A method for processing a protein sample for mass spectrometric detection

Abstract

This application discloses a method for processing protein samples for mass spectrometry detection. The method involves processing the protein sample into peptides using only ultrasound to obtain a protein sample suitable for mass spectrometry detection, without the use of enzymatic digestion. By precisely controlling the power, time, and mode of ultrasound, the high shear force generated by ultrasonic cavitation directly acts on the peptide bonds of the protein, achieving random and non-specific breakage. This method significantly reduces the protein sample pretreatment time from hours in existing enzymatic methods to minutes, eliminates the need for expensive enzymes, significantly reduces detection costs, simplifies the operation process, and can generate peptides of varying lengths covering the entire protein sequence, providing complementary protein sequence coverage information to enzymatic methods. This offers a novel and efficient solution for protein identification and structural analysis.

Description

Technical Field

[0001] This application relates to the field of protein mass spectrometry detection technology, and in particular to a method for processing protein samples for mass spectrometry detection. Background Technology

[0002] Currently, the standard method for processing proteins into peptides before mass spectrometry analysis is enzymatic digestion, most commonly using trypsin. The basic procedure involves reductive alkylation of the protein solution to open disulfide bonds and stabilize cysteine ​​residues, followed by incubation with trypsin at 37°C for several hours to overnight. Trypsin specifically cleaves at the C-terminus of lysine (Lys) and arginine (Arg), generating peptides of relatively uniform length suitable for mass spectrometry analysis. This method is the cornerstone of proteomics research.

[0003] Although enzymatic hydrolysis is widely used, it has the following inherent drawbacks:

[0004] Excessive time consumption: The enzymatic digestion process usually takes 4 to 18 hours, which is the most time-consuming step in the entire mass spectrometry sample pretreatment process, and seriously restricts the detection throughput.

[0005] High cost: Sequencing-grade trypsin is expensive, especially when conducting large-scale sample testing, the reagent cost is significant.

[0006] Incomplete digestion and specificity limitations: Enzymatic digestion efficiency is affected by various factors such as pH, temperature, and buffer composition, which may lead to incomplete digestion or non-specific cleavage, introducing complexity. Simultaneously, the specificity of enzyme cleavage sites means that peptides covering all regions of the protein cannot be obtained, especially in regions with long intervals between two cleavage sites.

[0007] The process is cumbersome: In order to obtain the best enzymatic hydrolysis effect, the protein usually needs to undergo multiple pretreatment steps such as desalting, quantification, and reductive alkylation. The process is complicated and has a high risk of introducing errors and sample loss.

[0008] Therefore, how to achieve a rapid, low-cost, simple sample processing method for protein mass spectrometry detection with higher protein sequence coverage remains a key research focus and challenge in the field of protein mass spectrometry detection technology. Summary of the Invention

[0009] The purpose of this application is to provide a novel method for processing protein samples for mass spectrometry detection.

[0010] To achieve the above objectives, this application adopts the following technical solution:

[0011] This application discloses a method for processing protein samples for mass spectrometry detection, which includes processing the protein sample to be tested into peptides by ultrasonic treatment alone to obtain a protein sample suitable for mass spectrometry detection, and the entire process does not use biological enzymes for enzymatic digestion.

[0012] It should be noted that this application achieves random, non-specific breakage of protein peptide bonds by precisely controlling the power, time, and mode of ultrasound, enabling the high shear force generated by ultrasonic cavitation to act directly on the peptide bonds of proteins. This method significantly reduces protein sample pretreatment time from hours to minutes using existing enzymatic methods, eliminates the need for expensive enzymes, significantly reduces detection costs, simplifies the operation process, and can generate peptides of varying lengths covering the entire protein sequence. It can provide protein sequence coverage information that complements enzymatic methods, offering a novel and efficient solution for protein identification and structural analysis.

[0013] It should also be noted that there are existing studies and reports on the use of ultrasound-assisted enzymatic digestion; however, these are merely ultrasound-assisted enzymatic digestion methods, and are essentially still enzymatic digestion methods. Furthermore, there are no existing studies or reports on the use of ultrasound alone to prepare protein mass spectrometry samples.

[0014] In one implementation of this application, the ultrasonic treatment time is 10~30 min.

[0015] It should be noted that the sonication time should not be too long or too short. If the sonication time is too short, for example, less than 10 minutes, the protein sample cannot be completely peptided. For example, in one implementation of this application, sonication for 5 minutes can still detect proteins that have not been peptided. On the other hand, the sonication time should not be too long, for example, more than 30 minutes. This not only does not further enhance the peptidedation of proteins, but also damages the protein sample, which is not conducive to subsequent detection.

[0016] In one implementation of this application, the power of the ultrasonic processing is greater than or equal to 80W.

[0017] It should be noted that, unlike existing ultrasound-assisted enzymatic digestion methods, this application uses ultrasound with a power of not less than 80W for processing alone, without the use of any enzymes, to obtain protein sequencing samples that can meet the requirements of mass spectrometry detection.

[0018] In one implementation of this application, the ultrasonic processing mode is focused ultrasound mode.

[0019] It should be noted that this application employs focused ultrasound mode. For example, ultrasonic energy is emitted by a bowl-shaped ultrasonic transducer. The bowl-shaped transducer concentrates the energy to a single point, resulting in high shear force. This high shear force acts directly on the protein, causing random shearing. Under this action, the peptide bonds of the protein break randomly and non-specifically, thereby obtaining a protein sequencing sample that meets the requirements for mass spectrometry detection. It is understood that the bowl-shaped ultrasonic transducer is only one specific ultrasonic device used in one implementation of this application. Ultrasonic devices capable of producing the same or similar protein shearing effects are also applicable to this application, and no specific limitation is made here.

[0020] In one implementation of this application, the ultrasonic treatment is performed at a temperature of 25~37 ℃.

[0021] In one implementation of this application, the processing method further includes adding UA buffer to maintain stability before sonicating the protein sample to be tested, followed by sequential alkylation and reduction, and then sonication.

[0022] In one implementation of this application, the UA buffer contains 8 mol / L of urea, and the solvent is Tris-HCl.

[0023] In one implementation of this application, alkylation includes adding 20 mmol / L dithiothreitol and incubating at 56 °C for 30 min.

[0024] In one implementation of this application, the reduction includes adding 10 mmol / L iodoacetamide and incubating at room temperature in the dark for 20 min.

[0025] In one implementation of this application, the processing method further includes sonicating the protein sample to be tested, desalting it using a C18 column, and then using it for mass spectrometry detection.

[0026] Due to the adoption of the above technical solutions, the beneficial effects of this application are as follows:

[0027] The processing method described in this application, through precise control of the power, time, and mode of ultrasound, allows the high shear force generated by the ultrasonic cavitation effect to directly act on the peptide bonds of proteins, achieving random and non-specific breakage. This method significantly shortens the protein sample pretreatment time from hours in existing enzymatic digestion methods to minutes, eliminates the need for expensive enzymes, significantly reduces detection costs, simplifies the operation process, and can generate peptides of varying lengths covering the entire protein sequence. It can provide protein sequence coverage information complementary to enzymatic digestion methods, offering a novel and efficient solution for protein identification and structural analysis. Attached Figure Description

[0028] Figure 1This is a peak diagram of the characteristic peak at 653 m / z in the enzymatic digestion group in the embodiments of this application;

[0029] Figure 2 This is a peak diagram showing the characteristic peak at 820 m / z in the enzymatic digestion group in the embodiments of this application;

[0030] Figure 3 This is a peak diagram showing the position of the characteristic peak at 653 m / z in the ultrasound group in the embodiments of this application;

[0031] Figure 4 This is a peak diagram showing the position of the characteristic peak at 820 m / z in the ultrasound group in the embodiments of this application;

[0032] Figure 5 The SDS-PAGE electrophoresis results are shown in the embodiments of this application. Detailed Implementation

[0033] To address the shortcomings and deficiencies of enzymatic digestion methods, such as long processing time, high cost, incomplete digestion, limited specificity, and cumbersome procedures, this application creatively develops a new method for processing protein samples for mass spectrometry detection.

[0034] The core concept of this application lies in abandoning traditional enzymatic hydrolysis and providing a novel physical fragmentation strategy. We have developed a method for rapidly fragmenting intact proteins into peptide mixtures suitable for mass spectrometry analysis directly in the liquid phase using high-intensity ultrasonic energy.

[0035] The problem-solving approach, core content or principle of this application, and the resulting technical effects are as follows:

[0036] By precisely controlling the power, time, and mode of ultrasound, the high shear force generated by ultrasonic cavitation directly acts on the peptide bonds of proteins, achieving random and non-specific breakage. This method significantly reduces sample pretreatment time (from hours to minutes), substantially lowers detection costs (eliminating the need for expensive enzymes), simplifies the operational process, and can generate peptides of varying lengths covering the entire protein sequence. It is expected to provide protein sequence coverage information complementary to enzymatic methods, offering a novel and efficient solution for protein identification and structural analysis.

[0037] The present application will be further described in detail below through specific embodiments. The following embodiments are only for further illustration of the present application and should not be construed as limiting the present application.

[0038] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0039] Example

[0040] I. Experimental Materials

[0041] Protein samples: Bovine serum albumin (BSA, Maclean's, purity >98%), urea, dithiothreitol (DTT), and iodoacetamide (IAA) were all purchased from Maclean's.

[0042] Instruments and equipment: BoFU-60 focused ultrasound instrument (Longlight), high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0043] II. Experimental Methods

[0044] 1. UA buffer: 8 M urea to maintain stability, solvent is Tris-HCl.

[0045] 2. Alkylation: Add 20 mM DTT and incubate at 56 °C for 30 min.

[0046] 3. Reduction: Add 10 mM IAM and incubate at room temperature in the dark for 20 min.

[0047] Then, enzymatic hydrolysis and ultrasonic treatment were performed separately, as follows:

[0048] 4-1. Enzyme digestion (control group): Take 100 µL of BSA sample treated with "3. Reduction" and add trypsin (purchased from Maclean, catalog number T6325) at a ratio of 1:50 (enzyme: protein sample), and digest overnight at 37 ℃.

[0049] 4-2. Ultrasonic fragmentation: Take 80 µL of the BSA sample after the "3. Reduction" treatment and put it into a 100 µL fragmentation tube. Use a BoFU-60 focused ultrasound instrument to sonicate for 30 min. The temperature is controlled at 25-37 ℃. Ultrasonic conditions: power 80 W, pulse width 5 ms, pulse period 2 ms.

[0050] The above peptided samples were desalted using a C18 column and then processed using a Thermo Fisher column. TM OrbitrapFusion TM Lumos TM Tribrid TM Mass spectrometry is used for mass spectrometry detection.

[0051] To test the effect of different sonication times on protein samples, this example tested the effects of sonication for 5 min, 10 min, and 30 min, with all other conditions being the same. The sonicated samples and the original BSA samples were then analyzed by SDS-PAGE electrophoresis.

[0052] SDS-PAGE uses self-prepared gels, with a separating gel concentration of 12% and a stacking gel concentration of 5%.

[0053] 10 mL of 12% separating gel: 4.0 mL of 30% Acr-Bis (29:1), 2.5 mL of 1.5M Tris, pH 8.8, 100 µL of 10% SDS, 100 µL of 10% ammonium persulfate, 4 µL of TEMED, and add H2O to 10 mL.

[0054] 5 mL of 5% concentration stacking gel: 830 µL of 30% Acr-Bis (29:1), 630 µL of 1.0M Tris, pH 6.8, 50 µL of 10% SDS, 50 µL of 10% ammonium persulfate, 3 µL of TEMED, and add H2O to 5 mL.

[0055] Acrylamide (Acr) was purchased from Maclean's, catalog number A6299; N-methyleneacrylamide (Bis) was purchased from Maclean's, catalog number M6024; Sodium dodecyl sulfonate (SDS) was purchased from Maclean's, catalog number S817668; Ammonium persulfate was purchased from Maclean's, catalog number A6295; N,N,N,N-Tetramethylethylenediamine (TEMED) was purchased from Maclean's, catalog number T6023; and glycine was purchased from Maclean's, catalog number C800883.

[0056] Mix the loading buffer (Biosharp, BL502A SDS-PAGE protein loading buffer (5×)) with the sample at a 1:1 ratio (10 µL each), heat at 100 °C for 10 min, centrifuge at 10000 rpm for 20 min, and then take the supernatant for electrophoresis.

[0057] The electrophoresis buffer was 10× glycine buffer, 1 L, with the following formula: 30.285 g Tris, 144.4 g glycine, 10 g SDS, and a final volume of 1 L. The electrophoresis buffer was diluted to 1× before use. The sample loading volume was 10 µL. The electrophoresis conditions were 80 V for 30 min, followed by 120 V for 50 min.

[0058] III. Experimental Results

[0059] 1. Mass spectrometry detection results

[0060] Mass spectrometry analysis of the sonicated BSA samples successfully identified multiple peptides. Compared with samples prepared by traditional trypsin digestion, the ultrasonic method produced peptides with a wider length distribution, and the identified peptide sequences covered multiple regions of BSA, demonstrating the effectiveness of this method. The entire pretreatment process (from intact protein to machine-ready peptides) can be completed in 60 minutes, which is far more efficient than enzymatic digestion. See Table 1 for details.

[0061] Table 1 Summary of mass spectrometry data

[0062] Enzymatic digestion group Ultrasonic treatment group Master Master Protein Master Protein Accession P02769 P02769 Description Albumin OS=Bos taurus OX=9913 GN=ALB PE=1 SV=4 Albumin OS=Bos taurus OX=9913 GN=ALB PE=1 SV=4 Coverage [%] 8 95 Peptides 5 907 PSMs 11 1223 Unique Peptides 5 907 AAs 607 607 MW [kDa] 69.2 69.2 calc. pI 6.18 6.18 Score Sequest HT 6.21 1405.21 Peptides: Sequest HT 5 907 Found in Sample: [S2] F2: Sample High High

[0063] In Table 1, Master indicates whether the row contains the master protein. In a proteome, multiple highly homologous proteins may be grouped into a single "proteome," with the most representative one labeled "Master Protein," indicating a high degree of reliability of the results.

[0064] Accession: P02769 is the standard accession number for bovine serum albumin (BSA). Both treatment methods can identify BSA protein.

[0065] Description: Protein description. OS: Species (Bos taurus); OX: Species classification number; GN: Gene name (ALB); PE: Protein presence evidence level (1-Experimental evidence); SV: Sequence version number; PE=1 indicates experimental evidence and reliable results.

[0066] Coverage [%]: Sequence coverage. The percentage of the protein's amino acid sequence covered by all identified peptide sequences. Higher is better; >20% is generally good, and >50% is excellent. Low coverage may be due to low protein abundance, excessive hydrophobicity, or poor enzymatic digestion efficiency. Sonication is superior to enzymatic digestion.

[0067] Peptides: Total number of peptides identified. Includes the number of all identified (unique + non-unique) peptides.

[0068] PSMs: Peptide spectral match number. A peptide may be fragmented multiple times, generating multiple PSMs. PSMs are indicators of abundance and the number of detections. Higher values ​​indicate more reliable identification and generally higher protein abundance. Sonication is superior to enzymatic digestion.

[0069] Unique Peptides: The number of peptides that uniquely identify with this protein (and not belong to any other protein). This is a core indicator of the reliability of protein identification; ≥ 1 is the minimum standard. Sonication is superior to enzymatic digestion.

[0070] AAs: The total number of amino acids in the protein.

[0071] MW[kDa]: The theoretical molecular weight of the protein.

[0072] calc. pI: The theoretical isoelectric point of this protein.

[0073] Sequest HT Score: Sequest HT is a search engine score representing the confidence score of a match. A higher score indicates a better match. Ultrasonic treatment is superior to enzymatic digestion.

[0074] Peptides (by Search Engine): Sequest HT: The number of peptides identified by a specific search engine, which should be consistent with or very close to the Unique Peptides value.

[0075] Found in Sample: [S2] F2: Sample: Abundance level in the sample, usually calculated based on the number of PSMs or spectra. “High” indicates high abundance.

[0076] Some mass spectrometry peaks are shown below. Figures 1 to 4 As shown, Figure 1 This is a peak diagram showing the characteristic peak at 653 m / z for the enzymatic digestion group. Figure 2 This is a peak diagram showing the characteristic peak at 820 m / z for the enzymatic digestion group. Figure 3 This is a peak diagram showing the characteristic peak at 653 m / z in the ultrasound group. Figure 4 This is a peak diagram showing the position of the characteristic peak at 820 m / z in the ultrasound group.

[0077] The results above show that the ultrasonic method is more efficient in this case, and characteristic peaks of BSA can be clearly seen at 653 m / z and 820 m / z.

[0078] 2. SDS-PAGE electrophoresis results

[0079] SDS-PAGE electrophoresis results are as follows: Figure 5 As shown. Figure 5 The results showed that, in the original state: the BSA signature main band, 66.430 kDa, with impurities, consistent with expectations; after 5 min of sonication: the main band decreased and small fragments appeared; after 10 min and 30 min of sonication: there was no main band, and all of them became peptides. Theoretically, peptides are less than 10 kDa and are not visible in SDS-PAGE.

[0080] It should be noted that this example uses ultrasound instead of enzymatic digestion for protein peptide fragmentation. The fundamental method lies in the role of ultrasound. Therefore, regardless of whether the protein peptide fragmentation is performed using focused ultrasound, water bath ultrasound, or probe-type ultrasound, it is applicable to this application. It can be understood that this example using BSA is applicable to the peptide fragmentation of all proteins / proteomes, and the processing method of this application is suitable for all types of proteins. Furthermore, the processing method of this application may not require alkylation and reduction treatments. In the examples, alkylation and reduction treatments were performed on the BSA samples for comparison with enzymatic digestion; however, the ultrasonic processing method of this application can eliminate the need for alkylation and reduction treatments, further shortening the processing steps and time.

[0081] The above description, in conjunction with specific embodiments, provides a further detailed explanation of this application and should not be construed as limiting the specific implementation of this application to these descriptions. Those skilled in the art to which this application pertains can make several simple deductions or substitutions without departing from the concept of this application.

Claims

1. A method for processing protein samples for mass spectrometry detection, characterized in that: This includes using only ultrasonic treatment to process the protein sample into peptides to obtain a protein sample suitable for mass spectrometry detection, without using biological enzymes for enzymatic digestion throughout the entire process.

2. The processing method according to claim 1, characterized in that: The ultrasonic treatment time is 10~30 min.

3. The processing method according to claim 1, characterized in that: The power of the ultrasonic treatment is greater than or equal to 80W.

4. The processing method according to claim 1, characterized in that: The ultrasonic processing mode is focused ultrasound mode.

5. The processing method according to any one of claims 1-4, characterized in that: The ultrasonic treatment was performed at a temperature of 25~37 ℃.

6. The processing method according to any one of claims 1-4, characterized in that: Before sonicating the protein sample, a UA buffer is added to maintain stability, followed by alkylation and reduction in sequence, and then sonication.

7. The processing method according to claim 6, characterized in that: The UA buffer contains 8 mol / L of urea, and the solvent is Tris-HCl.

8. The processing method according to claim 6, characterized in that: The alkylation process involves adding 20 mmol / L dithiothreitol and incubating at 56 °C for 30 min.

9. The processing method according to claim 6, characterized in that: The reduction process involves adding 10 mmol / L iodoacetamide and incubating at room temperature in the dark for 20 min.

10. The processing method according to any one of claims 1-4, characterized in that: This also includes desalting the protein sample after sonication using a C18 column before mass spectrometry detection.

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