Application of phosphorylation labeling reagent iSIPL in identification of protein N-terminal sequence

Abstract

The application belongs to the technical field of proteomics, and relates to application of a phosphorylation labeling reagent iSIPL in identification of a protein N-terminal sequence, which comprises the following steps: denaturation treatment is performed on a protein, and a disulfide bond of the protein is opened by a reducing agent; a sulfydryl group is blocked by using an alkylating reagent to prevent the sulfydryl group from re-forming a disulfide bond; an iSIPL reagent is used to modify an N-terminal amino group of the protein, and a hydroxylamine solution is added to terminate a reaction of the iSIPL reagent; the modified protein is subjected to enzymatic hydrolysis by using a protease to obtain a peptide mixture suitable for mass spectrometry analysis, a secondary mass spectrum is collected by using liquid chromatography tandem mass spectrometry; and a mass spectrum obtained is analyzed, and protein information and N-terminal sequence information of the protein are identified. The application labels the N-terminal of the protein by using the phosphorylation labeling reagent, improves accuracy and reliability of N-terminal peptide identification, effectively distinguishes the N-terminal peptide from internal peptide segments, simplifies a mass spectrum analysis process, and provides an efficient and accurate solution for N-terminal analysis of the protein.

Description

Technical Field

[0001] This invention relates to the field of proteomics technology, and in particular to the application of a phosphorylation labeling reagent iSIPL in the identification of the N-terminal sequence of proteins. Background Technology

[0002] The N-terminal sequence of a protein is a key component of its primary structure, directly reflecting the translation initiation site, post-translational processing state, and functional regulation pattern. It has irreplaceable application value in antibody drug structure confirmation, recombinant protein quality control, disease protease substrate screening, and proteomics research.

[0003] The traditional Edman degradation method is a classic method for protein N-terminal sequencing, with high accuracy in single amino acid identification, but it has significant limitations: it has strict requirements on sample purity and starting amount, and cannot handle complex mixed samples; it cannot identify proteins with N-terminal acetylation, pyroglutamylation and other blocking modifications; and it has low detection throughput and limited sequencing length, making it difficult to meet the needs of large-scale research.

[0004] Protein identification techniques based on liquid chromatography-tandem mass spectrometry (LC-MS / MS) are compatible with complex samples and various post-translational modifications, and have become the mainstream method for proteomics analysis. However, under label-free conditions, N-terminal peptides are low in abundance, easily masked, and lack characteristic markers to distinguish N-terminal peptides from internal peptides, resulting in high false positive rates and insufficient reliability. Existing isotope labeling reagents such as TMT and iTRAQ can modify the N-terminus, but their improvement on peptide ionization efficiency is limited, the detection sensitivity of low-abundance N-terminal peptides is insufficient, and the labeling simultaneously covers the N-terminus and lysine side chains, making it difficult to accurately distinguish N-terminal specific labels, complicating the data analysis process, and limiting the accuracy of identification. Summary of the Invention

[0005] The purpose of this invention is to provide a method for using the phosphorylation labeling reagent iSIPL in identifying the N-terminal sequence of proteins, aiming to solve the following problems:

[0006] 1. Overcoming the detection limitations of traditional Edman degradation and label-free mass spectrometry, this method leverages the high proton affinity of the phosphoryl group in the iSIPL reagent to significantly enhance the ionization efficiency and mass spectrometry response signal of N-terminal peptides, enabling sensitive detection of N-terminal peptides in low-abundance protein samples and improving the detection sensitivity and identification coverage of N-terminal sequencing.

[0007] 2. Establish a precise identification rule for N-terminal peptides based on characteristic mass shift. By introducing a fixed mass shift through iSIPL modification, the rule can clearly distinguish between N-terminal specific labeled peptides and lysine side chain labeled peptides. This allows for the specific screening of target N-terminal peptides from complex mixtures of enzymatically digested peptides, reducing internal peptide interference, simplifying the mass spectrometry analysis process, and improving the accuracy and reliability of N-terminal sequence identification.

[0008] 3. Construct two complementary pretreatment systems: FASP ultrafiltration and in-gel enzymatic digestion, to adapt to the high-throughput processing of soluble proteins and the analysis needs of complex samples, membrane proteins, and trace samples, respectively, thereby expanding the applicability of the method to different forms and types of protein samples and improving the method's scenario compatibility.

[0009] To achieve the above objectives, the present invention provides a method for using the phosphorylation labeling reagent iSIPL in identifying the N-terminal sequence of a protein, comprising the following steps:

[0010] The target protein is pretreated to obtain a sample protein, and the sample protein is then denatured to obtain a denatured protein.

[0011] The higher-order structure of the denatured protein is destroyed by using a reducing agent and an alkylating agent, and a phosphorylation labeling agent is added to carry out a labeling reaction. The corresponding characteristic mass shift is introduced at the modified site of the denatured protein to obtain the labeled protein.

[0012] The labeled protein was digested using proteinase 2 to generate a peptide mixture, and the peptide mixture was identified and analyzed by liquid chromatography to obtain a secondary mass spectrum.

[0013] The secondary mass spectra are analyzed to identify the N-terminal peptides in the peptide mixture and to determine the N-terminal sequence information of the target protein.

[0014] Further, the steps of preprocessing the target protein to obtain a sample protein and then denaturing the sample protein to obtain a denatured protein include:

[0015] Take an appropriate amount of the target protein into a centrifuge tube, add the corresponding protein 1 at a mass ratio of 1:50, mix well, incubate at 37℃ for 2 hours and perform enzyme digestion to obtain the sample protein.

[0016] The sample protein solution was placed in an ultrafiltration centrifuge tube and centrifuged for 10 minutes. After centrifugation, guanidine hydrochloride was added to denature the target protein solution after enzyme digestion to obtain denatured protein.

[0017] Furthermore, the higher-order structure of the denatured protein is disrupted using a reducing agent and an alkylating agent, and a phosphorylation labeling reagent iSIPL is added to perform a labeling reaction to obtain a labeled protein, including:

[0018] The disulfide bonds in the denatured protein were destroyed by adding a dithiothreitol solution to the protein and vortexing it at a constant temperature of 37°C for 2 hours.

[0019] After cooling to room temperature, add iodoacetamide alkylating agent and carry out alkylation reaction for 15 min under light-protected conditions to block free thiol groups and obtain denatured protein with exposed free amino groups;

[0020] Centrifuge at 11,000 rpm for 10 min to remove the reaction solution, including the dithiothreitol solution and the iodoacetamide alkylating reagent, and retain the denatured protein with exposed free amino groups. Then, centrifuge and wash the denatured protein with exposed free amino groups retained by the ultrafiltration membrane with 200 µL of 50 mM triethyl ammonium bicarbonate. Repeat the washing twice.

[0021] The denatured protein retained on the ultrafiltration membrane was resuspended in triethylammonium bicarbonate buffer, and then phosphorylation labeling reagent was added. The mixture was vortexed at room temperature for 30 min to label the exposed free amino groups in the denatured protein. After the free amino groups had fully reacted with the phosphorylation labeling reagent, hydroxylamine solution was added to terminate the labeling reaction, and the labeled protein was obtained.

[0022] Further, the labeled protein is enzymatically digested using proteinase 2 to generate a peptide mixture, and the peptide mixture is identified and analyzed by liquid chromatography to obtain a secondary mass spectrum, including:

[0023] Add proteinase 2 to the labeled protein and hydrolyze it at a constant temperature of 37°C for 2 hours to obtain the hydrolysate;

[0024] Centrifuge at 11,000 rpm for 10 min. After centrifugation, collect the enzymatic hydrolysate and dry it under vacuum to obtain a peptide mixture.

[0025] The peptide mixture was dissolved in Nano-LC mobile phase A, bottled, and loaded onto a liquid chromatography-tandem mass spectrometry (LC-MS) system. The peptides were fragmented by high-energy collision-induced dissociation. The full scan signal of the primary mass spectrometer and the secondary fragment signal of the peptides were acquired sequentially to obtain the secondary mass spectrum of the peptide mixture.

[0026] Wherein, mobile phase A is an aqueous solution containing formic acid, and mobile phase B is an aqueous solution of acetonitrile containing formic acid.

[0027] Further, analyzing the secondary mass spectra to identify the N-terminal peptides in the peptide mixture and determining the N-terminal sequence information of the target protein includes the following steps:

[0028] The secondary mass spectrum of the peptide mixture was analyzed to extract the parent ion mass and fragment ion information corresponding to each peptide.

[0029] Based on the aforementioned characteristic quality shift, all candidate peptides modified with iSIPL were screened out;

[0030] The fragment ion information of all candidate peptides is compared and matched with a protein sequence database to obtain the amino acid sequences of all candidate peptides and the target protein sequence.

[0031] When the modification site of iSIPL is located at the N-terminus of the candidate peptide, the corresponding candidate peptide is determined to be the N-terminal peptide of the target protein, and the N-terminal sequence information of the target protein is obtained.

[0032] Furthermore, the target protein can be pretreated for N-terminal sequence identification using an in-gel pretreatment method, including the following steps:

[0033] Proteinase 1 was added to the target protein for enzymatic digestion to obtain the sample protein, and the sample protein was mixed with non-reducing loading buffer.

[0034] Centrifuge at 11,000 rpm for 10 min, take the supernatant for sodium dodecyl sulfate and polyacrylamide gel electrophoresis to separate the protein components of the sample, and perform staining and destaining treatment to obtain the target protein strip;

[0035] The target protein strips are cut and broken to obtain protein gel blocks, which are then decolorized and alternately washed again.

[0036] A reducing agent is added to break the protein disulfide bonds in the protein gel block, and then an alkylating agent is added to block the protein thiol groups in the protein gel block;

[0037] A phosphorylation labeling reagent is added to the protein gel block to label the proteins in the protein gel block. After the reaction is complete, a hydroxylamine solution is added to terminate the labeling reaction.

[0038] Residual reagents were washed away, and protease 2 was added to the protein gel for in-gel enzymatic hydrolysis. The resulting peptide mixture was extracted and collected, and then dried by vacuum centrifugation to obtain a dried peptide mixture for mass spectrometry analysis.

[0039] Furthermore, the hydroxylamine solution is used to hydrolyze unreacted NHS ester groups;

[0040] The hydroxylamine solution has a mass fraction of 5% to 20%, and the hydrolysis of the NHS ester group is most effective when the mass fraction of the hydroxylamine solution is 11%.

[0041] Furthermore, the protease 2 is trypsin / chymotrypsin.

[0042] Furthermore, the phosphorylation labeling reagent iSIPL comprises: a phosphorimide reporter ion group, a balancing group, and an NHS ester reaction group.

[0043] Furthermore, the protease 1 is an IdeS enzyme.

[0044] The present invention provides a method for using the phosphorylation labeling reagent iSIPL in identifying the N-terminal sequence of proteins, which has the following beneficial effects:

[0045] (1) By specifically labeling the N-terminal amino group of a protein with iSIPL reagent and binding it with a phosphoryl group with high proton affinity, the ionization efficiency and mass spectrometry response signal of the N-terminal peptide can be significantly improved, so that the low abundance of N-terminal peptides can be sensitively detected, avoiding the problem that the N-terminal peptides in unlabeled samples are difficult to be specifically identified due to low signal intensity, and improving the detection sensitivity and identification coverage of N-terminal sequencing.

[0046] (2) By setting iSIPL modification (+264.0875Da) as a variable modification on both the N-terminus and lysine residues of a protein, the characteristic mass shift can be used to accurately distinguish the N-terminal labeled peptide from the labeled peptide that occurs only on lysine residues. This can specifically screen out the target N-terminal peptide from a complex mixture of enzymatically digested peptides, avoid interference from internal peptides, and improve the accuracy and reliability of N-terminal sequence identification.

[0047] (3) By adopting two complementary pretreatment methods, FASP and in-gel enzymatic digestion, FASP is suitable for high-throughput processing of soluble proteins, while in-gel enzymatic digestion is suitable for complex samples (tissue lysate, cell pellet) and membrane protein analysis. This invention can flexibly adapt to the N-terminal sequencing requirements of different types of protein samples and expand the applicability of the method.

[0048] (4) The reaction is terminated by hydroxylamine solution, which effectively quenches excess unreacted iSIPL reagent, reduces sample complexity, and the labeling reaction can be completed within 30 minutes at room temperature. The concentration of iSIPL reagent can be adjusted in the range of 1-20 μg / μL, and the reaction time of 15-60 minutes and the temperature from 0℃ to room temperature can be flexibly adjusted, which improves the ease of operation and parameter tolerance of the method.

[0049] (5) It is suitable for high-throughput and high-precision identification of protein N-terminal sequences in fields such as proteomics research, biomarker discovery, drug development and medical diagnosis, providing an efficient and accurate solution for protein N-terminal analysis. Attached Figure Description

[0050] Figure 1 This is a flowchart illustrating the application of the phosphorylation labeling reagent iSIPL in the identification of the N-terminal sequence of proteins.

[0051] Figure 2 This is an amino acid sequence coverage diagram of the antibody 5C4-Heavy-Fc band obtained using the FASP pretreatment method;

[0052] Figure 3 This is a map showing the amino acid sequence coverage of the N-terminus of the antibody 5C4-Light-Fab band, which was pretreated using the FASP method.

[0053] Figure 4 This is a secondary mass spectrometry image of the N-terminal peptide GPSVFIFPPKPK sequence of the antibody 5C4-Heavy-Fc band digested with trypsin using the FASP pretreatment method.

[0054] Figure 5 This is a secondary mass spectrum of the N-terminal peptide GPSVF sequence of the antibody 5C4-Heavy-Fc band digested with chymotrypsin using the FASP pretreatment method.

[0055] Figure 6 SDS-PAGE electrophoresis image of antibody 11F8Ides digested with enzymes;

[0056] Figure 7 Amino acid sequence coverage of antibody 11F8-Heavy-Fc band obtained using in-gel pretreatment method;

[0057] Figure 8 Amino acid sequence coverage of antibody 11F8-Heavy-Fc band obtained using in-gel pretreatment method;

[0058] Figure 9 Amino acid sequence coverage of antibody 11F8-Heavy-Fc band obtained using in-gel pretreatment method;

[0059] Figure 10 This is a map showing the unmodified amino acid sequence coverage of the N-terminus of the antibody 11F8-Heavy-Fab band obtained using an in-gel pretreatment method.

[0060] Figure 11 This is a map showing the unmodified amino acid sequence coverage of the N-terminus of the antibody 11F8-Heavy-Fab band obtained using an in-gel pretreatment method.

[0061] Figure 12 This is a map showing the unmodified amino acid sequence coverage of the N-terminus of the antibody 11F8-Heavy-Fab band obtained using an in-gel pretreatment method.

[0062] Figure 13 This is a secondary mass spectrum of the N-terminal peptide GPSVFIFPPK sequence of the antibody 11F8-Heavy-Fc band digested with trypsin using the in-gel pretreatment method.

[0063] Figure 14This is a secondary mass spectrum of the N-terminal peptide GPSVF sequence of the antibody 11F8-Heavy-Fc band digested with chymotrypsin, obtained using an in-gel pretreatment method. Detailed Implementation

[0064] The technical solutions of this invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are merely some embodiments of this application, and not all embodiments. The components of this application described and shown in the accompanying drawings can generally be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of this application provided in the accompanying drawings is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0065] Example 1, such as Figure 1 As shown, this invention proposes the application of a phosphorylation labeling reagent, iSIPL, in the identification of protein N-terminal sequences. Using iSIPL as the core, it labels proteins through specific covalent reactions and combines this with mass spectrometry analysis to achieve accurate identification of protein N-terminal sequences. Based on the labeling technology principle of iSIPL, the identification process can be broken down into five core stages: sample pretreatment, covalent labeling, enzymatic digestion and separation, mass spectrometry detection, and data analysis. Each stage is interconnected, working together to achieve specific recognition and sequence analysis of N-terminal peptides. The molecular structure of iSIPL contains three functional parts: a phosphorimide reporter ion group, a balancing group, and an NHS ester reactive group. The NHS ester reactive group can covalently bind to the free amino groups on the protein, immobilizing the iSIPL tag onto the protein. The phosphorimide reporter ion group has high proton affinity, which can significantly improve the ionization efficiency and mass spectrometry response signal of the labeled peptide, while introducing a fixed mass shift of +264.0875 Da as a characteristic identification marker for the N-terminal peptide of the protein. The balancing group is used to regulate the reactivity and physicochemical properties of the reagent, ensuring the stability and specificity of the labeling reaction. The free amino groups of the protein mainly include the N-terminal α-amino group and the lysine side chain ε-amino group. The iSIPL reagent can react with both types of amino groups simultaneously. By analyzing the secondary mass spectrum and determining the position of the modification site, the N-terminal specific labeled peptide and the lysine side chain labeled peptide can be accurately distinguished, thereby screening the target N-terminal peptide from a large number of internal peptides.

[0066] This invention proposes an application of the phosphorylation labeling reagent iSIPL in the identification of N-terminal sequences of proteins, comprising the following steps:

[0067] Step S1: Add protease 1 to the target protein for enzymatic digestion, and then denature the digested protein to obtain denatured protein.

[0068] In this embodiment, antibody 5C4 was used as the detection sample, and FASP ultrafiltration was used for pretreatment. The specific operation is as follows:

[0069] Place 40 μg of antibody 5C4 solution into a 1.5 mL centrifuge tube (EP tube). Add 0.8 μg of IdeS enzyme at an enzyme-to-antibody mass ratio of 1:50. Gently pipette to mix, then centrifuge at low speed for 3-5 seconds to allow all droplets adhering to the tube wall to settle to the bottom. Incubate the centrifuge tube in a 37°C metal bath for 2 hours. The IdeS protease specifically cleaves the peptide bonds in the hinge region of the antibody heavy chain, digesting the intact antibody into two independent protein fragments: the Fc fragment and the Fab fragment. After incubation, transfer the centrifuge tube to a 60°C water bath and heat for 10 minutes. Heat denaturation destroys the spatial structure of the IdeS protease and inactivates it, thus terminating the enzymatic digestion reaction. The enzyme-digested antibody 5C4 solution was divided into two equal portions, each containing 20 μg of antibody 5C4 solution. The portions were transferred to two ultrafiltration centrifuge tubes with a molecular weight cutoff of 10 kDa and centrifuged at 4 °C and 11,000 rpm for 10 min, and the filtrate was discarded. 150 µL of 6 M guanidine hydrochloride solution was added to each of the two ultrafiltration centrifuge tubes and mixed well to allow the higher-order structure of the antibody 5C4 to unfold, completing the denaturation treatment and obtaining the denatured protein.

[0070] Step S2: The higher-order structure of the denatured protein is destroyed using a reducing agent and an alkylating agent, and a phosphorylation labeling agent is added to carry out a labeling reaction. A corresponding characteristic mass shift is introduced at the modified site of the denatured protein to obtain a labeled protein.

[0071] The specific procedure is as follows: Add 15 µL of 10× dithiothreitol (DTT) solution to an ultrafiltration centrifuge tube containing denatured protein, and vortex continuously at 37°C for 2 hours to reduce the disulfide bonds in the denatured protein. This reduction process is achieved through a thiol-disulfide bond exchange reaction: the free thiol groups of DTT molecules first attack the disulfide bonds between cysteine ​​residues in the protein, forming a mixed disulfide bond intermediate state, which completely reduces the disulfide bonds of the denatured protein to free thiol groups (-SH), while DTT itself is oxidized to a stable cyclic disulfide compound; continuous vortexing, combined with the denaturing effect of guanidine hydrochloride, promotes the full unfolding of the higher-order structure of the denatured protein, allowing DTT to contact the disulfide bond sites inside the protein, achieving complete reduction, and further exposing the free amino groups on the peptide chain.

[0072] After the reaction system cooled to room temperature, 15 µL of 10× iodoacetamide (IAA) alkylating agent was added, and the reaction was carried out for 15 min at room temperature in the dark to block the free thiol groups on the denatured protein and inhibit the reformation of disulfide bonds.

[0073] The alkylation process is a specific nucleophilic substitution reaction: the active iodine group of the IAA alkylating agent binds directionally to the free thiol groups on the denatured protein, converting the thiol groups of cysteine ​​residues into stable carbamoyl methyl thioether structures, irreversibly blocking all free thiol groups, preventing them from being re-oxidized to form disulfide bonds during washing, labeling, and enzymatic digestion, continuously maintaining the unfolded state of the denatured protein, and ensuring the accessibility of the free amino group; the light-protected operation can prevent the IAA from decomposing in light and producing byproducts, reducing non-specific modification of the free amino group, and ensuring the specificity of the iSIPL labeling reaction.

[0074] DTT has a low redox potential, which allows it to efficiently reduce sterically hindered disulfide bonds within protein molecules. The reaction ultimately produces a stable six-membered cyclic oxidation product, ensuring complete reduction of protein disulfide bonds and preventing spontaneous renaturation. When used with guanidine hydrochloride solution, it can fully open the higher-order structure of proteins. Furthermore, DTT has extremely low volatility and no strong irritation, making it far superior to the traditional reducing agent β-mercaptoethanol. During high-speed centrifugation, it will not cause a decrease in concentration due to volatilization, nor will it cause system contamination, resulting in higher operational safety and stability. DTT can still maintain stable reducing activity in denaturing systems with high concentrations of guanidine hydrochloride. It does not conflict with alkylating reagents such as IAA, buffers such as triethylammonium bicarbonate (TEAB), and iSIPL labeling systems, and will not interfere with the covalent labeling reaction of free amino groups. It can exert optimal activity in a neutral environment at 37°C without causing protein peptide bond breakage or excessive degradation. While opening the higher-order structure of the target protein, it maintains the integrity of the target protein skeleton, providing the prerequisite for enzymatic digestion of the target protein and sequence identification.

[0075] Under neutral pH conditions, the IAA reagent preferentially reacts with thiol groups, exhibiting minimal non-specific modification of other groups such as amino and hydroxyl groups. It does not prematurely consume free amino groups in proteins, maximizing the preservation of iSIPL-labeled reaction sites and ensuring labeling efficiency and specificity. The carbamoyl methylation product generated from the reaction of thiol groups with iodoacetamide is chemically stable and will not detach or undergo group transfer during subsequent washing, enzymatic digestion, liquid chromatography separation, and mass spectrometry detection, ensuring sample consistency. Complete blocking can be achieved in 15 minutes at room temperature, demonstrating rapid reaction speed and a simple experimental procedure, eliminating the need for prolonged incubation and effectively shortening the overall experimental cycle. The +57.0215 Da mass shift corresponding to IAA reagent modification is a standard fixed modification in proteomics mass spectrometry searches, with a mature data analysis system that will not interfere with the identification results of N-terminal peptides.

[0076] After the alkylation reaction was completed, the mixture was centrifuged at 11,000 rpm for 10 min and the lower layer of reaction solution was discarded. Then, 200 µL of 50 mM TEAB buffer was added to each tube and the tubes were washed twice under the same centrifugation conditions to thoroughly remove residual guanidine hydrochloride, reducing agent and alkylating reagent.

[0077] After washing, 50 µL of 50 mM TEAB buffer was added to each ultrafiltration tube to resuspend the denatured protein trapped on the ultrafiltration membrane, followed by 50 µL of iSIPL acetonitrile solution with a concentration of 8 μg / µL. The tube was then vortexed at room temperature for 30 min to perform a covalent labeling reaction, so that the iSIPL tag was bound to the free amino group of the denatured protein and a characteristic mass shift of +264.0875 Da was introduced at the modified site.

[0078] After the labeling reaction was completed, 4 µL of 11% hydroxylamine solution was added to each tube, mixed well, and allowed to stand at room temperature for 5 min to terminate the labeling reaction. The hydroxylamine solution rapidly quenched the active NHS ester groups of the remaining free iSIPL reagent in the system through nucleophilic substitution, converting them into isohydroxamic acid derivatives without amino reactivity, thus avoiding non-specific modification of peptide amino groups by residual reagents in the enzymatic digestion step. At the same time, the hydroxylamine solution can selectively hydrolyze the non-specific O-ester bond byproducts formed by the side chain hydroxyl groups of serine and threonine residues of proteins with iSIPL, retaining only the stable amide bond modification formed by the amino group with iSIPL, further improving the site specificity of the labeling reaction.

[0079] After the quenching reaction was completed, the sample was centrifuged at 11,000 rpm for 10 min, and then washed twice with 200 µL of 50 mM TEAB buffer, centrifuged at 11,000 rpm for 10 min each time, to remove unreacted iSIPL reagent and reaction byproducts, and to obtain a stable labeled protein sample.

[0080] Step S3: The labeled protein is digested with protease 2 to generate a peptide mixture, and the peptide mixture is identified and analyzed by liquid chromatography to obtain a secondary mass spectrum.

[0081] The specific procedure is as follows: Replace with a new collection tube, and add 100 µL of proteinase 2 dissolved in 50 mM MTEAB buffer (1.5 μg trypsin and 1.5 μg chymotrypsin) to each of the two ultrafiltration tubes containing the labeled protein. Gently mix by pipetting, and incubate at 37°C for 2 hours. During incubation, the two proteases recognize specific amino acid sites on the denatured and unfolded protein peptide chains, directionally hydrolyze the corresponding peptide bonds, and progressively cleave the intact labeled protein into short peptide fragments of suitable length, forming a peptide mixture.

[0082] After the enzymatic hydrolysis reaction is completed, the mixture is centrifuged at 11,000 rpm for 10 minutes. Taking advantage of the molecular weight cutoff characteristics of the ultrafiltration membrane, the small peptide fragments produced by the enzymatic hydrolysis pass through the ultrafiltration membrane into the lower collection tube, while the large protein fragments that are not completely hydrolyzed are retained on the membrane. The lower filtrate is collected as the enzymatic hydrolysate. The two sets of enzymatic hydrolysates are combined and then vacuum centrifuged and dried to obtain a dry peptide mixture.

[0083] This embodiment employs a parallel enzymatic digestion strategy using trypsin and chymotrypsin, leveraging the complementary cleavage specificities of the two proteases to enhance sequence coverage of the N-terminal region of the target protein: trypsin cleaves the carboxyl termini of lysine and arginine, while chymotrypsin primarily cleaves the carboxyl termini of aromatic amino acids and leucine. When there is a lack of trypsin cleavage sites near the N-terminus of the labeled protein, chymotrypsin can provide an effective supplement.

[0084] The dried peptide mixture was redissolved in an aqueous solution containing 0.1% formic acid, bottled, and loaded for online liquid chromatography-tandem mass spectrometry (LC-MS) separation and detection. The sample was loaded onto a nanoViper C18 pre-column and washed with 20 µL for desalting. Using an Easyn LC1200 nano-liquid chromatography system, the sample was desalted via the pre-column and then subjected to gradient separation on a C18 reversed-phase analytical column. Mobile phase B consisted of 80% acetonitrile aqueous solution containing 0.1% formic acid, and linear gradient elution was performed for 60 min, with the volume percentage of mobile phase B increasing from 5% to 38%.

[0085] Mass spectrometry was performed using information-dependent acquisition (DDA) mode. The primary mass spectrometry scan resolution was 60,000 m / s, with a scan range of 350–1250 m / s and a maximum injection time of 100 ms. Each DDA cycle lasted 3 s. Precursor ions with charge states of 2+ to 5+ were selected for secondary fragmentation, with a maximum injection time of 50 ms for secondary mass spectrometry. The collisional dissociation method was high-energy collision-induced dissociation (HCD), with a collision energy of 28 eV and a dynamic exclusion time of 60 s. The spray voltage was 2.2 kV, and the ion transmission tube heating temperature was 250 °C. Secondary mass spectra of the peptide mixture were acquired.

[0086] Step S4: Analyze the secondary mass spectrum to identify the N-terminal peptide in the peptide mixture and determine the N-terminal sequence information of the target protein.

[0087] The specific operation is as follows: The raw mass spectrometry data obtained is analyzed using PEAKSStudio8.5 software to extract the parent ion mass and secondary fragment ion information corresponding to each peptide segment.

[0088] First, based on the +264.0875 Da characteristic mass shift corresponding to iSIPL modification, all peptides carrying iSIPL modification were screened from all collected peptides as candidate peptides. The candidate peptides cover all peptides that have undergone iSIPL covalent modification, including peptides with modified N-terminal α-amino groups and peptides with modified ε-amino groups on the internal lysine side chains.

[0089] The fragment ion information of all candidate peptides was compared and matched with the known amino acid sequence database of antibody 5C4 to determine the amino acid sequence of each candidate peptide and its corresponding target protein sequence.

[0090] The search parameters were set as follows: trypsin / chymotrypsin digestion, mass tolerance of 10 ppm for primary mass spectrometry, mass tolerance of 0.03 Da for secondary mass spectrometry, and a maximum number of missed cleavage sites of 4; the fixed modification was cysteine ​​iodoacetamide; the variable modifications were protein N-terminal acetylation, asparagine / glutamine deamination, methionine oxidation, glutamate pyroglutamylation, glutamine pyroglutamyl cyclization, and iSIPL-modified lysine residues, i.e., protein N-terminus.

[0091] After the search is completed, the identification results are screened: based on a characteristic mass shift of +264.0875 Da, all candidate peptides modified with iSIPL are screened from the peptide mixture; after comparing and matching the fragment ion information of the candidate peptides with the database sequences, if the peptide originates at the N-terminus of the protein sequence and the modification site is located at the N-terminal α-amino group of the peptide, then the peptide is determined to be the N-terminal peptide of the target protein, and thus the N-terminal sequence information of the target protein is obtained. This rule can accurately distinguish between N-terminal specific markers and lysine side chain markers, effectively eliminating interference from internal peptides.

[0092] The search results show that the method described in this embodiment can successfully identify the N-terminal peptides of the Fc segment of the heavy chain and the Fab segment of the light chain of antibody 5C4. The N-terminal peptide GPSVFIFPPKPK was obtained by trypsin digestion of the heavy chain Fc segment, and the N-terminal peptide GPSVF was obtained by chymotrypsin digestion. Both peptides have iSIPL modification at their N-terminus. The amino acid sequence coverage results corresponding to this embodiment are shown in [link to example]. Figure 1 , Figure 2 The secondary mass spectrum of the N-terminal peptide is shown in [reference needed]. Figure 3 , Figure 4 .

[0093] Example 2 differs from Example 1 in that an in-gel pretreatment method is used for sample preparation, and the sample to be tested is antibody 11F8.

[0094] Take 40 μg of antibody 11F8 solution and place it in a 1.5 mL EP tube. Add 0.8 μg of Ides enzyme at an enzyme to antibody mass ratio of 1:50. After mixing, incubate at a constant temperature of 37 °C for 2 h to carry out the enzymatic digestion reaction. Then heat the mixture of antibody 11F8 and Ides enzyme at a heating temperature of 60 °C for 10 min to terminate the reaction through the principle of thermal denaturation. Antibody 11F8, after enzymatic digestion and heat treatment, was mixed thoroughly with non-reducing loading buffer containing bromophenol blue indicator. The mixture was centrifuged at 11,000 rpm for 10 min to remove any insoluble antibody 11F8 aggregates. The supernatant was then thoroughly mixed with the non-reducing loading buffer and loaded into a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) in two lanes. Electrophoresis was performed at a constant voltage of 80 V for 90 min. During electrophoresis, the bromophenol blue indicator migrated towards the positive electrode along with the antibody 11F8 sample, remaining at the leading edge to visually indicate the electrophoresis progress. Electrophoresis was stopped when the bromophenol blue indicator reached near the bottom of the gel, separating the enzyme-digested antibody 11F8 fractions. After electrophoresis, Coomassie Brilliant Blue staining solution was added and stained at 37°C for 30 min. The staining was then decolorized until the antibody 11F8 protein bands were clear. The target Fab and Fc fragments were cut into gel strips, placed in 2 mL centrifuge tubes, and ground with steel balls to obtain protein gel blocks.

[0095] Add 500µL of Coomassie Brilliant Blue decolorizing solution to the centrifuge tube and decolorize at room temperature for 4 hours to remove the Coomassie Brilliant Blue dye bound in the gel. Continue decolorizing until the blue color fades and the gel becomes milky white and translucent. Centrifuge to remove the supernatant of the decolorizing solution. Then, wash the protein gel with ultrapure water and acetonitrile alternately, adding 500µL of the corresponding solution each time. Vortex for 5 minutes, then centrifuge at 8000 rpm to remove the supernatant. Repeat the alternating washing 2-3 times to thoroughly remove residual decolorizing solution components, salts, and soluble impurities from the gel. Acetonitrile also causes the gel to dehydrate and shrink, improving the penetration efficiency of subsequent reduction, alkylation, and labeling reagents. Add 150 µL of 1× dithiothreitol (DTT) solution to the protein gel block and reduce it in a 56°C water bath for 30 min. After centrifugation to remove the supernatant, add 150 µL of 2× iodoacetamide (IAA) solution and react at room temperature in the dark for 15 min to carry out the alkylation reaction. After centrifugation to remove the supernatant, elute twice again with ultrapure water and acetonitrile alternately to remove residual reducing agent and alkylating agent.

[0096] Add 50 µL of LTEAB buffer and 50 µL of acetonitrile solution containing 200 µL of iSIPL reagent to the protein gel block. Vortex at room temperature for 30 min to carry out the labeling reaction, so that the iSIPL reagent can fully covalently bind to the free amino groups of the protein in the gel. Add 4 µL of 11% hydroxylamine solution and let stand at room temperature for 5 min to quench the reaction. Hydroxylamine hydrolyzes unreacted NHS ester groups in the gel through nucleophilic interaction and removes nonspecific O-ester bond byproducts. After quenching, elute three times with ultrapure water and acetonitrile alternately to remove unreacted labeling reagent, reaction byproducts and residual hydroxylamine solution.

[0097] Add 50 µL of 25 mM ammonium bicarbonate buffer to the protein gel block. Divide the Fab and Fc segments into two groups, and add 1 µg of trypsin and 1 µg of chymotrypsin to each group, respectively. Perform intragel enzymatic hydrolysis overnight at 37°C. During the hydrolysis process, the proteases diffuse into the pores of the gel block, recognize and cleave the denatured proteins fixed in the gel, and hydrolyze them into short peptides. After the hydrolysis is completed, centrifuge and transfer the supernatant to a new EP tube. Then add 100 µL of peptide extraction solution to the protein gel block, and use sonication to release the residual peptides in the gel. Centrifuge and collect the supernatant, combine it with the previously obtained supernatant, and vacuum centrifuge to dry, obtaining a dried peptide mixture.

[0098] The dried peptide mixture was analyzed using the same liquid chromatography and mass spectrometry detection conditions and data retrieval parameters as in Example 1. During the analysis, all candidate peptides carrying iSIPL modification were first screened based on the characteristic mass shift of +264.0875 Da, and then compared and matched with the known amino acid sequence database of antibody 11F8. Finally, the N-terminal peptide was determined by the location of the modification site.

[0099] The search results show that the method of this embodiment can successfully identify the N-terminal peptides of the Fc and Fab segments of the heavy chain of antibody 11F8. The Fc segment of the heavy chain was digested with trypsin to obtain the N-terminal peptide GPSVFIFPPK, and digested with chymotrypsin to obtain the N-terminal peptide GPSVF. Both peptides have iSIPL modification at the N-terminus. The N-terminus of the Fab segment of the heavy chain has no natural blocking modification and can be effectively labeled and identified by iSIPL. The corresponding SDS-PAGE electrophoresis results of this embodiment are shown in [Figure number missing]. Figure 6 The amino acid sequence coverage results are shown in [link to relevant documentation]. Figures 7-12 The secondary mass spectrum of the N-terminal peptide is shown in [reference needed]. Figure 13 , Figure 14 .

[0100] The results show that the method of the present invention is suitable for both solution-state proteins and intracolloidal proteins, and can flexibly select the pretreatment scheme according to the sample type, thus having good versatility.

[0101] Verification of beneficial effects:

[0102] (1) High detection sensitivity: Due to the high proton affinity of the phosphoryl group in the phosphorylated labeling reagent iSIPL, the mass spectrometry response signal of the N-terminal peptide can be increased by more than one order of magnitude, and the N-terminal peptide of low-abundance protein can also be effectively detected, which is significantly better than the label-free direct detection scheme.

[0103] (2) High accuracy of identification: By determining the binding site position of the characteristic mass shift of +264.0875Da, the N-terminal peptide segment and the internal peptide segment can be accurately distinguished, which greatly reduces the false positive rate and the identification results are stable and reliable.

[0104] (3) Wide sample compatibility: It supports both FASP ultrafiltration and intragel enzymatic digestion as pretreatment methods, and can be adapted to various sample types such as purified proteins, cell lysates, and tissue extracts. It also has good applicability to membrane proteins and trace samples.

[0105] (4) High degree of standardization of operation: The parameters of the whole process are controllable, the reaction conditions are mild, and the reagents and instruments are all routine configurations for proteomics, which can be easily automated and processed in high throughput, greatly improving the efficiency of analysis;

[0106] (5) Wide range of applicable scenarios: It can be applied to multiple fields such as antibody drug structure confirmation, recombinant protein quality control, N-terminal proteomics analysis, and biomarker discovery, providing efficient and accurate technical solutions for protein N-terminal research.

[0107] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0108] It should be understood that the sequence number of each step in the above embodiments does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0109] The above-described embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application, and should all be included within the protection scope of this application.

Claims

1. The application of a phosphorylation labeling reagent iSIPL in the identification of the N-terminal sequence of proteins, characterized in that, Includes the following steps: The target protein is pretreated to obtain a sample protein, and the sample protein is then denatured to obtain a denatured protein. The higher-order structure of the denatured protein is destroyed by using a reducing agent and an alkylating agent, and a phosphorylation labeling agent is added to carry out a labeling reaction. The corresponding characteristic mass shift is introduced at the modified site of the denatured protein to obtain the labeled protein. The labeled protein was digested using proteinase 2 to generate a peptide mixture, and the peptide mixture was identified and analyzed by liquid chromatography to obtain a secondary mass spectrum. The secondary mass spectra are analyzed to identify the N-terminal peptides in the peptide mixture and to determine the N-terminal sequence information of the target protein.

2. The application of the phosphorylation labeling reagent iSIPL according to claim 1 in the identification of the N-terminal sequence of proteins, characterized in that, The steps of obtaining a sample protein by preprocessing the target protein and then denaturing the sample protein to obtain a denatured protein include: Take an appropriate amount of the target protein into a centrifuge tube, add the corresponding protein 1 at a mass ratio of 1:50, mix well, incubate at 37℃ for 2 hours and perform enzyme digestion to obtain the sample protein. The sample protein was placed in an ultrafiltration centrifuge tube and centrifuged for 10 minutes. After centrifugation, guanidine hydrochloride was added to denature the sample protein, resulting in denatured protein.

3. The application of the phosphorylation labeling reagent iSIPL according to claim 1 in the identification of the N-terminal sequence of proteins, characterized in that, The denatured protein's higher-order structure was disrupted using a reducing agent and an alkylating agent, and a phosphorylation labeling reagent, iSIPL, was added to perform a labeling reaction to obtain a labeled protein, including: The disulfide bonds in the denatured protein were destroyed by adding a dithiothreitol solution to the protein and vortexing it at a constant temperature of 37°C for 2 hours. After cooling to room temperature, add iodoacetamide alkylating agent and carry out alkylation reaction for 15 min under light-protected conditions to block free thiol groups and obtain denatured protein with exposed free amino groups; Centrifuge at 11,000 rpm for 10 min to remove the reaction solution, including the dithiothreitol solution and the iodoacetamide alkylating reagent, and retain the denatured protein with exposed free amino groups. Then, centrifuge and wash the denatured protein with exposed free amino groups retained by the ultrafiltration membrane with 200 µL of 50 mM triethyl ammonium bicarbonate. Repeat the washing twice. The denatured protein retained on the ultrafiltration membrane was resuspended in triethylammonium bicarbonate buffer, and then phosphorylation labeling reagent was added. The mixture was vortexed at room temperature for 30 min to label the exposed free amino groups in the denatured protein. After the free amino groups had fully reacted with the phosphorylation labeling reagent, hydroxylamine solution was added to terminate the labeling reaction, and the labeled protein was obtained.

4. The application of the phosphorylation labeling reagent iSIPL according to claim 1 in the identification of the N-terminal sequence of proteins, characterized in that, The labeled protein was digested using proteinase 2 to generate a peptide mixture, which was then identified and analyzed by liquid chromatography to obtain a secondary mass spectrum, including: Add proteinase 2 to the labeled protein and hydrolyze it at a constant temperature of 37°C for 2 hours to obtain the hydrolysate; Centrifuge at 11,000 rpm for 10 min. After centrifugation, collect the enzymatic hydrolysate and dry it under vacuum to obtain a peptide mixture. The peptide mixture was dissolved in Nano-LC mobile phase A, bottled, and loaded onto a liquid chromatography-tandem mass spectrometry (LC-MS) system. The peptides were fragmented by high-energy collision-induced dissociation. The full scan signal of the primary mass spectrometer and the secondary fragment signal of the peptides were acquired sequentially to obtain the secondary mass spectrum of the peptide mixture. Wherein, mobile phase A is an aqueous solution containing formic acid, and mobile phase B is an aqueous solution of acetonitrile containing formic acid.

5. The application of the phosphorylation labeling reagent iSIPL according to claim 1 in the identification of the N-terminal sequence of proteins, characterized in that, Analyzing the secondary mass spectra to identify the N-terminal peptides in the peptide mixture and determining the N-terminal sequence information of the target protein includes the following steps: The secondary mass spectrum of the peptide mixture was analyzed to extract the parent ion mass and fragment ion information corresponding to each peptide. Based on the aforementioned characteristic quality shift, all candidate peptides modified with iSIPL were screened out; The fragment ion information of all the candidate peptides is compared and matched with a protein sequence database to obtain the amino acid sequence of the candidate peptides and the target protein sequence. When the modification site of iSIPL is located at the N-terminus of the candidate peptide, the corresponding candidate peptide is determined to be the N-terminal peptide of the target protein, and the N-terminal sequence information of the target protein is obtained.

6. The application of the phosphorylation labeling reagent iSIPL according to claim 2 in the identification of the N-terminal sequence of proteins, characterized in that, The target protein can also be pretreated for N-terminal sequence identification using an in-gel pretreatment method, including the following steps: Proteinase 1 was added to the target protein for enzymatic digestion to obtain the sample protein, and the sample protein was mixed with non-reducing loading buffer. Centrifuge at 11,000 rpm for 10 min, take the supernatant for sodium dodecyl sulfate and polyacrylamide gel electrophoresis to separate the protein components of the sample, and perform staining and destaining treatment to obtain the target protein strip; The target protein strips are cut and broken to obtain protein gel blocks, which are then decolorized and alternately washed again. A reducing agent is added to break the protein disulfide bonds in the protein gel block, and then an alkylating agent is added to block the protein thiol groups in the protein gel block; A phosphorylation labeling reagent is added to the protein gel block to label the proteins in the protein gel block. After the reaction is complete, a hydroxylamine solution is added to terminate the labeling reaction. Residual reagents were washed away, and protease 2 was added to the protein gel for in-gel enzymatic hydrolysis. The resulting peptide mixture was extracted and collected, and then dried by vacuum centrifugation to obtain a dried peptide mixture for mass spectrometry analysis.

7. The application of the phosphorylation labeling reagent iSIPL according to claim 3 in the identification of the N-terminal sequence of proteins, characterized in that, The hydroxylamine solution is used to hydrolyze unreacted NHS ester groups; The hydroxylamine solution has a mass fraction of 5% to 20%, and the hydrolysis of the NHS ester group is most effective when the mass fraction of the hydroxylamine solution is 11%.

8. The application of the phosphorylation labeling reagent iSIPL according to claim 1 in the identification of the N-terminal sequence of proteins, characterized in that, The protease 2 is trypsin / chymotrypsin.

9. The application of the phosphorylation labeling reagent iSIPL according to claim 1 in the identification of the N-terminal sequence of proteins, characterized in that, The phosphorylation labeling reagent iSIPL comprises: a phosphorimide reporter ion group, a balancing group, and an NHS ester reaction group.

10. The application of the phosphorylation labeling reagent iSIPL according to claim 2 in the identification of the N-terminal sequence of proteins, characterized in that, The protease 1 is the IdeS enzyme.

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