Separation method of sperm head subcellular structure and application of sperm head subcellular structure

Abstract

The invention belongs to the technical field of biology, and particularly relates to a sperm head subcellular structure separation method and application thereof. According to the sperm head subcellular structure separation method provided by the invention, a specific ultrasonic and centrifugal treatment mode is adopted, more than 1500 proteins are identified from each component of the sperm head, the analysis reproducibility is relatively good, GO analysis shows that the proteins corresponding to the subcellular structures are also enriched to corresponding cellular components, and the separation efficiency of the sperm head subcellular structures is greatly improved. And each subcellular structure of the sperm head is effectively separated.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and specifically relates to a method for separating subcellular structures of sperm heads and applications thereof. Background Art

[0002] Sperm is composed of multiple structures such as acrosome, nucleus, flagellum, etc., among which the sperm head plays an important role in the growth and development of sperm. Among them, the substructures of the sperm head mainly include the following parts: nucleus, acrosome, implantation pit, nuclear vesicle, nuclear membrane, perinuclear sheath and nuclear plate.

[0003] The sperm head is mainly composed of the nucleus, which is a flat round structure located in the center of the head and contains genetic material; the acrosome is a flat membrane vesicle in the front of the sperm head, which can be divided into three parts: the inner acrosome membrane, the outer acrosome membrane and the septal membrane connecting the two. The acrosome contains a variety of hydrolases and glycoproteins. The implantation pit is a shallow pit on the posterior pole of the sperm nucleus, which is chimeric with the small head of the neck segment to enhance the connection between the head and the tail. The nuclear vesicle is an irregular translucent area that often exists in the dense nuclear chromatin, which may affect the fertilization ability of the sperm. The nuclear membrane is the nuclear membrane wrapped around the sperm nucleus. Most of the nuclear membranes have no nuclear pores, but there are more nuclear pores in the wrinkled area. The perinuclear sheath is a special cytoskeletal structure that tightly wraps the sperm cell nucleus. The nuclear plate is a layer of protein mesh on the inner surface of the nuclear membrane, called the nuclear plate, which forms the skeleton structure of the nuclear membrane and can fix the chromatin. These substructures work together to enable sperm to complete its main function of participating in fertilization.

[0004] However, there are no reports on the separation and proteomic analysis of the various structures in the sperm head. Summary of the invention

[0005] Based on this, an embodiment of the present application provides a method for separating the subcellular structure of a sperm head and its application.

[0006] On the one hand, the present application provides a method for separating the subcellular structure of the sperm head, comprising:

[0007] Provide sperm samples, perform ultrasonic treatment, and separate the sperm heads and tails;

[0008] The head is subjected to a first resuspension treatment and a first centrifugation treatment, and the supernatant is taken to obtain the acrosome component;

[0009] The precipitate after the first centrifugation is resuspended for the second time, and then centrifuged for the second time, the supernatant is taken, and the supernatant is centrifuged for the third time, and the supernatant is taken to obtain the perinuclear sheath component; the acrosome component and the perinuclear sheath component are the separated sperm head subcellular structures.

[0010] The parameters of the first centrifugation treatment include: temperature of 3.5°C to 4.5°C, centrifugal force of 400g to 600g, and time of 18min to 22min;

[0011] The parameters of the second centrifugation treatment include: temperature of 3.5°C to 4.5°C, centrifugal force of 1400g to 1600g, and time of 18 to 22min;

[0012] The parameters of the third centrifugal treatment include: temperature of 3.5°C to 4.5°C, rotation speed of 20000rpm to 30000rpm, and time of 25min to 35min.

[0013] In some embodiments, the solvent for the first resuspension treatment includes a 0.8M to 1.2M NaCl solution.

[0014] In some of the embodiments, the temperature of the first resuspension treatment is 3.5° C. to 4.5° C., the time of the resuspension treatment is 0.8 h to 1.2 h, and a magnetic stirrer is used for stirring during the resuspension.

[0015] In some embodiments, the conditions for the second resuspension treatment include: resuspending the precipitate after the first centrifugation treatment in 80 mM-120 mM NaOH solution.

[0016] In some embodiments, the resuspension conditions include 3.5°C to 4.5°C overnight.

[0017] On the other hand, the present application provides a method for extracting protein from the subcellular structure of the sperm head, comprising:

[0018] The sperm head subcellular structure separation method is used to separate the sperm head subcellular structure, and the protein in the sperm head subcellular structure is extracted.

[0019] In some embodiments, the method comprises: subjecting the protein to ultrasonic disruption, denaturation and reductive alkylation treatments, as well as enzymatic hydrolysis to obtain peptide fragments, and then subjecting the peptide fragments to desalting treatment.

[0020] In some of the embodiments, the protein is placed in a protein disruption solution for disruption treatment; the protein disruption solution comprises 25 mM to 100 mM TEAB buffer and dodecyl-β-D-maltoside with a final concentration of 0.08 w / v% to 0.12 w / v%.

[0021] In some of the embodiments, the sample is subjected to ultrasonic treatment at a temperature of 3.5° C. to 4.5° C., with an ultrasonic frequency of 100 W to 150 W and a time of 10 min to 20 min.

[0022] In some embodiments, the denaturing agent includes dithiothreitol at a final concentration of 80 mM to 120 mM.

[0023] In some embodiments, the temperature of the denaturation treatment is 70° C. to 80° C., and the time is 30 min to 40 min.

[0024] In some embodiments, the alkylation agent includes IAA at a final concentration of 80 mM to 120 mM.

[0025] In some embodiments, the alkylation treatment is carried out at a temperature of 20° C. to 30° C. and for a time of 20 min to 30 min.

[0026] In some embodiments, the enzymatic treatment agent includes trypsin.

[0027] In some embodiments, the enzymatic treatment time is 2 h to 16 h.

[0028] In some embodiments, the conditions for desalting treatment include: desalting the peptide using microchromatography;

[0029] In some of these embodiments, a ZipTip microchromatography column is used.

[0030] In some embodiments, the microchromatography column is washed 2 to 4 times with buffer B, and the microchromatography column is washed 2 to 4 times with buffer A.

[0031] In some embodiments, the peptide fragment is added to the microchromatographic column and then washed with buffer A for 2 to 4 times.

[0032] In some embodiments, the buffer A comprises: 0.08 v / v% to 0.12 v / v% formic acid aqueous solution.

[0033] In some embodiments, the buffer B comprises: 60 v / v% to 98 v / v% acetonitrile and 0.08 v / v% to 0.12 v / v% formic acid aqueous solution.

[0034] On the other hand, the present application provides a mass spectrometry analysis method for the subcellular structure of a sperm head, comprising: using the subcellular structure separation method of the sperm head to obtain the acrosome and the perinuclear sheath, using the protein extraction method of the subcellular structure of the sperm head to prepare a sample to be analyzed, and performing mass spectrometry analysis.

[0035] In some of the embodiments, the chromatographic gradient for peptide separation is: a linear gradient of 3% to 38% buffer B, and the time of the linear gradient is 100 min to 105 min.

[0036] In some embodiments, the equilibration is performed in 98% to 100% buffer B for 8 to 12 minutes.

[0037] In some of the embodiments, mass spectrometry analysis was performed using a mass spectrometer equipped with a FAIMS Pro interface, and the FAIMS was switched between CV values ​​of -35 V, -45 V, and -65 V, with a cycle time of 0.8 s to 2 s.

[0038] In some of these embodiments, the primary spectrum is collected using an Orbitrap and the secondary spectrum is collected using a linear ion trap or an Orbitrap.

[0039] The details of one or more embodiments of the present application are set forth in the description below, and other features, objects, and advantages of the present application will be apparent from the description and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0041] Figure 1 It is the result of identifying the components of the acrosome, perinuclear sheath and nucleus in the sperm head structure;

[0042] Figure 2 This is a heat map of the correlation between two repeated analyses of each component;

[0043] Figure 3 The cellular fractions in which the proteins identified in each fraction are enriched;

[0044] Figure 4 Schematic diagram of the separation and mass spectrometry analysis of the sperm acrosome subcellular structures. DETAILED DESCRIPTION

[0045] Below in conjunction with embodiment and example, the application is further described in detail.It should be understood that these embodiments and examples are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiments and examples is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0046] Unless otherwise defined, 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 application belongs.

[0047] the term

[0048] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0049] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0050] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0051] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0052] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0053] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0054] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0055] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0056] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0057] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0058] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0059] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0060] The term "subcellular structure" refers to the various tissues and organs inside the cell, which are displayed at different scales. These structures are the basis of cell function and include organelles, cytoskeleton, cell membrane system, etc.

[0061] The term "acrosome" is a flat sac-like structure covering the front 2 / 3 of the sperm head. It is a specialized lysosome. The acrosome contains a variety of hydrolases related to fertilization, such as hyaluronidase, acrosin, protease, phosphatase, etc. These enzymes are released during the acrosome reaction, helping sperm to pass through structures such as the zona pellucida and corona radiata on the surface of the egg cell, thereby achieving fertilization.

[0062] The term "perinuclear sheath" The perinuclear sheath (PT) is a special cytoskeletal structure surrounding the sperm nucleus. It can be structurally divided into two continuous parts: the subacrosomal plate and the postacrosomal region. The main function of the perinuclear sheath is to support and stabilize the sperm nucleus, and it is also involved in the morphological maintenance and functional regulation of sperm.

[0063] On the one hand, the present application provides a method for separating the subcellular structure of the sperm head, comprising:

[0064] Provide sperm samples, perform ultrasonic treatment, and separate the sperm heads and tails;

[0065] The head is subjected to a first re-suspension treatment and a first centrifugation treatment, and the supernatant is taken to obtain the acrosome component;

[0066] The precipitate after the first centrifugation is resuspended for the second time, and then centrifuged for the second time to obtain the supernatant, and the supernatant is centrifuged for the third time to obtain the perinuclear sheath component;

[0067] The acrosome component and the perinuclear sheath component are the separated subcellular structures of the sperm head.

[0068] The parameters of the first centrifugal treatment include: a temperature of 3.5°C to 4.5°C, a centrifugal force of 400g to 600g, and a time of 8min to 12min. For example, the temperature is 3.5°C, 3.6°C, 3.7°C, 3.8°C, 3.9°C, 4.0°C, 4.1°C, 4.2°C, 4.3°C, 4.4°C, 4.5°C, and any value therebetween. For example, the centrifugal force is 400g, 410g, 420g, 430g, 440g, 450g, 460g, 470g, 480g, 490g, 500g, 510g, 520g, 530g, 540g, 550g, 560g, 570g, 580g, 590g, or 600g, and any value therebetween. For example, the time is 18 minutes, 19 minutes, 20 minutes, 21 minutes or 22 minutes and any value in between.

[0069] The parameters of the second centrifugation treatment include: temperature of 3.5°C to 4.5°C, centrifugal force of 1400g to 1600g, and time of 18min to 12min. For example, the temperature is 3.5°C, 3.6°C, 3.7°C, 3.8°C, 3.9°C, 4.0°C, 4.1°C, 4.2°C, 4.3°C, 4.4°C, 4.5°C, and any value therebetween. For example, the centrifugal force is 1400g, 1500g, or 1600g, and any value therebetween. For example, the time is 18min, 19min, 20min, 21min, or 22min, and any value therebetween.

[0070] The parameters of the third centrifugal treatment include: temperature of 3.5°C to 4.5°C, rotation speed of 20000rpm to 30000rpm, and time of 25min to 35min. For example, 20000rpm, 21000rpm, 22000rpm, 23000rpm, 24000rpm, 25000rpm, 26000rpm, 27000rpm, 28000rpm, 29000rpm or 30000rpm and any value in between. For example, the temperature is 3.5°C, 3.6°C, 3.7°C, 3.8°C, 3.9°C, 4.0°C, 4.1°C, 4.2°C, 4.3°C, 4.4°C, 4.5°C and any value in between. For example, the time is 25min, 26min, 27min, 28min, 29min, 30min, 31min, 32min, 33min, 34min or 35min and any value in between.

[0071] In some embodiments, the conditions of the first resuspension treatment include: mixing the sperm heads with a buffer solution containing 0.8M to 1.2M NaCl and stirring the mixture, for example, the concentration of NaCl is 0.8M, 0.9M, 1.0M, 1.1M or 1.2M and any value therebetween.

[0072] In some embodiments, the stirring treatment temperature is 3.5°C to 4.5°C, and the time is 0.8h to 2h. For example, the temperature is 3.5°C, 3.6°C, 3.7°C, 3.8°C, 3.9°C, 4.0°C, 4.1°C, 4.2°C, 4.3°C, 4.4°C, or 4.5°C and any value therebetween. The time is 0.8h, 0.9h, 1.0h, 1.1h or 1.2h and any value therebetween.

[0073] In some embodiments, the buffer comprises one or more of TBS buffer, TBST buffer, PBS buffer and PBST buffer.

[0074] In some embodiments, the conditions for the second resuspension treatment include:

[0075] The precipitate after the first centrifugation is resuspended in 80mM to 120mM alkali solution at a temperature of 3.5°C to 5°C. For example, the concentration of the alkali solution is 80mM, 90mM, 100mM, 110mM or 120mM and any value therebetween. For example, the temperature is 3.5°C, 3.6°C, 3.7°C, 3.8°C, 3.9°C, 4.0°C, 4.1°C, 4.2°C, 4.3°C, 4.4°C or 4.5°C and any value therebetween.

[0076] In some embodiments, the alkaline solution includes NaOH solution.

[0077] On the other hand, the present application provides a method for extracting proteins from the subcellular structure of a sperm head, comprising: using the above-mentioned method for separating the subcellular structure of a sperm head to obtain the acrosome and the perinuclear sheath, and extracting proteins from the acrosome and the perinuclear sheath.

[0078] In some embodiments, the method comprises: subjecting the proteins of the acrosome and the perinuclear sheath to ultrasonic disruption, followed by denaturation and reductive alkylation.

[0079] Perform enzymatic hydrolysis to obtain peptide fragments, and then perform peptide fragment desalting treatment.

[0080] In some of the embodiments, the proteins of the acrosome and the perinuclear sheath are placed in a protein disruption solution for disruption; the protein disruption solution includes 25mM to 100M TEAB buffer and dodecyl-β-D-maltoside with a final concentration of 0.08w / v% to 0.12w / v%.

[0081] For example, the concentration of TEAB buffer is 25mM, 30mM, 35mM, 40mM, 42mM, 44mM, 46mM, 48mM, 50mM, 52mM, 54mM, 56mM, 58mM, 60mM, 62mM, 64mM, 66mM, 68mM, 70mM, 72mM, 74mM, 76mM, 78mM, 80mM, 82mM, 84mM, 86mM, 88mM, 90mM, 92mM, 94mM, 96mM, 98mM or 100mM and any number in between.

[0082] For example, the concentration of dodecyl-β-D-maltoside is 0.08 w / v%, 0.09 w / v%, 0.10 w / v%, 0.11 w / v% or 0.12 w / v% and any values ​​therebetween.

[0083] In some embodiments, the frequency of ultrasound is 100W-150W and the duration is 10min-20min.

[0084] For example, the frequency of ultrasound is 100 W, 110 W, 120 W, 130 W, 140 W or 150 W and any value in between.

[0085] For example, the ultrasound time is 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min or 20 min and any value in between.

[0086] In some embodiments, the conditions for the denaturation-reduction treatment include: the reagent for the denaturation-reduction treatment includes dithiothreitol with a final concentration of 80 mM to 120 mM, the temperature is 70° C. to 80° C., and the time is 30 min to 50 min.

[0087] For example, the concentration of dithiothreitol is 80 mM, 90 mM, 100 mM, 110 mM or 120 mM and any values ​​therebetween. For example, the temperature is 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76° C., 77° C., 78° C., 79° C. or 80° C. For example, the time is 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min, 40 min, 41 min, 42 min, 43 min, 44 min, 45 min, 46 min, 47 min, 48 min, 49 min or 50 min and any values ​​therebetween.

[0088] The conditions for the alkylation treatment include: the reagent for the alkylation treatment includes IAA with a final concentration of 80 mM to 120 mM, the temperature is 22° C. to 26° C., and the time is 25 min to 35 min.

[0089] For example, the concentration of IAA is 80 mM, 90 mM, 100 mM, 110 mM or 120 mM and any values ​​therebetween. For example, the temperature is 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C or 30°C and any values ​​therebetween. For example, the time is 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min, 40 min, 41 min, 42 min, 43 min, 44 min, 45 min, 46 min, 47 min, 48 min, 49 min or 50 min and any values ​​therebetween.

[0090] In some embodiments, the conditions of the enzymatic treatment include: the reagent for the enzymatic treatment includes trypsin, and the time is 2 hours to 16 hours. For example, the time is 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours or 16 hours and any value therebetween.

[0091] In some embodiments, the peptide desalting conditions include: using a microchromatographic column to desalt the peptide.

[0092] In some embodiments, the microchromatography column comprises a ZipTip microchromatography column, which is a tool for biomolecule concentration and purification, and is particularly suitable for sample preparation prior to analytical techniques such as mass spectrometry, high performance liquid chromatography (HPLC), and capillary electrophoresis.

[0093] In some embodiments, the microchromatography column is washed 2 to 4 times with buffer B, and the microchromatography column is washed 2 to 4 times with buffer A. For example, the number of times is 2 times, 3 times, or 4 times.

[0094] In some embodiments, the peptide fragments are added to the microchromatography column and then washed with buffer A for 2 to 4 times.

[0095] In some embodiments, buffer A comprises: 0.08 v / v% to 0.12 v / v% formic acid aqueous solution. For example, the concentration of the formic acid aqueous solution is 0.08 v / v%, 0.09 v / v%, 0.10 v / v%, 0.11 v / v% or 0.12 v / v% and any value in between.

[0096] In some embodiments, buffer B comprises: 60 v / v% to 98 v / v% acetonitrile and 0.08 v / v% to 0.12 v / v% formic acid aqueous solution. For example, the acetonitrile concentration is 60 v / v%, 61 v / v%, 62 v / v%, 63 v / v%, 64 v / v%, 65 v / v%, 66 v / v%, 67 v / v%, 68 v / v%, 69 v / v%, 70 v / v%, 71 v / v%, 72 v / v%, 73 v / v%, 74 v / v%, 75 v / v%, 76 v / v%, 77 v / v%, 78 v / v%, 79 v / v%, The concentration of the formic acid aqueous solution is 0.08 v / v%, 0.09 v / v%, 0.10 v / v%, 0.11 v / v% or 0.12 v / v% and any value therein.

[0097] On the other hand, the present application provides a mass spectrometry analysis method for the subcellular structure of the sperm head, comprising: using the above-mentioned sperm head subcellular structure separation method to obtain the acrosome and perinuclear sheath, and the above-mentioned sperm head subcellular structure protein extraction method to prepare the sample to be analyzed, and perform mass spectrometry analysis.

[0098] In some embodiments, the parameters of mass spectrometry analysis include a separation column length of 20 cm to 30 cm, for example, a separation column length of 20 cm, 21 cm, 22 cm, 23 cm, 24 cm, 25 cm, 26 cm, 27 cm, 28 cm, 29 cm or 30 cm and any value therebetween.

[0099] In some embodiments, the sample to be analyzed is mixed with buffer A and then loaded onto the column.

[0100] In some embodiments, 3% to 38% buffer B is used for linear gradient separation with a time of 100 min to 105 min, for example, the time is 100 min, 101 min, 102 min, 103 min, 104 min or 105 min and any value in between.

[0101] In some embodiments, the equilibration time is 8 min to 12 min in 98% to 100% buffer B. For example, the time is 8 min, 9 min, 10 min, 11 min or 12 min and any value therebetween.

[0102] In some embodiments, buffer A includes 0.08 v / v% to 0.12 v / v% formic acid aqueous solution, and buffer B includes 80 v / v% to 82 v / v% acetonitrile and 0.08 v / v% to 0.12 v / v% formic acid aqueous solution. For example, the concentration of acetonitrile is 80 v / v%, 81 v / v% or 82 v / v% and any value in between. For example, the concentration of formic acid aqueous solution is 0.08 v / v%, 0.09 v / v%, 0.10 v / v%, 0.11 v / v% or 0.12 v / v% and any value in between.

[0103] In some embodiments, mass spectrometry analysis is performed using a mass spectrometer equipped with a FAIMS Pro interface, and FAIMS switches between CV values ​​of -35V, -45V, and -65V, with a cycle time of 0.8s to 2s. For example, the cycle time is 0.8s, 0.9s, 1.0s, 1.1s, 1.2s, 1.3s, 1.4s, 1.5s, 1.6s, 1.7s, 1.8s, 1.9s, or 2.0s, and any value in between.

[0104] In some embodiments, the primary spectrum is collected using an Orbitrap, and the secondary spectrum is collected using a linear ion trap or an Orbitrap. An Orbitrap is an ion trap mass analyzer that consists of two outer electrodes and a central electrode and can be used as both an analyzer and a detector. Ions entering the Orbitrap are captured by "electric squeezing", after which the ions oscillate around the central electrode and between the two outer electrodes. Different ions oscillate at different frequencies and are thus separated.

[0105] The linear ion trap is a mass spectrometry technique that consists of two sets of hyperbolic poles and two plates at both ends. Similar to a quadrupole mass spectrometer, in a linear ion trap, ions are confined to a strip space arranged along the center axis of the trap. By applying an alternating voltage, ions are captured in the trap and move stably. By adjusting the electric field parameters, ions of a specific mass can be made to enter an unstable state and ejected from the narrow slit.

[0106] The present application provides a method for separating subcellular structures of sperm heads. By using specific ultrasonic and centrifugal treatment methods, more than 1,500 proteins were identified from various components of the sperm head, and the analysis reproducibility was good. GO analysis showed that the proteins of the corresponding subcellular structures were also enriched in the corresponding cellular components, thereby effectively separating the subcellular structures of the sperm head.

[0107] In addition, after the subcellular structures are separated by the sperm head subcellular structure separation method of the present application, proteomic sample preparation and mass spectrometry analysis methods can be further performed to separate the various components such as the acrosome, perinuclear sheath, and cell nucleus in the sperm head structure and perform proteomic analysis, which will be beneficial for further in-depth research on the mechanism of action of sperm in various fertilization events.

[0108] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0109] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0110] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean 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 the present application.

[0111] Example 1

[0112] This embodiment provides a method for separating subcellular structures of sperm heads.

[0113] 1. Separation of sperm head subcellular structure:

[0114] (1) Ultrasonicate human / mouse sperm in PBS buffer at 150W for 4s to separate the sperm head and tail, and observe the separation under a microscope.

[0115] (2) Use 45% and 90% SpermGrad (sperm gradient separation solution) to perform density gradient centrifugation on the sperm separated from the head and tail, and take the precipitate for microscopic examination. If the proportion of sperm head exceeds 80%, the next step of the test can be carried out.

[0116] (3) Centrifuge the sperm heads at 500 g for 10 min and keep the pellet.

[0117] (4) The separated sperm head components were resuspended in 0.2% Triton X-100 TBS and stirred with a magnetic stirrer at 4°C for 1 h.

[0118] (5) After centrifugation at 2500 g for 10 min, rinse with PBS and resuspend in 1 M NaCl, stir for 1 h at 4 °C, and centrifuge at 1500 g for 10 min. The supernatant is the acrosome fraction.

[0119] (6) After washing, the precipitate was resuspended in 100 mM NaOH solution at 4°C overnight and centrifuged at 25,000 rpm for 30 min to neutralize the NaOH. The supernatant was the perinuclear sheath component.

[0120] The results of the identification of the acrosome, perinuclear sheath, and nucleus components in the specific sperm head structure are as follows Figure 1 As shown, Figure 1 A in the figure is the number of proteins identified in the acrosome, perinuclear sheath, and nucleus (n=2). Figure 1 B in the figure is the number of peptides identified in the acrosome, perinuclear sheath, and nucleus (n=2).

[0121] The heat map of the correlation between the two repeated analyses of each component is shown in Figure 2 As shown, it can be seen that the correlation between the two replicates of each component is very good, indicating that the analytical reproducibility of the method is high.

[0122] Example 2

[0123] This embodiment provides a method for extracting protein from the subcellular structure of sperm head, comprising:

[0124] 1. Separation of sperm head subcellular structure:

[0125] (1) Ultrasonicate human or mouse sperm in PBS buffer at 150W for 4s to separate the sperm head and tail, and observe the separation under a microscope.

[0126] (2) Use 45% and 90% SpermGrad (sperm gradient separation solution) to perform density gradient centrifugation on the sperm separated from the head and tail, and take the precipitate for microscopic examination. If the proportion of sperm head exceeds 80%, the next step of the test can be carried out.

[0127] (3) Centrifuge the sperm heads at 500 g for 10 min and keep the pellet.

[0128] (4) The separated sperm head components were resuspended in 0.2% Triton X-100 TBS and stirred with a magnetic stirrer at 4°C for 1 h.

[0129] (5) After centrifugation at 2500 g for 10 min, rinse with PBS and resuspend in 1 M NaCl, stir for 1 h at 4 °C, and centrifuge at 1500 g for 10 min. The supernatant is the acrosome fraction.

[0130] (6) After washing, the precipitate was resuspended in 100 mM NaOH solution at 4°C overnight, and the supernatant was separated by ultracentrifugation at 25,000 rpm for 30 min to neutralize the NaOH. The supernatant was the perinuclear sheath component.

[0131] 2. Protein extraction:

[0132] (1) Each component was added to a DDM solution prepared with 50 mM TEAB to a final DDM concentration of 0.1%, and the cells were disrupted and protein extracted by ultrasonication for 15 min.

[0133] (2) Protein denaturation reduction alkylation: Add 0.5 μL 100 mM DTT to the PCR tube and react at 75°C for 40 min; then add 0.5 μL 100 mM IAA and react at room temperature in the dark for 30 min.

[0134] (3) Proteolysis: Add mass spectrometry grade modified trypsin to the alkylated protein solution, digest it overnight, and then centrifuge to obtain the supernatant to obtain the hydrolyzed peptides.

[0135] (4) Peptide desalting: Use ZipTip to desalt the peptides after enzymatic hydrolysis to remove NaCl. Use 100 μL of buffer B (80% ACN, 19.9% ​​H2O, 0.1% formic acid) to rinse the ZipTip column 3 times, and then use 100 μL of buffer A (99.9% H2O, 0.1% formic acid) to rinse the ZipTip column 3 times to balance the ZipTip column. After the peptide solution is transferred to the ZipTip column, use 100 μL of buffer A to rinse 3 times to desalt. Finally, use 200 μL of buffer B to elute the desalted peptides, evaporate to dryness and leave for mass spectrometry analysis.

[0136] Figure 3 The cellular components in which the proteins identified in each component are enriched, Figure 3 A in the figure is the cellular fraction where the protein identified in the acrosome is enriched; Figure 3 B in the figure is the cellular component where the proteins identified in the perinuclear sheath are enriched; Figure 3 C in the figure indicates the cellular fractions where the proteins identified in the nucleus are enriched.

[0137] Depend on Figure 3 The results show that the GO enrichment analysis of each component showed that the proteomic identification results of each component were well enriched in the regions corresponding to the sperm components, which indirectly reflected that the subcellular structures of the sperm head were effectively separated.

[0138] Example 3

[0139] This embodiment provides a mass spectrometry analysis method for sperm head subcellular structure, comprising:

[0140] 1. Separation of sperm head subcellular structure:

[0141] (1) Ultrasonicate human / mouse sperm in PBS buffer at 150W for 4s to separate the sperm head and tail, and observe the separation under a microscope.

[0142] (2) Use 45% and 90% SpermGrad (sperm gradient separation solution) to perform density gradient centrifugation on the sperm separated from the head and tail, and take the precipitate for microscopic examination. If the proportion of sperm head exceeds 80%, the next step of the test can be carried out.

[0143] (3) Centrifuge the sperm heads at 500 g for 10 min and keep the pellet.

[0144] (4) The separated sperm head components were resuspended in 0.2% Triton X-100 TBS and stirred with a magnetic stirrer at 4°C for 1 h.

[0145] (5) After centrifugation at 2500 g for 10 min, rinse with PBS and resuspend in 1 M NaCl, stir for 1 h at 4 °C, and centrifuge at 1500 g for 10 min. The supernatant is the acrosome fraction.

[0146] (6) After washing, the precipitate was resuspended in 100 mM NaOH solution at 4°C overnight, and the supernatant was separated by ultracentrifugation at 25,000 rpm for 30 min to neutralize the NaOH. The supernatant was the perinuclear sheath component.

[0147] 2. Protein extraction:

[0148] (1) Each component was added to a DDM solution prepared with 50 mM TEAB to a final DDM concentration of 0.1%, and the cells were disrupted and protein extracted by ultrasonication for 15 min.

[0149] (2) Protein denaturation reduction alkylation: Add 0.5 μL 100 mM DTT to the PCR tube and react at 75°C for 40 min; then add 0.5 μL 100 mM IAA and react at room temperature in the dark for 30 min.

[0150] (3) Proteolysis: Add mass spectrometry grade modified trypsin to the alkylated protein solution, digest it overnight, and then centrifuge to obtain the supernatant to obtain the hydrolyzed peptides.

[0151] (4) Peptide desalting: Use ZipTip to desalt the peptides after enzymatic hydrolysis to remove NaCl. Use 100 μL of buffer B (80% ACN, 19.9% ​​H2O, 0.1% formic acid) to rinse the ZipTip column 3 times, and then use 100 μL of buffer A (99.9% H2O, 0.1% formic acid) to rinse the ZipTip column 3 times to balance the ZipTip column. After the peptide solution is transferred to the ZipTip column, use 100 μL of buffer A to rinse 3 times to desalt. Finally, use 200 μL of buffer B to elute the desalted peptides, evaporate to dryness and leave for mass spectrometry analysis.

[0152] 3. Mass spectrometry analysis. Specific analysis diagram is as follows Figure 4 shown.

[0153] The evaporated peptides were reconstituted with 25 μL buffer A and transferred to a loading bottle for mass spectrometry analysis using a Thermo Fisher Orbitrap Eclipse Tribrid mass spectrometer equipped with a FAIMS Pro interface. The enzymatically hydrolyzed peptides were directly loaded onto a 2 cm PEPMAP Trap column (ThermoFisher Scientific) for online desalting. They were then separated for 102 min using a linear gradient from 3% to 38% buffer B (80% ACN, 19.9% ​​H2O, 0.1% formic acid) on a 25 cm PepMap C18 separation column (ThermoFisher Scientific) and equilibrated in 100% buffer B for 10 min. FAIMS switched between CV values ​​of -35 V, -45 V, and -65 V with a cycle time of 1 s.

[0154] The primary spectrum was collected using Orbitrap (Resolution: 60k; AGC target: Standard; MaxIT: Auto; RF lens: 50%; mass range: 350 to 1,500), and the dynamic exclusion time was 40 s.

[0155] Secondary spectra were collected using a linear ion trap (isolation window: (6) m / z; scan rate: rapid; AGQ target: standard; MaxIT: Auto; HCD CE: 35%; data type: centroid).

[0156] 4. Data analysis: Proteome Discoverer software (version 2.5, San Jose, CA) was used for mass spectrometry data library search and proteome label-free relative quantitative analysis. The UniProt human database (downloaded on June 27, 2022, containing 79,435 sequences) was used for library search. Methionine oxidation and protein N-terminal acetylation were set as variable modifications, and cysteine ​​alkylation was set as a fixed modification. The mass tolerances for precursor ions and fragment ions were 10 ppm and 0.6 Da, respectively. The minimum and maximum peptide lengths were 6 and 144 amino acids, respectively. Two missed cleavage sites were allowed for each peptide segment. The FDR was set to 0.01. The other parameters of Proteome Discoverer used the default values.

[0157] The results showed that more than 1,500 proteins were identified from each component of the sperm head, and the analysis was reproducible. GO analysis showed that the proteins of the corresponding subcellular structures were also enriched in the corresponding cellular components, which indirectly reflected that a better separation was achieved.

[0158] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A method for separating subcellular structures of sperm heads, characterized in that: include: Providing sperm samples, performing ultrasonic treatment and the first centrifugation treatment, taking sediments, and separating the heads and tails of sperms; The head is subjected to a first re-suspension treatment and a second centrifugation treatment, and the supernatant is taken to obtain the acrosome component; The precipitate after the second centrifugation is resuspended for the second time, and then centrifuged for the third time, and the supernatant is taken to obtain the perinuclear sheath component; The acrosome component and the perinuclear sheath component are the separated sperm head subcellular structures; The parameters of the first centrifugation treatment include: temperature of 3.5°C to 4.5°C, centrifugal force of 400g to 600g, and time of 18min to 22min; The parameters of the second centrifugation treatment include: temperature of 3.5°C to 4.5°C, centrifugal force of 1400g to 1600g, and time of 18min to 22min; The parameters of the third centrifugal treatment include: temperature of 3.5°C to 4.5°C, rotation speed of 20000rpm to 30000rpm, and time of 25min to 35min.

2. The method for separating the sperm head subcellular structure according to claim 1, characterized in that: The first resuspension process meets one or more of the following conditions: (1) The solvent for the resuspension treatment includes a 0.8M to 1.2M NaCl solution; (2) The resuspension temperature is 3.5°C to 4.5°C, the resuspension time is 0.8h to 1.2h, and a magnetic stirrer is used for stirring during the resuspension period; Optionally, the second resuspension treatment satisfies one or more of the following conditions: (1) Resuspend the precipitate after the first centrifugation in 80 mM to 120 mM NaOH solution; (2) The resuspension temperature is 3.5℃~4.5℃ overnight.

3. A method for extracting protein from the subcellular structure of sperm head, characterized in that: include: The sperm head subcellular structure is separated by the method for separating the sperm head subcellular structure according to any one of claims 1 to 2, and the protein in the sperm head subcellular structure is extracted.

4. The method for extracting protein from the subcellular structure of sperm head according to claim 3, characterized in that: include: The protein is subjected to ultrasonic disruption, denaturation, reductive alkylation, and enzymatic hydrolysis to obtain peptide fragments, and then the peptide fragments are subjected to desalting treatment.

5. The method for extracting protein from the subcellular structure of sperm head according to claim 4, characterized in that: Ultrasonication treatment meets one or more of the following conditions: (1) placing the protein in a protein disruption solution for disruption; the protein disruption solution comprises 25 mM to 100 mM TEAAB buffer and dodecyl-β-D-maltoside with a final concentration of 0.08 w / v% to 0.12 w / v%; (2) The sample is subjected to ultrasonic treatment at a temperature of 3.5°C to 4.5°C, with an ultrasonic frequency of 100W to 150W and a time of 10min to 20min.

6. The method for extracting protein from the subcellular structure of sperm head according to claim 4, characterized in that: The conditions for the denaturation reduction treatment meet one or more of the following conditions: (1) The reagent for the denaturation reduction treatment includes dithiothreitol at a final concentration of 80 mM to 120 mM; (2) The temperature of the denaturation reduction treatment is 70°C to 80°C, and the time is 30min to 40min.

7. The method for extracting protein from the subcellular structure of sperm head according to claim 4, characterized in that: The conditions of the alkylation treatment meet one or more of the following conditions: (1) the reagent for alkylation treatment includes IAA at a final concentration of 80 mM to 120 mM; (2) The temperature of the alkylation treatment is 20°C to 30°C, and the time is 20min to 30min.

8. The method for extracting protein from the subcellular structure of the sperm head according to any one of claims 3 to 7, characterized in that: The conditions for the enzymatic treatment meet one or more of the following conditions: (1) The reagents for enzymatic treatment include trypsin; (2) The enzymatic hydrolysis time is 2h to 16h.

9. The method for extracting protein from the subcellular structure of the sperm head according to any one of claims 3 to 7, characterized in that: The conditions for desalting treatment included: peptide desalting using microchromatography; Optionally, the microchromatography method satisfies one or more of the following conditions: (1) Using ZipTip microchromatography columns; (2) Rinse the microchromatography column 2 to 4 times with buffer B and rinse the microchromatography column 2 to 4 times with buffer A; (3) adding the peptide fragment to the microchromatographic column and then washing it with buffer A for 2 to 4 times; (4) The buffer A comprises: 0.08 v / v% to 0.12 v / v% formic acid aqueous solution; (5) The buffer solution B comprises: 60 v / v% to 98 v / v% acetonitrile and 0.08 v / v% to 0.12 v / v% formic acid aqueous solution.

10. A mass spectrometry analysis method for sperm head subcellular structure, characterized in that: include: The method for separating the subcellular structure of the sperm head according to any one of claims 1 to 2 is used to obtain the acrosome and the perinuclear sheath, and the method for extracting the protein of the subcellular structure of the sperm head according to any one of claims 3 to 9 is used to prepare the sample to be analyzed, and mass spectrometry analysis is performed; Optionally, the mass spectrometry analysis satisfies one or more of the following conditions: (1) The chromatographic gradient for peptide separation is: a linear gradient of 3% to 38% buffer B, and the linear gradient time is 100 min to 105 min; (2) Equilibrate in 98% to 100% buffer B for 8 to 12 minutes; (3) mass spectrometry analysis was performed using a mass spectrometer equipped with a FAIMS Pro interface, with the FAIMS switching between CV values ​​of -35 V, -45 V, and -65 V with a cycle time of 0.8 s to 2 s; and, (4) The primary spectrum was collected using an Orbitrap, and the secondary spectrum was collected using a linear ion trap or an Orbitrap.

Images