CN115372513A

CN115372513A - Differential alkylation identification method of disulfide bonds in glycoprotein

Info

Publication number: CN115372513A
Application number: CN202210997212.6A
Authority: CN
Inventors: 杨嘉明; 邓钦培; 邓巧春; 谢佳倩; 容宜欣; 关欣; 路勇
Original assignee: Livzon Mabpharm Inc
Current assignee: Livzon Mabpharm Inc
Priority date: 2022-08-19
Filing date: 2022-08-19
Publication date: 2022-11-22
Anticipated expiration: 2042-08-19
Also published as: CN115372513B

Abstract

The invention relates to a differential alkylation identification method of disulfide bonds in glycoprotein. The process of the present invention comprises the use of a differential alkylation process comprising the steps of: (1) sample preparation: taking a glycoprotein sample to be detected for denaturation treatment; (2) Reducing with tris (2-carbonyl ethyl) phosphate solution, and reacting with N-ethyl maleimide solution; (3) Desalting, denaturing, enzyme cutting to eliminate sugar modification and alcohol precipitating; (4) Dissolving the guanidine hydrochloride into the precipitate, reducing the precipitate with dithiothreitol solution, reacting with iodoacetamide solution, and performing UPLC-MS analysis. The method provided by the invention can be used for accurately, simply and efficiently identifying the protein disulfide bond sites.

Description

Differential alkylation identification method of disulfide bonds in glycoprotein

Technical Field

The invention relates to the field of medicines, in particular to a differential alkylation identification method for disulfide bonds in glycoprotein.

Background

Human Chorionic Gonadotropin (HCG), which is a non-covalent combination of alpha and beta subunits. Two subunits each contain a cluster of cysteine-knot (Cystine-knots, the main mode is Cys-X-Gly-X-Cys). This also increases the complexity of disulfide bond identification.

At present, the multi-wavelength anomalous scattering Method (MAD) and the differential alkylation method (Stepwise reduction) are mainly used for disulfide bond analysis. The theoretical disulfide-bond linkage pattern of hCG retrieved from NCBI databases was obtained by crystal diffraction methods, in which the MAD method requires excision of sugar modifications and appropriate reduction and modification of the product to obtain the desired crystals, and determination of protein conformation, which is complicated in procedure and requires a large amount of samples. In addition to this method, there is also a method for studying the disulfide bond pairing of proteins, which is a conventional differential alkylation method, also called partial reduction method, in which proteins are first partially reduced with a reducing agent, and then opened cysteines are blocked with an alkylating agent or a cyanating agent, etc. Then, the reduction condition is strengthened, and the subsequently opened disulfide bond is modified by a labeling reagent which can be distinguished from the former. For example, mise et al (Journal of Biological Chemistry,1980,255 (18): 8516-8522, journal of Biological Chemistry,1981,256 (13): 6587-6592): the protein was first partially reduced with DTT and uhCG was alkylated with 14C-labelled iodoacetic acid. After this time, complete reduction was carried out and the opened disulfide bond was alkylated with unlabeled iodoacetic acid. And (3) obtaining peptide fragment information through edman degradation, counting the isotopic labeling content of each cysteine position, and taking cysteine with consistent labeling content as a pair of disulfide bonds. The authors identified all disulfide bond pairs in the uhCG molecule by this method, but the identification was different from later hCG crystal structure study data (Nature, 1994,369 (6480), 455-461), such as the C10-C32, C28-C60 disulfide bonds contained in the alpha-subunit reported by Mise et al, and crystal structure studies showed that the two disulfide bonds should be linked: C10-C60, C32-C28. By examining and optimizing the method, the later people find that the reduction of the protein under the environment with the pH value more than 5 can cause the rearrangement of disulfide bonds. This may explain why the hCG disulphide bond identified by Mise et al, above, does not match the crystal results.

The complexity of disulfide bond identification is seen. Therefore, it is necessary to provide a method for identifying the disulfide bond sites of proteins accurately, simply and efficiently.

Disclosure of Invention

Based on the above, the invention aims to provide a method for accurately, simply and efficiently identifying protein disulfide bond sites.

The specific scheme is as follows:

a method for identifying disulfide bonds in a glycoprotein comprising using a differential alkylation process comprising the steps of:

(1) Sample preparation: taking a glycoprotein sample to be detected for denaturation treatment;

(2) Reducing with tris (2-carbonyl ethyl) phosphate solution, and reacting with N-ethyl maleimide solution;

(3) Desalting, denaturing, enzyme cutting to eliminate sugar modification and alcohol precipitating;

(4) Dissolving the guanidine hydrochloride into the precipitate, reducing the precipitate with dithiothreitol solution, reacting the reduced precipitate with iodoacetamide solution, and performing UPLC-MS analysis;

the reagent used for the denaturation treatment comprises a guanidine hydrochloride solution having a pH of (3. + -. 0.5).

In some of these embodiments, the glycoprotein is human chorionic gonadotropin (hCG).

In some embodiments, in step (1), the concentration of the guanidine hydrochloride solution is 1-10mol/L.

In some embodiments, in step (1), the glycoprotein sample to be tested is concentrated and then denatured; further, the concentration is performed by using an ultrafiltration centrifugal tube.

In some of these embodiments, the concentration of the tris (2-carbonylethyl) phosphonium hydrochloride (i.e., TCEP) solution of step (2) is from 0.01 to 1mol/L; the dosage ratio of the glycoprotein sample to be detected to the tris (2-carbonylethyl) phosphate is 0.2mg: (0.001-0.005) mmol.

In some embodiments, the reduction time in step (2) is 5-65min.

In some embodiments, the concentration of the N-ethylmaleimide (i.e., NEM) solution in step (2) is 0.2-0.3mol/L, and the ratio of the amount of the glycoprotein sample to be tested to the amount of the N-ethylmaleimide is 0.2mg: (0.001-0.01) mmol.

In some embodiments, the N-ethylmaleimide solution in step (2) is reacted for 20-40min at room temperature in a dark environment.

In some embodiments, the desalting of step (3) is desalting with a desalting column; the denaturation in step (3) comprises adding a denaturing lysis buffer for denaturation.

In some embodiments, the reagents used for sugar modification on the protein cleaved by the enzyme of step (3) comprise: NP40, glycob buffer2 and PNGaseF enzymes; the time for the enzyme to remove the sugar modification on the protein in the step (3) is 10-20 hours.

In some embodiments, the alcohol precipitation of step (3) comprises: precipitating with precooled ethanol, standing at (-20 + -5) deg.C, and centrifuging to obtain precipitate.

In some embodiments, the conditions for reducing the dithiothreitol solution of step (4) comprise: reducing at 30-60 deg.C for 30-60min; the concentration of the dithiothreitol solution is 1mol/L, and the dosage ratio of the glycoprotein sample to be detected to the dithiothreitol is as follows: 0.2mg: (0.002-0.006) mmol.

In some embodiments, the concentration of the iodoacetamide solution in step (4) is 1mol/L, and the dosage ratio of the glycoprotein sample to the iodoacetamide is: 0.2mg: (0.005-0.02) mmol.

In some embodiments, the conditions for reacting the iodoacetamide solution of step (4) comprise: and reacting for 10-40min at room temperature in a dark place.

In some embodiments, after the reaction with the iodoacetamide solution in step (4), the reaction product is desalted, treated with urea, and digested with trypsin, and subjected to UPLC-MS analysis after the reaction is terminated.

In some embodiments, in the UPLC-MS analysis of step (4), the mobile phase of UPLC is: the organic phase is 0.05-0.2wt% formic acid solution; the organic phase is 0.05-0.2wt% formic acid solution in acetonitrile; the elution gradient for UPLC was:

0min: the volume percentage of the organic phase is 2-4%;

0-26min: the volume percentage of the organic phase is increased from 2-4% to 8-12%;

26-30min: the volume percentage of the organic phase is increased from 8-12% to 18-22%;

30-60min: the volume percentage of the organic phase is increased from 18-22% to 28-31%;

60-70min: the volume percentage of the organic phase is increased from 28-31% to 31.5-34%;

70-72min: the volume percentage of the organic phase is increased from 31.5-34% to 98-100%;

72-75min: the volume percentage of the organic phase is 98-100%;

75-77min: the volume percentage of the organic phase is reduced from 98-100% to 1-5%;

77-80min: the volume percentage of the organic phase is 1-5%.

The invention also provides another identification method of disulfide bonds in glycoprotein, which comprises adopting a differential alkylation method and a thermophilic bacteria protease cleavage method after partial reduction of TCEP; wherein the differential alkylation process is as described above; the thermophilic protease cleavage method after partial reduction of TCEP comprises the following steps:

(S1) taking a glycoprotein sample to be detected, and performing denaturation treatment;

(S2) reducing by using a tris (2-carbonyl ethyl) phosphate solution, adding N-ethyl maleimide for reaction, and desalting;

(S3) adding deglycosylase, and measuring the protein concentration after ultrafiltration centrifugal desalination;

(S4) adding thermolysin;

(S5) UPLC-MS analysis is carried out.

In some embodiments, the reagent used in the denaturation treatment in step (S1) comprises a guanidine hydrochloride solution, the pH of the guanidine hydrochloride solution is (3 +/-0.5), and the concentration of the guanidine hydrochloride solution is 1-10mol/L.

In some embodiments, in step (S1), the glycoprotein to be tested is concentrated and then denatured; the concentration is performed by using an ultrafiltration centrifugal tube.

In some embodiments, in step (S2), the concentration of the solution of tris (2-carbonylethyl) phosphate in the solution of tris (2-carbonylethyl) phosphate is 30 to 50mM, and the conditions for reducing the solution of tris (2-carbonylethyl) phosphate comprise: reacting for 10-30min at 30-60 ℃.

In some embodiments, in the step (S2), the conditions of the N-ethylmaleimide reaction include: and reacting for 20-40min at room temperature in dark.

In some embodiments, in step (S3), the Deglycosylation enzyme is Deglycosylation mix ii; adding deglycosylase, standing at room temperature, heating to 35-40 deg.C for 20-30 hr, desalting, and determining protein concentration.

In some embodiments, in step (S4), the conditions under which thermolysin cleaves comprise: enzyme digestion is carried out for 15-20 hours at the temperature of 60-70 ℃.

In some of these embodiments, the mobile phase of UPLC in step (S5) is: the organic phase is 0.05-0.2wt% formic acid solution; the organic phase is 0.05-0.2wt% formic acid solution in acetonitrile; the elution gradient conditions for UPLC were:

0-60min: the volume percentage of the organic phase is increased from 1-5 percent to 35-40 percent;

60-63min: the volume percentage of the organic phase is increased from 35-40% to 98-100%;

63-65min: the volume percentage of the organic phase is 98-100%;

65-68min: the volume percentage of the organic phase is reduced from 98-100% to 1-5%;

68-72min: the volume percentage of the organic phase is 1-5%.

Compared with the prior art, the invention has the following beneficial effects:

the method optimizes the differential alkylation method, particularly optimizes the reduction pH condition in the sample pretreatment, reduces the probability of mismatching of the reduced disulfide bond, improves the identification accuracy, and finally identifies 4 pairs of disulfide bonds: C7-C31, C59-C87 located in the alpha subunit; at C26-C110, C93-C100 of the beta subunit.

In addition, the method of the invention also combines a differential alkylation method with a thermolysin cleavage method after partial reduction of TCEP, and the thermolysin cleavage method after partial reduction of TCEP can identify 3 pairs of disulfide bonds: C9-C57, C23-C72, C26-C110; furthermore, based on the MS results, it was identified that the β subunit of the hCG protein contains both the remaining 2 pairs of disulfide-linked peptide fragments (IC = AGYCPT = LSCQC) in the form of C34-C88, C38-C90 or C34-C90, C38-C88. Finally, the optimized differential alkylation method is combined with the thermophilic bacteria protease cleavage method and the MS method after partial reduction of TCEP, the two methods are complementary with high efficiency, the number of the identified disulfide bonds is increased as much as possible, and 8 pairs of disulfide bonds are identified in total.

Drawings

FIG. 1 shows the theoretical linkage of disulfide bonds between the alpha and beta subunits of hCG protein; wherein A represents the theoretical connection mode of an alpha subunit disulfide bond, and B represents the theoretical connection mode of a beta subunit disulfide bond.

FIG. 2 is a schematic diagram of the differential alkylation method for determining disulfide bond pairing.

FIG. 3 shows the percentage content of each cysteine-carrying NEM modified peptide fragment in the alpha subunit of hCG protein.

FIG. 4 shows the percentage of each cysteine-carrying NEM modified peptide fragment in the beta subunit of hCG protein.

FIG. 5 shows the theoretical disulfide bond linkage of the human chorionic gonadotropin beta-subunit and the Thermolysin protease cleavage site.

FIG. 6 is a secondary map of the 1.

Detailed Description

Experimental procedures according to the invention, in which no particular conditions are specified in the following examples, are generally carried out under conventional conditions, or under conditions recommended by the manufacturer. The various chemicals used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The terms "comprises" and "comprising," as well as any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, article, or apparatus that comprises a list of steps is not limited to only those steps or modules recited, but may alternatively include other steps not recited, or may alternatively include other steps inherent to such process, method, article, or apparatus.

The "plurality" referred to in the present invention means two or more. "and/or" describes the association relationship of the associated object, indicating that there may be three relationships, for example, a and/or B, which may indicate: a exists alone, A and B exist simultaneously, and B exists alone. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.

The present invention will be described in further detail with reference to specific examples.

The reagents used in the examples of the present invention are, unless otherwise specified, conventional commercially available products or prepared by conventional methods. For example, the hCG protein of the present invention refers to human chorionic gonadotropin, without any particular limitation, and further refers to human chorionic gonadotropin which is artificially synthesized or artificially isolated, without any particular limitation, and the protein sequence thereof may be the same as or different from that of the wild type.

Example 1 identification of disulfide bonds in hCG protein by differential alkylation

1.1 sample preparation

A sample of hCG protein (V20180601) 3mg was taken, concentrated using a 3K ultrafiltration concentrator tube, and the solution was replaced into 6M guanidine hydrochloride solution at pH 3. The volume of the concentrated solution was measured by pipette and the hCG protein was approximately 1.3mL. A sample of LZM003 (86. Mu.L/tube, 15 tubes) was taken and 14. Mu.L of 6M guanidine hydrochloride solution (pH 3) was added. To each of the above samples, 10. Mu.L of TCEP (tris (2-carbonylethyl) phosphate) solution (0.2M) was added, and the mixture was reduced at 40 ℃ for 5min, 20min, 35min, 50min and 65min, respectively. Three samples were prepared in parallel for each time point. After completion, all samples were removed, and 30. Mu.L of 0.25M NEM (N-ethylmaleimide) solution was added, and the mixture was protected from light at room temperature for 30min. Desalting with desalting column (Spin G-25 column), centrifuging at 800 Xg for 2min, and replacing the sample with ultrapure water. Add 10. Mu.L of Denaturing buffer and denature in 100 ℃ water bath for 10min. After cooling, 10. Mu.L NP40 solution, 10. Mu.L Glycobuffer2, 1. Mu.L LPNGaseF enzyme were added and cleaved at 37 ℃ for 17 hours. The next day, taking out, adding cold anhydrous ethanol for alcohol precipitation, and standing in refrigerator at-20 deg.C for 30min. Centrifuge at 12,000 Xg for 5min and discard the supernatant. Standing the precipitate, and volatilizing the surface liquid. The precipitate was dissolved by adding 80. Mu.L of 6M guanidine hydrochloride solution. mu.L of 1M DTT solution was added and reduced at 50 ℃ for 45min. After completion, 12. Mu.L of 1M Iodoacetamide (IAM) solution was added, and the mixture was protected from light at room temperature for 30 minutes. Desalting was performed using a desalting column, and the sample was replaced into a 1M urea solution. mu.L of 40. Mu.L of trypsin was added and the mixture was digested at 37 ℃ for about 17 hours. The reaction was stopped by the addition of 5. Mu.L acetonitrile (containing 0.1% formic acid). Centrifuged at 12000 Xg for 5min, and the supernatant was collected and analyzed by UPLC-MS (Q-TOF).

1.2UPLC-MS conditions

The chromatographic method comprises the following steps: CSH 150mm 80min distifide

And (3) chromatographic column: waters Acquity UPLC column, CSH C18,1.7 μm, 2.1X 150mm (SEQ ID NO: 01323527115724);

mobile phase: A. water +0.1% formic acid; B. acetonitrile +0.1% formic acid;

flow rate: 0.2mL/min; sample injection amount: 5 mu L of the solution; column temperature: 50 ℃;

elution gradient:

before sample detection, the mass axis of the mass spectrum is corrected by sodium iodide (range: 100-2500 Da); and at the same time of detection, leucine enkephalin (leucine enkephalin,200 pg/mu L) is used for real-time correction. Detection mode: positive ion sensitivity mode; cone voltage (Sampling Cone): 20.0V; desolventization temperature (Desolvation temperature): 350; cone gas flow (Cone gas flow): 50.0L/Hr; desolventizing gas (Desolvation gas flow): 600.0L/Hr; scan range (Scan range): 100-2500Da; scanning time: 2s; the quadrupole rod is arranged: 300. 600 and 900.

1.3 data processing

The experimental data were processed using Biopharmalynx software from Waters. The amino acid sequence of hCG protein is a theoretical sequence derived from gene sequence. The "Peptide Mapping" mode is selected, and the method parameters are as follows: instrument Resolution: auto; lock Mass for Charge 1 (Da): 556.2771; lock Mass Tolerance:0.25; MS ion intensity threshold (Counts): 500; MSE ion intensity threshold (counts): 200 of a carrier; MS mass tolerance:20ppm; MSE mass tolerance:20ppm; missed clearages: 1for Trypsin. The optional modifications retrieved are shown in table 1 below. Software obtains an identification result by comparing the accurate mass number of the related peptide fragment with the theoretical value and the experimental value of fragment ions. And after the data are calculated and matched, the quality of the MS/MS spectrum is confirmed by a manual comparison method.

Table 1 software search for added embellishments

1.4 peptide fragment identification

After reducing the sample with 20mM TCEP at 40 ℃ for 5min, 20min, 35min, 50min, 65min, respectively, in a pH3 environment, the opened thiol group is alkylated with NEM. Thereafter, excess NEM reagent was removed by desalting and alcohol precipitation after sugar cutting. The protein was then fully reduced with DTT and alkylated by IAM. The NEM and IAM alkylation modifications differ in mass number (NEM modification increases the mass number of the peptide fragment by 125.0477Da, IAM modification increases the mass number of the peptide fragment by 57.0215 Da), and therefore, can be used to distinguish the type of alkylation modification carried by cysteine at a site.

And (3) collecting the enzymolysis peptide fragments of the sample through LC-ESI-MS, and searching and matching the enzyme digestion peptide fragments by using Biopharmalynx 1.3.4 software. The matched peptide stretches covered all cysteines in the hCG protein. The peptide fragments in which these cysteines are located are again manually identified (identifying the type and site of the alkylation modification) by binding to the secondary (b/y) ion map of each peptide fragment. The identified cysteine with partial NEM modification on the alpha subunit has C7, C31, C59, C60 and C87, and other cysteine has CAM modification; the partially NEM modified cysteines on the β subunit included C9, C26, C57, C72, C93, C100, C110, and all other cysteines were modified with CAM, as shown in table 2.

TABLE 2hCG protein partial reductive alkylation peptide fragment search

Note: camC in the "Modifiers" column is a modification of cysteine by IAM alkyl methylation (Carbamidomethyl C); deam N is a Deamidation modification of asparagine (Deamidation N); NEM is the alkylation of cysteine introduced by NEM modification, with the red cysteine underlined in the table indicating the site where the NEM modification occurred, and the suffix number indicating the position in the sequence. There are multiple off-times for some NEM modified peptide fragments because the alkylation induced by NEM action will be sterically heterogeneous, peaking at multiple retention times.

1.5 measurement of content

According to the peptide fragment identification result, the NEM modification percentage content of each cysteine site is counted: the peak areas of the peptide fragments are first summarized. The peak intensities of the NEM modified peptides are then summed for the same cysteine and then ratioed to the sum of the peak intensities of all alkylated peptides at that site. The calculation formula is shown below, and the calculation results are shown in tables 3 to 6. Theoretically, the cysteines constituting the same disulfide bond pair will have the same chance of carrying NEM alkylation modifications at the cysteines at these two sites after partial reduction by TCEP, and the percentage of NEM modified peptide fragments introduced should be substantially identical, the principle of which is shown in fig. 2, by which the disulfide bonds are paired.

The formula:

note: x represents a cysteine site and NEM represents a modification introduced by NEM alkylation; CAM indicates modification introduced by IAM alkylation; the numbers following the NEM or CAM indicate the number of the corresponding alkylated modified peptide fragment, since the same alkylation modification may introduce a steric isomer, thereby peaking at different times to form two or more peptide fragments.

It is calculated that the content of C7, C31, C59 and C87-NEM modifications in the alpha subunit of hCG protein changes with the extension of partial reduction time, and the content of C60-NEM modified peptide fragment is less, about 0-3.8%. Cysteines at other sites on the alpha subunit all carry CAM modifications, indicating that these sites are not opened during the previous partial reduction. Only the disulfide bonds consisting of C7, C31, C59 and C87 are easy to open. The statistics of the percentage of NEM modifications in the four cysteines are shown in tables 3-4, and plotted against the partial reduction time (FIG. 3), from which it can be seen that the trends of the percentage of C7 and C31-NEM modified peptides at each time point of reduction are substantially the same, and are significantly higher than the C59 and C87-NEM modifications. For example, when the hCG protein sample is reduced for 65min, the C7 and C31-NEM are about 30-50%, and the C59 and C87-NEM are about 20% or less. C7 and C31 are illustrated as a pair of disulfide bonds, and C59 and C87 constitute a pair of disulfide bonds. The identification result is consistent with the literature report (Nature, 1994,369 (6480), 455-461.; structure,1994,2 (6): 545-558.). The amount of C59 and C87-NEM modifications at each time point and between replicates varied slightly, presumably because N-sugar modifications in the peptide stretch prevented the binding of the alkylating agent to cysteine, thereby showing different alkylation levels. In addition, the C7-C31 disulfide bond is opened in the hCG protein at a significantly higher level than the other disulfide bonds with increasing reduction time, indicating that the C7-C31 disulfide bonds are located outside the molecular space of the hCG protein or are in an exposed hydrophilic region after denaturation. This indirectly suggests that the α subunits have some similarity in spatial structure.

TABLE 3 percent NEM modification of cysteines at each position in the hCG protein alpha-subunit

Note: 1. numbers in the reducing conditions represent the numbers of three parallel samples; 2. since the chromatographic peak at 51.0min is the co-outflow peak of the peptide fragments of C7-NEM and C31-NEM, the peak area of the peptide fragment is counted when the contents of C7 and C31-NEM are respectively calculated; and 3. Comparing the marked data in three parallel samples to obtain extremely low or extremely high conditions, and temporarily not determining the type of the error, wherein the statistical time is one, and the error is reflected in a trend analysis chart.

It was calculated that the amount of C26, C93, C100, C110-NEM modifications in the β -subunit of hCG protein varied with the partial reduction time, the amount of C9, C57, C72-NEM modified peptide fragments was smaller, less than 1% at most of the time points, and were difficult to be reduced and opened, and the amount was higher in individual time points, such as in individual replicates at 40 ℃ for 35min, and is not representative. In addition, C34, C38, C88 and C90 in the beta-subunit form a cysteine knot (cysteine knottt) structure, which is not opened in the partial reduction process, and the four cysteines in the search result only carry CAM modification, and no NEM modification is identified. Therefore, only the contents of 2C26, 2C93, 2C100 and 2C110-NEM are taken for analysis, the contents are plotted against partial reduction time (figure 4), and as can be seen from the figure, in the hCG protein, the contents of C93 and C100-NEM modified peptide fragments are obviously higher than those of C26 and C110, the change trends are basically consistent, and the contents are basically more than 25%. It is presumed that C93 and C100 constitute a pair of disulfide bonds. The percent NEM modification of C26 and C110 varied less with time, reaching only about 3% to 7% at 65min. Therefore, C26 and C110 are assumed to be a pair of disulfide bonds. In summary, the two pairs of C93-C100, C26-C110 disulfide bonds in the beta subunit of hCG protein are relatively open when reduced compared to the other disulfide bonds, suggesting that they may be located outside the molecular structure or exposed to the outer hydrophilic region after denaturation.

TABLE 4 percentage NEM modification of cysteines at each position in the hCG protein beta-subunit

Note: 1. numbers in reducing conditions represent three parallel samples; 2. the 2C100-NEM modification content was corrected for in the calculation. The correction reason is that the peak emergence time of 2T11 of the 2C100-CAM modified peptide fragment is about 1.6min, and more salt peaks interfere and inhibit the strength of the peptide fragment, so that the peak strength is extremely low, and the 2C100-NEM modified content calculated according to the peak emergence time is higher. In the 2T11-12 peptide fragment containing 2C100, the peptide fragments are all modified with CAM, and the content of NEM modification is equal to 0. Therefore, the percentage of 2C100-NEM in 2T11 and 2T11-12 peptides thereof was added and averaged to obtain the NEM percentage of the site; the data are compared among three parallel samples to show extremely low or high conditions, the type of error is not determined, and the data are counted and embodied in a trend analysis chart.

The invention reduces the disulfide bond in hCG protein by stages through an improved differential alkylation method, respectively uses NEM and IAM to carry out differential labeling on cysteine which is partially reduced and completely reduced, and collects the enzymolysis peptide segment of the cysteine through LC-ESI-MS, thereby obtaining the NEM modification content of partial cysteine, further quantifying the opening rate of the disulfide bond, and confirming the matching of the disulfide bond according to the consistency of the content. By this method, the disulfide bond of the α -subunit and β -subunit in the hCG protein has been confirmed. In the alpha subunit, C7 and C31 are a pair of disulfide bonds, and C59 and C87 constitute a pair of disulfide bonds; in the β subunit, C26 and C110 are a pair of disulfide bonds, and C93 and C100 are a pair of disulfide bonds. Consistent with literature reports. Other cysteines were not matched because the content of open in this partial reduction condition was very low and did not change significantly with the extension of the reduction time.

Example 2 differential alkylation method in combination with thermal cleavage after partial reduction of TCEP to identify the disulfide bond of HCG

2.1 differential alkylation Process

The same as in example 1.

2.2 enzymatic cleavage of Thermolysin after partial reduction of TCEP

2.2.1 sample treatment

Reduction and alkylation: 1mg of hCG protein (V20180601) was taken, concentrated using a 3K ultrafiltration concentrator tube, and the solution was replaced into 6M guanidine hydrochloride solution at pH3 for 3 times in total. mu.L (about 200. Mu.g protein) of the sample was added with 31. Mu.L of 6M guanidine hydrochloride solution (pH 3), mixed well, and then the reducing agent TCEP was added to a final concentration of 40mM, and the mixture was subjected to water bath at 40 ℃ for 20min, and another portion of the sample prepared in the same manner as above was reduced in water bath at 65 ℃ for 30min. After cooling, 72. Mu.L of 0.25M NEM solution was added and alkylation was carried out at room temperature for 30min in the absence of light. The solution was replaced into ultrapure water using a desalting column (Spin-G25 column, GE).

Sugar cutting: add 10 x glycosylation Mix buffer1 (P6044S, NEB) 20. Mu.L, glycosylation Mix II 20. Mu.L, and make up ultra pure water to a final volume of 200. Mu.L. After addition of the sample as described above, the mixture was left at room temperature for 30 minutes and then transferred to a 37 ℃ water bath to incubate for 24 hours. The following day, the buffer was removed and replaced with 50mM Tris-HCl solution 4 times (3K ultrafiltration concentrator tubes, 400. Mu.L Tris-HCl solution added each time) to determine protein concentration.

Enzyme digestion: mu.g of the sample was added to 50mM Tris-HCl solution to a final volume of 37.5. Mu.L, and 2.5. Mu.L of LThermolysin enzyme (cat. RTM., manufactured) was added thereto, and digested in a water bath at 65 ℃ for 17 hours.

Sampling: the reaction was stopped by the addition of 5. Mu.L acetonitrile (containing 0.1% formic acid). Centrifuged at 12000 Xg for 5min, and the supernatant was collected and analyzed by UPLC-MS (Q-TOF).

2.2.2UPLC-MS conditions

The chromatographic method comprises the following steps:

a chromatographic column: aquity UPLC CSH C18.7 μm, 2.1X 150mm (SEQ ID NO: 01323527115721)

Mobile phase: A. water +0.1% formic acid; B. acetonitrile +0.1% formic acid;

flow rate: 0.2mL/min; sample introduction amount: 2 mu L of the solution; column temperature: 40 ℃;

elution gradient:

Time(min)	0	60	63	65	68	72
							％B	3％	38％	100％	100％	3％	3％

2.2.3 data processing

The experimental data were processed using Biopharmalynx software from Waters. The amino acid sequence of hCG protein is a theoretical sequence derived from the gene sequence. The "Peptide Mapping" mode is selected, and the method parameters are as follows: instrument Resolution: auto; lock Mass for Charge 1 (Da): 556.2771; lock Mass Tolerance:0.25; MS ion intensity threshold (Counts): 100; MS (Mass Spectrometry) ^E ion intensity threshold(counts)：50；Retention Time Range:5.0-26.0min；MS mass tolerance：30ppm；MS ^E mass tolerance:30ppm; missed clearages: 2for thermolysin, thermolysin cleavage site: F. v, I, A, M, L-N terminal.

The cysteines in the sequence were selectively linked according to the theoretical disulfide linkage of the β -subunit (see figure 1for details) and were searched. Software compares the accurate mass number of the relevant disulfide bond connecting peptide segment with the theoretical value and experimental value of fragment ions to obtain an identification result. And after the data are calculated and matched, the quality of the MS/MS spectrum is confirmed by a manual comparison method.

2.2.4 results

According to the theoretical cleavage site of Thermolysin enzyme (amino acids underlined in fig. 5), the β -subunit is cleaved to form disulfide peptides. Matching was performed by software to retrieve 4 disulfide bond peptides and 1 trisulfide bond peptide, as shown in fig. 1 and table 5. The identification result is further confirmed manually, and the connection mode of C23-C72 and C26-C110 can be identified in the sample reduced at 40 ℃ for 20 min. By further enhancing the reduction conditions, samples reduced at 65 ℃ for 30min can be searched, and five disulfide bond pairing modes of C9-C57, C23-C72, C26-C110, C34-C88 or C34-C90, C38-C88 or C38-C90 can be searched (the yellow highlighted peptide segment in the figure 5 is the detected disulfide bond connecting peptide segment). Briefly described as follows: peptide fragments 1, F1-2-1 and 1, F19-21, are directed to disulfide bond pairing of C9-C57. Taking the secondary map of 1. In the same manner, we have identified all of the above-mentioned peptide fragments containing a pair of disulfide bonds. In addition, the C34-C90 and C38-C88 (or C38-C88 or C38-C90) connecting peptide fragments are two disulfide-linked peptide fragments consisting of three peptide fragments, the secondary b/y ions of the disulfide-linked peptide fragments are few, and only 1y2 ions exist, so that the specific connection mode cannot be judged through a secondary map.

TABLE 5 results of peptide fragment search of Thermolysin enzyme after 40mM TCEP reduction

TABLE 6.1F2-1F19-21 (C9-C57) peptide fragment secondary b/y ion assignment

The invention identifies C7-C31 and C59-C87 positioned in alpha subunit in hCG protein by differential alkylation method; 4 pairs of disulfide bonds at C26-C110, C93-C100 of the beta subunit; partial reduction conditions are controlled to open partial disulfide bonds on beta-subunits in the product, so that the molecular structure of the product is changed. A large amount of sugar modification on molecules is cut by sugar-cutting mixed enzyme, and then the enzymatic digestion is carried out by using thermolysin under the high-temperature condition, so as to obtain rich disulfide bond peptide sections. The enzyme digestion peptide fragment is identified by LC-MS/MS, and MS/MS confirms that the pairing of 3 pairs of disulfide bonds in the hCG protein beta subunit is consistent with theory and is respectively C9-C57, C26-C110 and C23-C72; in addition, a peptide fragment containing two disulfide bonds was identified for the beta subunit (IC = AGYCPT = LSCQC), and the indicated pairing pattern may be C34-C88, C38-C90, or C34-C90, C38-C88.

All possible combinations of the technical features of the above embodiments may not be described for the sake of brevity, but should be considered as within the scope of the present disclosure as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A method for identifying disulfide bonds in a glycoprotein comprising using a differential alkylation process comprising the steps of:

2. The method of claim 1, wherein the glycoprotein is human chorionic gonadotropin; and/or, in the step (1), the concentration of the guanidine hydrochloride solution is 1-10mol/L.

3. The method according to claim 1, wherein in step (1), the sample of the glycoprotein to be tested is concentrated and then denatured; further, the concentration is performed by using an ultrafiltration concentration tube.

4. The method according to claim 1, wherein the concentration of the tris (2-carbonylethyl) phosphate solution in the step (2) is 0.01 to 1mol/L; the dosage ratio of the tris (2-carbonylethyl) phosphate to the glycoprotein sample to be detected is (0.001-0.005) mmol:0.2mg;

and/or, the concentration of the N-ethylmaleimide solution in the step (2) is 0.2-0.3mol/L, and the dosage ratio of the glycoprotein sample to be detected to the N-ethylmaleimide is 0.2mg: (0.001-0.01) mmol.

5. The method of any one of claims 1 to 4, wherein the reagent for sugar modification on the enzyme cleavage protein of step (3) comprises: NP40, glycob buffer2 and PNGaseF enzymes; the time for the enzyme to remove the sugar modification on the protein in the step (3) is 10-20 hours.

6. The method according to any one of claims 1 to 4, wherein the concentration of the iodoacetamide solution in step (4) is 1mol/L, and the ratio of the amount of the glycoprotein sample to the iodoacetamide is: 0.2mg: (0.005-0.02) mmol.

7. A method for identifying disulfide bonds in glycoproteins is characterized by comprising a differential alkylation method and a thermophilic bacteria protease cleavage method after partial reduction of TCEP; wherein the differential alkylation process is as described in any one of claims 1 to 6; the thermophilic protease cleavage method after partial reduction of TCEP comprises the following steps:

(S2) reducing the solution by using a tris (2-carbonylethyl) phosphate solution, adding an N-ethylmaleimide solution for reaction, and desalting;

(S3) adding deglycosylase, desalting and determining the protein concentration;

(S4) adding thermolysin;

(S5) UPLC-MS analysis is carried out.

8. The method according to claim 7, wherein the reagent used in the denaturing treatment in step (S1) comprises a guanidine hydrochloride solution having a pH of (3. + -. 0.5) and a concentration of 1 to 10mol/L.

9. The method according to claim 7, wherein in the step (S2), the concentration of the tris (2-carbonylethyl) phosphate solution is 30 to 50mM, and the conditions for reducing the tris (2-carbonylethyl) phosphate solution include: reacting for 10-30min at 30-60 ℃.

10. The method according to any one of claims 7 to 9, wherein in the step (S2), the conditions for the N-ethylmaleimide reaction include: reacting at room temperature in dark for 20-40min;

and/or, in the step (S4), the conditions for performing enzyme digestion by thermolysin comprise: enzyme digestion is carried out for 15-20 hours at the temperature of 60-70 ℃.