Washing composition, kit and application thereof

Abstract

The invention belongs to the field of surfactant removal, particularly relates to a washing composition and application thereof, and more particularly relates to a washing composition for removing a surfactant and application thereof. The invention provides a washing composition. The washing composition comprises the following reagents and reagent combinations: a first reagent: potassium salt or sodium salt; and a second reagent combination: trichloroacetic acid, acetonitrile, cyclodextrin or a cyclodextrin derivative. By using the washing composition disclosed by the invention, the surfactant used in the protein extraction process can be furthest removed through the synergistic interaction between the reagents and the reagent combination, and meanwhile, the loss of low-abundance protein and difficulty in redissolving the protein in the preparation process of other methods are avoided.

Description

Technical Field

[0001] This invention belongs to the field of surfactant removal, specifically, it relates to a washing composition and its use, and more specifically, it relates to a washing composition for removing surfactants and its use. Background Technology

[0002] In proteomics sample preparation, protein extraction from lysed biological samples is a crucial step. Common surfactants used for lysing biological samples, even mild ones like dodecyl-β-D-maltose (DDM), can adversely affect mass spectrometry (MS / MS), especially since DDM has weak lysing ability, particularly for tissue samples. Stronger surfactants, such as sodium dodecyl sulfate (SDS), bind to proteins and peptides, hindering the enzymatic breakdown of proteins into peptides and affecting peptide ionization efficiency at the MS / MS end, severely reducing peptide detection rates. Furthermore, residual SDS rapidly reduces column efficiency and damages the MS / MS spectrometer. Therefore, removing surfactants from the crude protein extract after lysation is extremely important.

[0003] Existing methods for removing surfactants include ultrafiltration centrifugation, acetone precipitation, anion exchange chromatography, and affinity chromatography. However, methods like ultrafiltration centrifugation, which rely on a specific membrane size (e.g., 20 kDa) to retain proteins, inevitably result in the loss of some proteins, especially low-molecular-weight proteins. For instance, acetone precipitation is extremely cumbersome and time-consuming, requiring approximately two days of repeated washing of the crude protein extract to obtain surfactant-free protein. However, this method suffers from incomplete protein precipitation and the difficulty in resolving the precipitate, leading to significant protein loss, particularly of low-abundance proteins. If the protein sample after acetone precipitation is used for subsequent proteomics sample pretreatment via in-solution enzymatic digestion, an additional two days are required. In other words, preparing peptides from the crude protein extract after surfactant cleavage via acetone precipitation and in-solution enzymatic digestion takes four days.

[0004] Therefore, there is a need in the art for a washing composition that can remove surfactants in a short time while minimizing sample loss. Summary of the Invention

[0005] In view of the above, in a first aspect, the present invention provides a washing composition comprising the following reagents and combinations thereof:

[0006] First reagent: potassium or sodium salt; and

[0007] The second reagent combination consists of trichloroacetic acid, acetonitrile, cyclodextrin, or cyclodextrin derivatives.

[0008] Using the washing composition of the present invention, the surfactants used in the protein extraction process can be removed to the maximum extent through the synergistic effect between the reagents and the reagent combination, while avoiding the loss of low-abundance proteins and the difficulty in resolvating proteins in other preparation methods.

[0009] Furthermore, the potassium or sodium salt may be a chloride, citrate, dihydrogen phosphate, dihydrogen phosphate, acetate, sulfate, or sorbate.

[0010] Specifically, the potassium salt may be potassium chloride, potassium citrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium acetate, potassium sulfate, or potassium sorbate.

[0011] Specifically, the sodium salt may be sodium chloride, sodium citrate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium acetate, sodium sulfate, or sodium sorbate.

[0012] Preferably, the first reagent is a potassium salt. When using a potassium salt, it can be combined with the second reagent to obtain a higher peptide recovery rate.

[0013] Further, the cyclodextrin or cyclodextrin derivative may be α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, (2-hydroxypropyl)-α-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, (2-hydroxypropyl)-γ-cyclodextrin, α-cyclodextrin sodium sulfate hydrate, β-cyclodextrin sodium sulfate hydrate, γ-cyclodextrin sodium sulfate hydrate, carboxymethyl-α-cyclodextrin, carboxymethyl-β-cyclodextrin, carboxymethyl-γ-cyclodextrin, methyl-α-cyclodextrin, methyl-β-cyclodextrin, methyl-γ-cyclodextrin, etc. Quaternary ammonium-α-cyclodextrin, quaternary ammonium-β-cyclodextrin, quaternary ammonium-γ-cyclodextrin, sulfobutyl-α-cyclodextrin, sulfobutyl-β-cyclodextrin, sulfobutyl-γ-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-α-cyclodextrin, 3A-amino-3A-deoxy-(2AS,3AS)-β-cyclodextrin hydrate, 3A-amino-3A-deoxy-(2AS,3AS)-γ-cyclodextrin hydrate, (2-hydroxy-3-N,N,N-trimethylamino)propyl-α-cyclodextrin chlorine Compounds, (2-hydroxy-3-N,N,N-trimethylamino)propyl-β-cyclodextrin chloride, (2-hydroxy-3-N,N,N-trimethylamino)propyl-γ-cyclodextrin chloride, 6-aminomethyl-6-deoxy-α-cyclodextrin, 6-aminomethyl-6-deoxy-β-cyclodextrin, 6-aminomethyl-6-deoxy-γ-cyclodextrin, 6-tert-butyldimethylsilane-α-cyclodextrin, 6-tert-butyldimethylsilane-β-cyclodextrin, 6-tert-butyldimethylsilane-γ-cyclodextrin, mono-(6 One or more of the following: (6-hexamethylenediamino-6-deoxy)-α-cyclodextrin, mono-(6-hexamethylenediamino-6-deoxy)-β-cyclodextrin, mono-(6-hexamethylenediamino-6-deoxy)-γ-cyclodextrin, 6-acetamido-α-cyclodextrin, 6-acetamido-β-cyclodextrin, 6-acetamido-γ-cyclodextrin, 2,3-dimethyl-6-tert-butyldimethyl-α-cyclodextrin, 2,3-dimethyl-6-tert-butyldimethyl-β-cyclodextrin, and 2,3-dimethyl-6-tert-butyldimethyl-γ-cyclodextrin.

[0014] Preferably, the cyclodextrin or cyclodextrin derivative is (2-hydroxy-3N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate.

[0015] Further, the concentration of the sodium salt is 10mM to 1500mM, for example, 10mM, 50mM, 100mM, 200mM, 500mM, 1000mM, 1500mM, preferably 10mM to 1000mM, more preferably 10mM to 700mM, and most preferably 100mM to 500mM.

[0016] Further, the concentration of the potassium salt is 10mM to 1500mM, for example, 10mM, 50mM, 100mM, 200mM, 500mM, 1000mM, 1500mM, preferably 10mM to 1000mM, more preferably 10mM to 700mM, and most preferably 100mM to 500mM.

[0017] Further, the final mass / volume concentration of trichloroacetic acid in the second reagent combination is 0.1% to 20% (w / v), for example, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, preferably 0.1% to 10% (w / v), and more preferably 0.1% to 5% (w / v).

[0018] Further, the concentration of the acetonitrile in the second reagent combination is 0.1% to 90% (v / v), for example, 0.1%, 1%, 10%, 20%, 30%, 40%, 46%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, preferably 20% to 90% (v / v), more preferably 20% to 80% (v / v), and most preferably 20% to 50% (v / v).

[0019] Further, the final concentration of the cyclodextrin or cyclodextrin derivative in the second reagent combination is 1 to 300 mM, for example, 1, 5, 10, 20, 50, 60, 100, 150, 200, 250, 300 mM, preferably 1 to 200 mM, more preferably 5 to 100 mM.

[0020] Secondly, the present invention provides the use of the above composition in the preparation of a protein extraction kit.

[0021] Furthermore, the protein is a proteome.

[0022] Furthermore, the protein is a polypeptide.

[0023] The present invention also provides the use of the above-described washing composition in a kit for removing surfactants.

[0024] Thirdly, the present invention provides a protein extraction kit comprising the washing composition described above.

[0025] Furthermore, the kit also includes a protein extraction kit.

[0026] Furthermore, the kit may be a solid-phase extraction column. Further, the solid-phase extraction column comprises one or both of a strong cation exchange resin and a strong anion exchange resin mixture, and a solid-phase extraction membrane.

[0027] Furthermore, the solid-phase extraction column includes a column tube, with the mixed packing material located at the lower end of the column tube and above the solid-phase extraction membrane.

[0028] In one specific implementation, the solid-phase extraction column is as follows: Figure 1 The structure is shown.

[0029] Furthermore, the kit also includes a third reagent and a fourth reagent, wherein the third reagent is a 0.1-10% (v / v) acidic solution, including but not limited to one or more combinations of formic acid, acetic acid, etc.; and the fourth reagent is a 5-100% organic reagent, including but not limited to one or more combinations of methanol, ethanol, acetonitrile, isopropanol, etc.

[0030] Furthermore, the protein is a proteome.

[0031] Furthermore, the protein is a polypeptide.

[0032] Fourthly, the present invention provides a method for extracting proteins, comprising the following steps:

[0033] S1. Protein lysis: Add surfactant to the sample to induce lysis;

[0034] S2, Protein pre-enrichment; Precipitate surfactants using the first reagent as described above, and enrich proteins;

[0035] S3, Surfactant Removal: The surfactant is removed using the second reagent combination as described above;

[0036] S4. Extract protein.

[0037] Furthermore, the surfactant may be hexadecyltrimethylammonium bromide (CTMAB), hexadecyltrimethylammonium chloride (CTMAC), sodium dodecyl sulfate (SDS), lithium dodecyl sulfate, sodium dodecyl sulfonate (SBS), sodium dodecylbenzene sulfonate (SDBS), etc.

[0038] Furthermore, the sample may be a protein solution, cells, large pieces of animal tissue, large pieces of plant tissue, animal tissue slices, plant tissue slices, plasma, serum, saliva, urine, cerebrospinal fluid, etc.

[0039] Furthermore, the pH value of step S1 is 7 to 10.

[0040] Furthermore, step S1 also includes heating and / or sonication for lysis of the sample.

[0041] Furthermore, step S2 also includes a protein extraction kit, which may be a solid-phase extraction column.

[0042] Furthermore, the pH value of step S2 is 0.1 to 3.

[0043] Furthermore, step S3, in addition to the second reagent combination, also includes a 0.1-10% (v / v) acidic solution, including but not limited to one or more combinations of formic acid, acetic acid, etc.; and 5-100% organic reagents. Organic reagents include, but are not limited to, one or more combinations of methanol, ethanol, acetonitrile, isopropanol, etc.

[0044] Further, step S4 includes one or more of the following steps: reduction, alkylation and enzymatic hydrolysis, transfer of peptides, desalting of peptides, elution of peptides, or drying of peptides. Attached Figure Description

[0045] Figure 1 This is a schematic diagram of the solid-phase extraction column of the present invention, wherein 1-column tube, 2-mixed packing of strong cation exchange resin (SCX) and / or strong anion exchange resin (SAX), 3-C18 membrane, and 4-collection tube;

[0046] Figure 2 The absorbance values ​​of SDS and three groups of samples at wavelengths of 420-500 nm are shown for the concentration range of 0-0.01% (w / v) of the SDS standard curve. Detailed Implementation

[0047] The present invention will be described in detail below with reference to specific implementation schemes and embodiments, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific implementation schemes and embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0048] Example 1: Protein Extraction Method

[0049] The method of the present invention

[0050] (1) Protein extraction: 2% (w / v) sodium dodecyl sulfate (SDS), 20 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, and protease inhibitors were added to the seven biological samples after liquid nitrogen grinding, and the pH was adjusted to 7-9. The biological samples were thoroughly lysed by contact (amplifier) ​​sonication to obtain crude protein extracts, and the protein concentration was quantified using the dioctanic acid (BCA) method.

[0051] (2) Protein pre-enrichment:

[0052] 5-30 μL of the first reagent of the washing composition of the present invention (such as potassium citrate or sodium citrate, containing 50-1120 mM potassium citrate, pH 1-3) is loaded into a pre-activated solid-phase extraction column; the crude extract containing 1-10 μg of protein in (1) is diluted to 30 μL with 0-0.1% SDS solution and loaded into the solid-phase extraction column; then 5-30 μL of the first reagent of the washing composition of the present invention (such as potassium citrate or sodium citrate, 50-1120 mM potassium citrate, pH 1-3) is added above the packing material of the solid-phase extraction column; the protein is enriched by centrifugation.

[0053] (3) Cleaning: The solid phase extraction column was cleaned sequentially with 80 μL of 0.1% (v / v) formic acid and 80 μL of acetonitrile.

[0054] (4) Removal of SDS: SDS is removed using the second reagent combination of the washing composition of the present invention;

[0055] (5) Cleaning: Use 60 μL of acetonitrile to clean twice to remove residual washing composition of the present invention;

[0056] (6) Reduction: Add 30 μL of reducing reagent (containing 14 mM potassium citrate, 10 mM TCEP, pH 2-3) and react at room temperature for 15 min.

[0057] (7) Alkylation and enzymatic hydrolysis: Add 8 μL of alkylation and enzymatic hydrolysis reagent to the solid phase extraction column, centrifuge to allow the reagent to enter the packing material, and react at 37°C for 1 h to enzymatically hydrolyze the protein in the packing material into peptides.

[0058] (8) Transfer of peptides: 60 μL of a high-salt solution containing 0.5-2 mol / L NaCl and 10 mM formate is added to the solid-phase extraction column, and the peptides in the packing are transferred to the solid-phase extraction membrane by centrifugation.

[0059] (9) Desalting of peptides: Add 60 μL of 0.1% formic acid and wash the solid phase extraction column by centrifugation to remove residual salt. Repeat the washing once.

[0060] (10) Elution: Add 60 μL of elution buffer containing 0.5% acetic acid and 80% acetonitrile, and collect the peptides into a centrifuge tube by centrifugation.

[0061] (11) Drying: The polypeptide solution collected in the above steps is placed in a freeze-concentrating centrifuge and dried at 35°C for 2 hours to prepare polypeptide powder.

[0062] (12) Add 20 μL of 0.1% FA to reconstitute.

[0063] In-solution enzymatic hydrolysis method

[0064] (1) Protein extraction: A protein extraction solution containing 0.4-5% (w / v) sodium dodecyl sulfate (SDS), 20 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, protease inhibitor, and pH 7-9 was added to the seven biological samples after grinding with liquid nitrogen. The biological samples were thoroughly lysed by contact sonication to obtain crude protein extract, and the protein concentration was quantified using BCA.

[0065] (2) Based on the concentration measured in step (1) above, take 50 μg of crude protein extract and dilute it to 1 μg / μL with 2% SDS lysis buffer so that the total volume of each tube is 50 μL.

[0066] (3) Add 200 μL (4 times the sample volume) of pre-cooled 80% acetone to each tube, vortex mix, and place in a -20°C refrigerator overnight;

[0067] (4) Centrifuge at 20000g for 10 min at 4℃;

[0068] (5) Remove the centrifuge tube and aspirate the supernatant;

[0069] (6) Add 1 mL of pre-cooled 80% acetone and blow the precipitate to disperse it;

[0070] (7) Centrifuge at 20000g for 10 min at 4℃;

[0071] (8) Repeat steps (5) to (7) three times, remove the centrifuge tube, and aspirate the supernatant;

[0072] (9) Invert the centrifuge tube on filter paper and dry for 5-15 minutes;

[0073] (10) Add 50 μL of 8M urea (containing 50 mM ammonium bicarbonate, pH 8) to reconstitute;

[0074] (11) Use an ultrasonic cleaning tank to sonicate for 30 seconds, centrifuge for 3-5 seconds, and repeat this step 3 times.

[0075] (12) Reduction: Add 5 μL (10% of the sample volume) of 100 mM (tris(2-carboxyethyl)phosphine hydrochloride (TCEP, pH 8) to the sample tube until the final concentration of TCEP is 10 mM, and reduce at 37°C in the dark for 40 min.

[0076] (13) Alkylation: Add 5.5 μL (10% of sample volume) of 200 mM chloroacetamide (CAA, pH 8) to the sample tube until the final concentration of CAA is 20 mM, and alkylate at 37°C in the dark for 40 min.

[0077] (14) Enzymatic hydrolysis: Add 339.5 μL of 50 mM NH4HCO3 solution (to dilute the urea concentration to 1 M) to the sample tube, add 1 μL of Trypsin enzyme solution (1 μg / μL, pH 8), and incubate overnight at 37°C in the dark.

[0078] (15) Terminate the enzymatic reaction: Add 44.4 μL of 10% FA solution (final FA concentration is 1%) to the sample tube;

[0079] (16) Desalting Tip Assembly: Place the SEP-PAK desalting column on a 15mL centrifuge tube;

[0080] (17) Activation: Add 1 mL of methanol to make all the liquid flow out;

[0081] (18) Equilibrium: Add 1 mL of Elute to allow all the liquid to flow out;

[0082] (19) Equilibrium: Add 1 mL of 1% FA to allow all the liquid to flow out, and repeat this step once;

[0083] (20) Loading the sample: Replace the waste liquid tube with a 15mL sample receiving tube, add all the enzymatically digested sample, let the liquid flow out slowly by gravity, collect the outflowing liquid and load the sample again.

[0084] (21) Clean the sample: Add 1 mL of 1% FA and let the liquid flow out completely. Repeat this step once.

[0085] (22) Collecting peptides: Replace the waste liquid collection tube with a 15 mL sample collection tube, add 0.5 mL of Elute, and let the liquid flow out slowly by gravity.

[0086] (23) Drying and reconstitution of peptide samples: Transfer the peptide solution in the sample collection tube to a 1.5 mL centrifuge tube, place it in a freeze centrifuge concentrator, and dry it at 35 °C for 12 h to prepare peptide powder;

[0087] (24) Add 200 μL of 0.1% FA to the 1.5 mL centrifuge tube of (23) above to reconstitute the polypeptide.

[0088] Example 2: SDS Residue Detection Method

[0089] The peptide powder obtained in Example 1 was reconstituted by adding 20 μL of PBS working solution (containing 1.4 mM phosphate buffer, 19.6 mM sodium chloride, 0.4 mM potassium chloride, pH 7.4). Then, 10 μL of the sample solution was taken and 75 μL of Stains-All working solution (containing 0.1 mg / mL Stains-All) was added. The absorbance at 453 nm was measured using a microplate reader. The SDS content was calculated using the standard curve (detection range 0-0.01% SDS) shown in Table 1, and the full spectrum scan was exported. The results are as follows: Figure 2 As shown.

[0090] Table 1

[0091]

[0092]

[0093] like Figure 2 As shown, the absorbance of the 0-0.01% (w / v) SDS standard curve at a wavelength of 453 nm shows a linear increase; the Pos-ctrl group is a peptide solution containing 0.4% (w / v) SDS diluted 100 times to a peptide solution containing 0.004% (w / v) SDS; while the Neg-ctrl group is a peptide solution without SDS; the Sample group is a peptide solution obtained by the protein extraction method of this invention from a sample containing 0.4% (w / v) SDS and 4 μg / μL protein.

[0094] The spectra show that the absorbance values ​​of the Pos-ctrl group essentially overlap with those of the 0.004% (w / v) SDS in the standard curve, and the absorbance at 453 nm is almost identical to that of the 0.004% (w / v) SDS at 453 nm. The Neg-ctrl group and the Sample group, however, completely overlap with those of the 0% (w / v) SDS group, and the absorbance at 453 nm is exactly the same as that of the 0% (w / v) SDS at 453 nm. This result indicates that the peptides obtained by the protein extraction method of this invention do not contain SDS.

[0095] Example 3: Detection results of washing samples with different compositions of the present invention

[0096] To further clarify the advantages of the washing composition of the present invention, a washing composition with the following system was designed and extracted according to the method described in Example 1 (the sample used was 293T cell lysis buffer):

[0097] Group A:

[0098] First reagent: 30 μL of 140 mM sodium citrate, pH 1;

[0099] Second reagent combination: Wash B: containing 1% (w / v) trichloroacetic acid, 46% (v / v) acetonitrile, and 60 mM (2-hydroxy-3N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate;

[0100] Group B:

[0101] First reagent: 30 μL of 140 mM potassium citrate, pH 1;

[0102] Second reagent combination: Wash B: containing 1% (w / v) trichloroacetic acid, 46% (v / v) acetonitrile, and 60 mM (2-hydroxy-3N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate;

[0103] Group C:

[0104] First reagent: 30 μL of 140 mM potassium citrate, pH 1;

[0105] Second reagent combination: Wash C: containing 1% (w / v) formic acid, 46% (v / v) acetonitrile, and 60 mM (2-hydroxy-3N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate;

[0106] Group D:

[0107] First reagent: 30 μL of 140 mM potassium citrate, pH 1;

[0108] Second reagent combination: Wash D: containing 1% (w / v) trichloroacetic acid, 46% (v / v) methanol, and 60 mM (2-hydroxy-3N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate;

[0109] Group E:

[0110] First reagent: 30 μL of 140 mM potassium citrate, pH 1;

[0111] Second reagent combination: Wash E: containing 1% (w / v) trichloroacetic acid, 46% (v / v) acetonitrile, and 60 mM hydroxypropyl-α-cyclodextrin;

[0112] Group F:

[0113] First reagent: 30 μL of 140 mM potassium citrate, pH 1;

[0114] Second reagent combination: Wash F: containing 46% (v / v) acetonitrile and 60 mM (2-hydroxy-3N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate;

[0115] Group G:

[0116] First reagent: 30 μL of 140 mM potassium citrate, pH 1;

[0117] Second reagent combination: Wash G: containing 1% (w / v) trichloroacetic acid and 60 mM (2-hydroxy-3N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate;

[0118] Group H:

[0119] First reagent: 30 μL of 140 mM potassium citrate, pH 1;

[0120] Second reagent combination: Wash H: containing 1% (w / v) trichloroacetic acid and 46% (v / v) acetonitrile;

[0121] Group I:

[0122] First reagent: 30 μL of 140 mM potassium citrate, pH 1;

[0123] Second reagent combination: Wash I: containing 1% (w / v) trichloroacetic acid;

[0124] Group J:

[0125] First reagent: 30 μL of 140 mM potassium citrate, pH 1;

[0126] Second reagent combination: Wash J: containing 46% (v / v) acetonitrile;

[0127] Group K:

[0128] First reagent: 30 μL of 140 mM potassium citrate, pH 1;

[0129] Second reagent combination: Wash K: contains 60 mM (2-hydroxy-3N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate;

[0130] The peptide content and SDS content in the peptide solution were measured separately, and the experimental results are shown in Table 2 below.

[0131] Table 2

[0132]

[0133] All data values ​​for the above groups were obtained by averaging the data values ​​of two samples. As can be seen from the table above, the peptide recovery rate of all groups was greater than 40%; only groups A and B had no SDS residue, and the SDS removal rate reached 100%; although the residual SDS detected in group F was only 10 ng, it would still be affected by SDS for extremely sensitive mass spectrometry instruments, such as Bruker's timsTOF PRO.

[0134] The results from Group A and Group B show that, regardless of whether the acidification solution is composed of potassium citrate or sodium citrate, as long as the component of washing solution A is WashB, the SDS removal rate can reach 100%. However, when the acidification solution is composed of potassium citrate (Group B), the peptide recovery rate is about 10% higher than that of the sodium citrate group (Group A). ​​Therefore, potassium citrate is superior to sodium citrate.

[0135] Example 4: Comparison of peptide recovery rate and protein identification amount between the present invention and other methods

[0136] The content of peptide powder obtained by the two extraction methods in Example 1 was measured using the BCA method, and the peptide recovery rate was calculated. As shown in Table 3 below, the data values ​​for all groups were obtained by averaging the data values ​​of three samples.

[0137] Table 3

[0138]

[0139] 0.1% formic acid was added to the peptide powder to reconstitute it, and then LC-MS analysis was performed. The instrument conditions are shown in Table 4 below.

[0140] Table 4

[0141]

[0142] The experimental results are shown in Table 5.

[0143] Table 5

[0144]

[0145]

[0146] All data values ​​in the above groups were obtained by averaging the data values ​​of three samples. The experimental results show that the method of the present invention only requires less than one-tenth (4μg / 50μg) of the sample starting amount. For various animal and plant tissues, the number of protein identifications and peptide identifications can reach 97%-101% and 91%-106% of the results of in-solution methods, respectively. This shows that the identification results of the present invention are comparable to those of in-solution methods.

[0147] Meanwhile, using this invention, SDS does not require overnight precipitation with acetone, thus saving a significant amount of time, allowing the peptide preparation process to be completed in just over two hours.

[0148] Example 5: Detection results of washing samples with different compositions of the present invention

[0149] The following washing composition was designed and extracted according to the method described in Example 1 (samples were prepared using 293T cell lysis buffer):

[0150] A. First reagent: 140mM sodium citrate;

[0151] Second reagent combination: 1% (w / v) trichloroacetic acid, 46% (v / v) acetonitrile, 60 mM (2-hydroxy-3N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate;

[0152] B. First reagent: 140mM sodium chloride;

[0153] Second reagent combination: 1% (w / v) trichloroacetic acid, 20% (v / v) acetonitrile, 60 mM (2-hydroxy-3N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate;

[0154] C. First reagent: 140mM sodium sulfate;

[0155] Second reagent combination: 0.1% (w / v) trichloroacetic acid, 46% (v / v) acetonitrile, 60 mM (2-hydroxy-3N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate;

[0156] D. First reagent: 140mM potassium citrate;

[0157] Second reagent combination: 0.1% (w / v) trichloroacetic acid, 20% (v / v) acetonitrile, 60 mM (2-hydroxy-3N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate;

[0158] E. First reagent: 140mM potassium chloride;

[0159] Second reagent combination: 1% (w / v) trichloroacetic acid, 46% (v / v) acetonitrile, 100 mM (2-hydroxy-3N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate;

[0160] F. First reagent: 140mM potassium sulfate;

[0161] Second reagent combination: 0.1% (w / v) trichloroacetic acid, 20% (v / v) acetonitrile, 5 mM (2-hydroxy-3N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate;

[0162] The residual SDS was detected simultaneously, and the results are shown in Table 6. As can be seen from the table, the washing compositions of the present invention with different ratios can effectively remove SDS.

[0163] Table 6

[0164]

[0165] Example 6: Detection results of washing SDS samples of different concentrations with the composition of the present invention.

[0166] Furthermore, the results of washing samples with different mass concentrations of SDS using the composition of the present invention to remove SDS residue were investigated. Table 7 shows the SDS residue results after adding different mass concentrations of SDS. As can be seen from Table 7, even when the mass concentration of SDS used for pyrolysis is as high as 1%, the composition of the present invention can still completely remove SDS residue.

[0167] Table 7

[0168]

[0169] Example 7: Preparation of peptides by washing protein samples of different concentrations using the method of the present invention.

[0170] The following samples were tested using the method described in Example 1. The samples were prepared as follows: 293T cells were lysed with 0.4% SDS (buffered with 20 mM 4-hydroxyethylpiperazine ethanesulfonic acid, pH 9, and a protease inhibitor) to obtain crude protein extracts. Appropriate amounts of the crude protein extract were diluted with 0.4% SDS to create sample groups containing 1 ng, 10 ng, 100 ng, 500 ng, 1 μg, and 4 μg of protein per 30 μL. The instrumentation conditions are shown in Table 8, and the results are shown in Table 9. The washing composition of this invention can be used to prepare clean peptides for proteomics research, processing protein samples as low as 1 ng.

[0171] Table 8

[0172]

[0173] Table 9

[0174]

[0175] Example 7: Extraction of samples processed using the method of the present invention for different types of samples.

[0176] Samples: Tissues (fresh mouse kidneys and mouse kidneys soaked in formalin for 50 days) were ground in liquid nitrogen and then subjected to 2% (w / v) SDS (both buffers containing 20 mM 4-hydroxyethylpiperazine ethanesulfonic acid, pH 9, and a protease inhibitor). The samples were thoroughly lysed by contact sonication to obtain crude protein extracts. The samples were then subjected to proteomics pretreatment according to the method of this invention to obtain clean peptides. The conditions for proteomics testing are shown in Table 3 (mice), and the results are shown in Table 10. As can be seen from Table 10, the clean peptides prepared using the washing composition of this invention for proteomics research yielded excellent protein identification levels for both fresh and formalin-soaked samples.

[0177] Table 10

[0178]

[0179]

Claims

1. A washing composition comprising the following reagents and reagent combinations: a first reagent:a potassium salt or a sodium salt; and a second reagent combination:trichloroacetic acid, acetonitrile, a cyclodextrin or a cyclodextrin derivative.

2. The washing composition according to claim 1, characterized in that, The cyclodextrin or cyclodextrin derivative is (2-hydroxy-3N, N, N-trimethylamino) propyl chloride-β-cyclodextrin hydrate.

3. The washing composition according to claim 1, characterized in that, The potassium salt and sodium salt are chloride, citrate, dihydrogen phosphate, hydrogen phosphate, acetate, sulfate, sorbate.

4. The washing composition according to claim 1, characterized in that, The concentration of the first reagent is 10-1500 mM.

5. The washing composition according to claim 1, characterized in that, In the second reagent combination, the mass / volume final concentration of trichloroacetic acid in the second reagent combination is 0.1-20%; the concentration of acetonitrile is the volume / volume final concentration in the second reagent combination, which is 0.1-90% (v / v) ; the final concentration of cyclodextrin or cyclodextrin derivative in the second reagent combination is 1-300 mM. 6.Use of the washing composition according to any one of claims 1-5 for a kit for removing surfactant. 7.A protein extraction kit comprising the washing composition according to any one of claims 1-5.

8. The protein extraction kit of claim 7, wherein, The kit further comprises a kit for extracting protein.

9. The protein extraction kit according to claim 7 or 8, characterized in that, The kit further comprises a third reagent and a fourth reagent, wherein the third reagent is a 0.1-10% (v / v) acidic solution, and the fourth reagent is a 5-100% organic reagent. 10.A method for extracting protein, comprising the following steps: S1, protein lysis:adding surfactant to the sample for lysis; S2, protein pre-enrichment; precipitating the surfactant and enriching the protein using the first reagent of the washing composition according to any one of claims 1-5; S3, surfactant removal:removing the surfactant using the second reagent combination of the washing composition according to any one of claims 1-5; S4, extracting protein.

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