tube, and the size of the adhesive strip is 0.5*1cm, with one adhesive strip inside each plastic tube.

[0020] Furthermore, the grinding media is stainless steel balls, the number of which is 3 to 5, the diameter of which is 1 to 5 mm, and the protective liquid is 0.5 mL of 75% ethanol.

[0021] The number of steel balls can be limited based on the space of the centrifuge tube and the diameter of the steel balls themselves. In existing test examples, the space of a 1.5mL centrifuge tube is limited. If there are more than 5 steel balls, the steel balls will not have enough space to move effectively. If there are too few, their movement path is simple and they cannot fully contact the sample.

[0022] Furthermore, the number of steel balls is 3, and the specifications are different; the adhesive strip is SDS-PAGE adhesive.

[0023] SDS-PAGE gels are the most commonly used gels in proteomics research, but they are easily broken.

[0024] Furthermore, the steel balls are available in three sizes: large, medium, and small, with the large ball having a diameter of 4mm, the medium ball having a diameter of 2mm, and the small ball having a diameter of 1mm; the SDS-PAGE adhesive is an 8-16% gradient adhesive.

[0025] 8-16% gradient gels are more suitable for automated gel cutting, as they have the highest recovery rates for peptide and protein identification. This is likely because the concentration gradient of an 8-16% gradient gel is just right—neither too hard nor too soft—making it easier to agitate and homogenize. The peptides and proteins cut from the gel are also more easily and fully extracted, resulting in better identification results.

[0026] Furthermore, the instrument used for oscillation is a high-throughput tissue grinder, with a frequency of 40 Hz and an oscillation time of 15 seconds during gel cutting.

[0027] High-throughput tissue grinding and crushing machines can simultaneously vibrate and cut multiple centrifuge tubes. For example, in some methods, a 48-channel high-throughput tissue grinding and crushing machine can simultaneously vibrate and crush 48 pieces of rubber strips.

[0028] In some methods, different oscillation frequencies and matching oscillation times can be selected, all of which can efficiently complete automatic glue cutting.

[0029] A 2006 Nature protocols (In-gel digestion for mass spectrometric characterization of proteins and proteomes) paper reported a method for manually cutting gel strips to obtain peptides. This method required manually cutting the gel strips into 1mm × 1mm particles, taking approximately 5 minutes per sample. In contrast, our new method reduces the shaking and disruption process to just 15 seconds. Even including the subsequent washing time for each centrifuge tube, the washing time for each tube is no more than 1 minute. This demonstrates a significant improvement in cutting efficiency, while also reducing air exposure time, minimizing keratin contamination, eliminating the risk of cross-contamination between different samples, and reducing human error. The results are remarkable.

[0030] On the other hand, the present invention provides the use of the method described above for preparing samples for identification by gel strip mass spectrometry.

[0031] Using the automated gel cutting sample provided by this invention, the number of peptide and protein identified by mass spectrometry is significantly higher than that of traditional manually cut gel samples.

[0032] Compared with existing technologies, the advantages of the automatic glue-cutting method provided by this invention are as follows:

[0033] 1) It takes only 15 seconds for the oscillation and crushing process to be completed, and it can also be processed in batches using high-throughput oscillation equipment;

[0034] 2) The obtained adhesive strip fragments are more uniform;

[0035] 3) It can reduce the time spent exposed to air;

[0036] 4) Reduce keratin contamination;

[0037] 5) There is no risk of cross-contamination when cutting different samples;

[0038] 6) Mechanized operation reduces human error;

[0039] 7) Facilitates color removal, shortening the color removal time to within 3 hours;

[0040] 8) Mass spectrometry analysis of automatically cut gel samples showed significantly better results in identifying both peptides and proteins compared to traditional manual gel cutting. Attached Figure Description

[0041] Figure 1 A photograph of the high-throughput tissue grinder / disruptor used in Example 1;

[0042] Figure 2 This is a photograph of the 420 stainless steel beads in the centrifuge tube in Example 1;

[0043] Figure 3 This is a photograph of the centrifuge tube after automatic gel cutting in Example 1;

[0044] Figure 4 This is a schematic diagram of the gel running results of the sample in Example 5 in fixed concentration gel and gradient gel, where from left to right are the self-made 12% fixed concentration gel, the self-made 4-20% gradient gel, the commercial 12% fixed concentration gel, and the commercial 4-20% gradient gel. Detailed Implementation

[0045] The preferred embodiments of the present invention will be described in further detail below with reference to the accompanying drawings. It should be noted that the following embodiments are intended to facilitate the understanding of the present invention and are not intended to limit it in any way. All features disclosed in the embodiments of the present invention, or all steps in all methods or processes disclosed, except for mutually exclusive features and / or steps, can be combined in any way.

[0046] Example 1: Automatic glue cutting method provided by the present invention

[0047] I. Instruments and Materials:

[0048] 1. High-throughput tissue grinder / disruptor (purchased from Shanghai Jingxin Industrial Development Co., Ltd., model JXFSTPRP-CL), see Figure 1 ;

[0049] 2. 420 stainless steel balls (large diameter 4mm, medium diameter 2mm, small diameter 1mm) (purchased from Yaster Steel Ball Sales Co., Ltd., model 420 stainless steel), see Figure 2 .

[0050] II. Operating Procedures:

[0051] 1. Preparation of SDS-PAGE adhesive:

[0052] 1) Place a special acrylic glass trough on the plate-making support. Insert the lower part of the fixed glass plate into the trough, and fix the middle part to the vertical plate of the support with two stationery clips. Pour fully melted agar into the trough, and seal the bottom of the cavity after it solidifies.

[0053] 2) Connect the gradient mixer, constant flow pump, and gel mold using a polyethylene pipe with a diameter of approximately 2 mm;

[0054] 3) Preparation of 20% glue solution: Take a 50mL beaker, add 5.2mL of water, 16.4mL of 30% acrylamide solution (acrylamide and methylenebisacrylamide mass ratio 29:1), 7.5mL of 1.5M Tris-HCl (pH 8.8), 0.3mL of 10% SDS solution, and 0.3mL of 10% ammonium persulfate. Vacuum under reduced pressure for 10min.

[0055] 4) Preparation of 4% gel solution: Take a 50mL beaker, add 14.2mL of water, 5.4mL of 30% acrylamide solution (acrylamide and methylenebisacrylamide mass ratio 29:1), 6.2mL of 1.5M Tris-HCl (pH 8.8), 0.24mL of 10% SDS solution, and 0.24mL of 10% ammonium persulfate. Vacuum under reduced pressure for 10min.

[0056] 5) To prepare the gradient gel, add 15 μl of TEMED to each of the two gel solutions, shake well, and pipe 18 mL of each solution into the corresponding mixing cup of the gradient mixer. Turn on the magnetic stirrer and gradient mixer, and connect the constant flow pump to slowly pour the gel solution from the gradient mixer into the gel chamber. Initially, the infusion tube outlet should extend from the center of the top opening of the gel chamber towards the bottom. As the liquid level in the gel chamber rises, gradually raise the infusion tube so that the tube opening remains close to the liquid surface without penetrating the liquid. The pouring flow rate should be appropriate, ideally so that the liquid flowing from the tube outlet does not impact the liquid surface.

[0057] 6) After the glue is poured, carefully add about 0.5cm of 25% ethanol to the liquid surface to seal the glue surface, and let it stand for about half an hour to polymerize;

[0058] 7) After gel polymerization, pour off the ethanol solution, blot dry the upper residual liquid with filter paper strips, take 5 mL of 4% gel solution, add 5 μl of TEMED, gently shake in a beaker, add to the gradient gel, quickly insert the sample comb, the lower edge of the sample comb should just touch the gradient gel surface, and let it stand to polymerize.

[0059] 2. SDS-PAGE adhesive running process:

[0060] The gel strips used in this embodiment are 4-20% gradient SDS-PAGE gels with a size of 0.5*1cm. The sample used is E. coli with a protein content of 10μg. The specific conditions and parameters are described below:

[0061] The preparation of the test samples is the same as that of the standard protein. 20 μl of the prepared sample (10 μg protein) is applied to each sample cell using a microsyringe. The upper cell is the negative electrode, and the lower cell is the positive electrode. Connect the electrophoresis apparatus and perform electrophoresis at a constant voltage (100V). After the indicator has dissipated from the gel, increase the voltage to 150V and continue electrophoresis for approximately 1.5 hours. After peeling off the gel, wash the gel with distilled water and then immerse it in staining solution for 4-5 hours or overnight. After removal, rinse briefly with tap water and place in destaining solution with shaking. Change the destaining solution several times during the process until the background is clear.

[0062] 3. Automatic glue cutting:

[0063] After rinsing the gel twice, add steel balls (one large, one medium, and one small) to a 1.5 mL tube, insert one gel strip, and add 0.5 mL of 50% ethanol. Use a high-throughput tissue homogenizer (capable of processing 48 gel blocks simultaneously) to agitate at 40 Hz for 15 seconds to break up the gel blocks (see...). Figure 3 ).

[0064] 4. LC-MS / MS identification:

[0065] The gel strips were chopped and decolorized using 50% acetonitrile containing 50 mM ammonium bicarbonate. The gel was then dehydrated by incubation in 100% acetonitrile for 5 minutes. The liquid phase was then removed, and a 10 mM dithiothreitol solution was added, followed by incubation at 37°C. The gel was then incubated again with 100% acetonitrile for further dehydration. After removing the liquid phase, 55 mM iodoacetamide was added, and the gel was incubated at room temperature in the dark for 45 minutes. The gel was then washed with 50 mM ammonium bicarbonate and incubated again with 100% acetonitrile for dehydration. Finally, the gel was resuspended in 50 mM ammonium bicarbonate containing 10 ng / μl trypsin and incubated on ice for 1 hour. After removing excess solution from the sample, the gel was enzymatically digested overnight at 37°C. The digested peptides were extracted sequentially using 50% acetonitrile / 5% formic acid and 100% acetonitrile to extract the gel blocks. The peptide solutions were then freeze-dried and stored for later use.

[0066] The peptides were dissolved in mobile phase A and then separated using an EASY-nLC 1000 ultra-high performance liquid chromatography system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile, and mobile phase B was an aqueous solution containing 0.1% formic acid and 98% acetonitrile. The liquid phase gradient settings were: 0–16 min 6%–25% B phase; 16–22 min 25%–40% B phase; 22–26 min 40%–80% B phase; 26–30 min 80% B phase, with the flow rate maintained at 450 nL / min.

[0067] After separation by an ultra-high performance liquid chromatography system, the peptide fragments are injected into an NSI ion source for ionization and then fed into ThermoScientific TM Q ExactiveTM Mass spectrometry analysis was performed. The ion source voltage was set to 2.2 kV, and high-resolution Orbitrap was used to detect and analyze peptide precursor ions and their secondary fragments. The primary mass spectrometry scan range was set to 350-1800 m / z with a scan resolution of 70,000; the Orbitrap scan resolution was set to 17,500. Data acquisition mode used a data-dependent scanning (DDA) procedure, in which the top 20 peptide precursor ions with the highest signal intensity were selected after the primary scan and sequentially entered the HCD collision cell for fragmentation at 28% of the fragmentation energy, and then sequentially analyzed by secondary mass spectrometry. To improve the efficiency of mass spectrometry, automatic gain control (AGC) was set to 5E4, the signal threshold was set to 10,000 ions / s, the maximum injection time was set to 100 ms, and the dynamic exclusion time for tandem mass spectrometry scans was set to 15 seconds to reduce repeated scans of precursor ions.

[0068] Example 2: The Influence of Steel Ball Quantity and Specification on Rubber Cutting Effect

[0069] In this embodiment, the gel strips were prepared using the method provided in Example 1. The sample was protein extracted from E. coli. The gel was run first and then automatically cut. The gel size (cm*cm) was 0.5*1, the centrifuge tube size was 1.5mL, and the shaking time was 15s. Different numbers and sizes of steel balls were used during gel cutting to investigate the effect of the automatically cut sample on the subsequent LC-MS / MS identification, thereby examining the influence of the number and size of steel balls on the gel cutting effect. The results are shown in Table 1.

[0070] Table 1. Effect of steel ball quantity and specifications on rubber cutting effect

[0071]

[0072] As can be seen from Table 1, the number and size of the steel balls not only affect the shearing effect, but also directly affect the accuracy of peptide and protein identification during subsequent LC-MS / MS. Therefore, it is necessary to select an appropriate number and size of steel balls.

[0073] After gel cutting, the obtained gel particles need to be relatively uniform (uniformity observed with the naked eye) and of appropriate size to avoid clogging the pipette tip and excessive gel powder being drawn into the tip. If the particles are too small, gel powder will be generated, easily drawn into the pipette, and may damage the integrity of peptides and proteins, affecting peptide and protein identification results. If the particles are too large, incomplete extraction will occur, and the pipette tip will be clogged, also affecting peptide and protein identification results. Peptide recovery rate indicates the total amount of peptides recovered from a gel block containing 10 μg of protein; the concentration was determined using the A280 method. A higher peptide recovery rate indicates a better method.

[0074] Studies have shown that a number of steel beads of 3 to 5 is ideal. With fewer than 3 beads, the movement path of the beads is singular, failing to fully contact the sample, resulting in unevenly cut gel particles and easy clogging of the pipette tip. This also directly affects the identification results of peptides and proteins, leading to low recovery rates. With more than 5 beads, the limited volume of the centrifuge tube results in insufficient space for the beads to move effectively, also leading to poor gel cutting, easy clogging of the pipette tip, and directly affecting the identification results of peptides and proteins, leading to low recovery rates.

[0075] Furthermore, the size and specifications of the steel balls directly affect the gel cutting effect, thus influencing the identification results of peptides and proteins. Comparing the gel cutting effects of samples 3 and 4, it can be seen that although relatively uniform gel particles that do not clog the pipette tip or get sucked in are obtained after cutting, excessive fragmentation during cutting may affect the integrity of peptides or proteins in the gel strip, resulting in poor recovery rates and lower identification results. Comparing samples 6 to 9, it can be seen that although all use 3 steel balls, the gel cutting effect varies greatly depending on the size of the steel balls. Only when using one large, one medium, and one small steel ball can a better gel cutting effect be obtained, resulting in uniform gel particles that do not clog the pipette tip or get sucked in. At the same time, the recovery rate of peptides and proteins is higher, and the identification results are more accurate.

[0076] Of course, it is understandable that different numbers and sizes of steel balls may be required for containers of different sizes or shapes. For the 1.5mL centrifuge tube in this embodiment, it is most preferable to use one large, one medium, and one small steel ball.

[0077] Example 3: Effect of different vibration times on rubber cutting effect

[0078] This embodiment uses the method provided in Example 1 to prepare the gel strips. The sample is protein extracted from E. coli. After gel running, the gel is automatically cut. The gel size (cm*cm) is 0.5*1. The centrifuge tube specification is 1.5mL. The steel balls are of one large, one medium, and one small size. The shaking time during gel cutting is 10, 15, 20, 25, and 30s, respectively. The effect of shaking time on the state after gel cutting and on subsequent LC-MS / MS identification is investigated, thereby examining the effect of the number and size of steel balls on the gel cutting effect. The results are shown in Table 2.

[0079] Table 2. Effect of vibration time on rubber cutting effect

[0080]

[0081] As shown in Table 2, too short a shaking time leads to uneven gel cutting, easily clogging the pipette tip and affecting the accuracy of subsequent peptide and protein identification. Too long a shaking time results in excessively small gel particles that are easily sucked into the pipette tip, also directly affecting the accuracy of subsequent peptide and protein identification. Therefore, it is essential to select an appropriate shaking time. Studies have shown that a shaking time of 15 seconds yields the best gel cutting effect, producing uniform gel particles that neither clog the pipette tip nor are sucked into it.

[0082] Example 4: The Influence of Different Media Environments on Rubber Cutting Effect

[0083] This embodiment uses the method provided in Example 1 to prepare the gel strips. The sample is protein extracted from E. coli. After gel running, the gel is automatically cut. The gel size (cm*cm) is 0.5*1. The centrifuge tube is 1.5mL. The steel balls are one large, one medium, and one small. Liquid nitrogen (inside or outside the tube), ultrapure water, 25% ethanol, 50% ethanol, 75% ethanol, and anhydrous ethanol are added respectively. The gel is then automatically cut again, and the shaking time is 15s. The effects of different media or environments on the state of the gel after cutting and on subsequent LC-MS / MS identification are investigated. The results are shown in Table 3.

[0084] Table 3. Effects of different media or environments on rubber cutting effect

[0085]

[0086] As shown in Table 3, different media or environments significantly affect the gel cutting effect. Using liquid nitrogen for gel cutting leads to fragile centrifuge tubes, causing the gel to shrink into paper-like pieces that are difficult to cut. Using ultrapure water as the medium results in no decolorization of the cut gel particles, with pigments severely contaminating the mass spectrometer, and identification shows low peptide and protein recovery rates. Ethanol provides better decolorization and further improves peptide and protein recovery rates. While anhydrous ethanol exhibits excellent decolorization, its peptide and protein recovery rates show a decreasing trend, potentially affecting the integrity of peptides and proteins in the gel particles. Therefore, 75% ethanol is the optimal choice, as it ensures complete sample decolorization.

[0087] In addition, compared with the blank control without medium, although the strips can be cut evenly, the shaking process may damage the peptides and proteins in the strips, thus significantly reducing the identification results. It can be seen that 75% ethanol can also play a good protective role, effectively decolorizing and protecting the integrity of peptides and proteins in the particles, thus resulting in higher identification results and higher recovery rate of peptides and proteins.

[0088] Example 5: Cutting effect of different adhesives

[0089] This embodiment uses the method provided in Example 1 to prepare adhesive strips and automatically cut them. The sample used is protein extracted from E. coli. The adhesive strips are prepared using a self-made 12% fixed concentration adhesive (preparation method see the experimental steps in Example 1) and a commercially available 12% fixed concentration adhesive (purchased from Nanjing Genscript Biotech Co., Ltd., model SurePAGE). TM Pre-cast 12% fixed concentration gel), homemade 4-20% gradient gel (preparation method: experimental steps in Example 1), commercially available 4-20% gradient gel (purchased from Nanjing GenScript Biotech Co., Ltd., model SurePAGE). TM Pre-cast gels (4-20% gradient gels), in which samples are run in both fixed-concentration gels and gradient gels, such as... Figure 4 As shown in the table (from left to right: self-made 12% fixed concentration gel, self-made 4-20% gradient gel, commercial 12% fixed concentration gel, commercial 4-20% gradient gel), the gel size (cm*cm) is 0.5*1, the centrifuge tube specification is 1.5mL, the steel ball specifications are one large, one medium, and one small, 0.5mL of 75% ethanol is added, and then the gel is automatically cut, and the shaking time is 15s. The cutting effect of different gels is investigated, and the results are shown in Table 4.

[0090] Table 4. Cutting effect of different adhesives

[0091]

[0092] As shown in Table 4, when using the method provided by this invention for automated gel cutting with different types and concentrations of gels, there was no significant difference in the recovery rates of peptide and protein identification after extraction of the gel-cut samples. Comparing gradient gels of different concentrations, it is evident that the 8-16% gradient gel is more suitable for automated gel cutting, exhibiting the highest recovery rates for peptide and protein identification. This is likely because the concentration gradient of the 8-16% gradient gel is more suitable—neither too stiff nor too soft—making it easier to homogenize by shaking. Furthermore, the peptides and proteins after gel cutting are more easily and fully extracted, resulting in better identification results.

[0093] Example 6: Comparison of parallelism between automatic and manual glue cutting

[0094] In this embodiment, the adhesive strips prepared in Example 1 were used. The adhesive strips were cut automatically and manually. The manual cutting method was to cut the adhesive block into 1mm*1mm particles with a scalpel under a clean bench. The automatic cutting method was as shown in Example 1. The peptide recovery rates of the two cutting methods were compared, and the results are shown in Table 5.

[0095] Table 5. Comparison of parallelism between automatic and manual glue cutting

[0096]

[0097]

[0098] As shown in Table 5, the peptide recovery rate of automated gel cutting is higher, and the parallelism is significantly better than that of manual gel cutting.

[0099] Example 7: Efficiency Comparison of Automatic and Manual Glue Cutting

[0100] This embodiment uses the adhesive strip prepared in Example 1. The adhesive strips are cut automatically and manually. The manual cutting method is as follows: under a clean bench, the adhesive block is cut into 1mm*1mm granules with a scalpel. After cutting, the granules are placed in 75% ethanol for decolorization. The automatic cutting method is as shown in Example 1. The cutting efficiency of the two methods is compared, and the results are shown in Table 6.

[0101] Table 6. Comparison of efficiency between automatic and manual glue cutting

[0102]

[0103] As shown in Table 6, the efficiency of automatic glue cutting is significantly improved. The efficiency of automatic glue cutting is more than 4 times that of manual glue cutting. In the complete glue strip sample preparation process, 30% of the operation time can be saved for 20 samples and 26% of the operation time can be saved for 40 samples.

[0104] Manual gel cutting can only cut one piece of gel at a time, and each piece takes 5 minutes. If 48 pieces are cut, it takes 240 minutes. However, automatic gel cutting can cut up to 48 pieces at a time, and the entire process of breaking the gel only takes 1 minute. If we include the 1 minute for cleaning each centrifuge tube, it only takes 1 + 48 * 1 = 49 minutes, saving 3.2 hours and increasing efficiency by 4.9 times. Moreover, automatic gel cutting can reduce air exposure time and keratin contamination. When cutting different samples, there is no risk of cross-contamination. Mechanized operation reduces human error and improves parallelism. 75% ethanol can be added during automatic gel cutting to keep the gel strips in a normal state and allow them to move freely in the centrifuge tube. It can also shorten the decolorization time to within 3 hours.

[0105] Using the same 75% ethanol for decolorization, the automatic cutting method decolorizes faster, taking a maximum of 3 hours, while the manual cutting method takes 12 hours. The main reason is the difference in the degree of rubber breakage. Manually cut rubber is not uniform or fine enough, requiring more time for decolorization, while the automatic cutting method is more uniform and finer, resulting in a faster decolorization speed.

[0106] Example 8: Comparison of mass spectrometry detection results between automatically and manually cut gels

[0107] This embodiment uses the adhesive strips prepared in Example 1. The adhesive strips are cut automatically and manually. The manual cutting method involves cutting the adhesive block into 1mm*1mm particles using a scalpel under a clean bench. The platform and scalpel need to be wiped repeatedly before and after each cutting process. After cutting, the adhesive is placed in 75% ethanol for decolorization. The automatic cutting method is as shown in Example 1. The peptide and protein identification results of the two cutting methods are compared, and the results are shown in Table 7.

[0108] Table 7. Comparison of mass spectrometry detection results between automated and manual gel cutting.

[0109]

[0110] As shown in Table 7, the number of peptides and proteins identified by mass spectrometry in automatically cut gel samples is significantly better than that of traditional gel cutting methods, and the parallelism is also better. This is because the use of automatic gel cutting can reduce air exposure time, mechanize the operation, reduce human error, and the addition of 75% ethanol can effectively and quickly decolorize while protecting the integrity of peptides and proteins in the gel particles, thereby effectively reducing loss and improving the recovery rate of peptides and proteins.

[0111] The embodiments described above provide a detailed explanation of the technical solution of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An automated mechanical cutting method for adhesive strip samples, characterized in that, The adhesive strip is placed in a container with abrasive media and broken up by vibration on an instrument capable of vibration. The abrasive media exerts shear force on the adhesive strip during vibration. The container also contains a protective liquid, which is 75% ethanol. The abrasive media consists of stainless steel balls, with one large, one medium, and one small ball each. The large ball has a diameter of 4 mm, the medium ball has a diameter of 2 mm, and the small ball has a diameter of 1 mm. The adhesive strip is made of SDS-PAGE adhesive, which is an 8-16% gradient adhesive. The container is a 1.5 mL centrifuge tube, and the adhesive strip measures 0.5*1 cm, with one adhesive strip in each plastic tube.

2. The method as described in claim 1, characterized in that, The instrument capable of oscillation is any one of a tissue grinder, mechanical grinder, oscillating grinder, or mechanical oscillator.

3. The method as described in claim 2, characterized in that, The instrument used for oscillation is a high-throughput tissue grinder, with a frequency of 40 Hz and an oscillation time of 15 seconds during gel cutting.

4. The use of the method according to any one of claims 1 to 3 for preparing samples for identification by gel strip mass spectrometry.

5. The application of the steel ball specifications and quantity in the automated mechanical cutting process of adhesive strip samples to prevent adhesive particles from clogging the nozzle and to improve peptide recovery rate, characterized in that... The steel balls are of three sizes: large (4 mm), medium (2 mm), and small (1 mm). The automated mechanical cutting method for the adhesive strip samples is as follows: the adhesive strips are placed in a container with abrasive media and crushed by vibration on an instrument capable of vibration. The abrasive media exerts shear force on the adhesive strips during vibration. The container also contains a protective liquid, which is 75% ethanol. The abrasive media are steel balls made of stainless steel. The adhesive strips are SDS-PAGE adhesive, which is an 8-16% gradient adhesive. The container is a 1.5 mL centrifuge tube, and the adhesive strips are 0.5*1 cm in size, with one adhesive strip in each plastic tube. The use of 6.75% ethanol in the preparation of a protective solution to improve peptide recovery during automated mechanical gel cutting of gel strip samples, characterized in that... The automated mechanical cutting method for the adhesive strip samples is as follows: the adhesive strip is placed in a container with abrasive media and crushed by vibration on an instrument capable of vibration. The abrasive media generates shear force on the adhesive strip during vibration. The container also contains a protective liquid; the protective liquid is 75% ethanol; the abrasive media consists of stainless steel balls, with one large, one medium, and one small ball each, wherein the large ball has a diameter of 4 mm, the medium ball has a diameter of 2 mm, and the small ball has a diameter of 1 mm; the adhesive strip is SDS-PAGE adhesive, and the SDS-PAGE adhesive is an 8-16% gradient adhesive; the container is a 1.5 mL centrifuge tube, the size of the adhesive strip is 0.5*1 cm, and one adhesive strip is placed in each plastic tube.

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