A proteome extraction pretreatment combination, and kit thereof

Abstract

This invention belongs to the field of proteomics extraction, specifically relating to a proteomics extraction pretreatment combination, and more specifically, to a proteomics extraction pretreatment combination for extracting trace amounts of protein (nanogram level). This invention provides a proteomics extraction pretreatment combination comprising: a first reagent: cyclodextrin or a cyclodextrin derivative; a second reagent combination: an organic solvent, an acid, and (2-hydroxypropyl)-α-cyclodextrin and / or (2-hydroxy-3-N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate; and a third reagent: sodium 3-[(2-methyl-2-undecyl-1,3-dioxolane-4-yl)methoxy]-1-propanesulfonate. Through the synergistic effect between the reagents and the reagent combination, the proteome of samples that are difficult to extract using existing technologies can be extracted. In particular, it can be used for nanogram-level proteomics samples. The combination of this invention achieves a peptide recovery rate of over 60% while simultaneously improving the depth of protein identification.

Description

Technical Field

[0001] This invention belongs to the field of proteomics extraction, specifically, it relates to a proteomics extraction pretreatment combination, and more specifically, it relates to a proteomics extraction pretreatment combination for extracting trace amounts of protein (nanogatari level). Background Technology

[0002] Proteomics is the study of the structure, function, interactions, and dynamic changes of all proteins in cells or organisms. Its core objective is to comprehensively understand the types, functions, and regulation of proteins in biological systems. Proteomics has wide applications in many fields, primarily including disease research, drug development, biomarker discovery, and systems biology. Therefore, proteomics plays a vital role in biological research.

[0003] Proteomics studies the proteome; therefore, extracting the proteome from a sample is crucial for proteomics research. However, current techniques are insufficient for extracting the proteome from samples, particularly those that are difficult to extract from, such as tissue sections. This is especially true for extracting proteomes from samples with very low proteome content, often resulting in insufficient samples for subsequent research.

[0004] Therefore, there is a need in the art for a proteome extraction pretreatment assembly that can be used to extract proteomes, particularly to extract samples that are difficult to extract, such as tissue sections, and more particularly, to extract proteomes at the microscale (nanogram level). Summary of the Invention

[0005] In view of this, in a first aspect, the present invention provides a proteomics extraction pretreatment assembly, comprising:

[0006] First reagent: cyclodextrin or cyclodextrin derivative;

[0007] The second reagent combination consists of an organic solvent, an acid, and (2-hydroxypropyl)-α-cyclodextrin and / or (2-hydroxy-3-N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate.

[0008] Third reagent: Sodium 3-[(2-methyl-2-undecyl-1,3-dioxolane-4-yl)methoxy]-1-propanesulfonate.

[0009] Furthermore, the first reagent, the second reagent combination, and the third reagent are packaged separately.

[0010] Using the proteomics extraction pretreatment combination of the present invention, the proteome of samples that are difficult to extract in the prior art can be extracted through the synergistic effect between reagents and reagent combinations. In particular, it can be used for nanogram-level proteomics samples. The combination of the present invention enables extraction with a peptide recovery rate of more than 60% while improving the depth of protein identification.

[0011] Further, the cyclodextrin or cyclodextrin derivative mentioned in the first reagent can 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. Cyclodextrin, 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 Chlorinated 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-( 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.

[0012] Furthermore, in the first reagent, the cyclodextrin or cyclodextrin derivative is preferably (2-hydroxypropyl)-α-cyclodextrin or (2-hydroxy-3-N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate.

[0013] Furthermore, in the second reagent combination, the organic solvent can be one or more of methanol, ethanol, acetonitrile, and isopropanol, preferably methanol.

[0014] Furthermore, in the second reagent combination, the acid can be one or more of formic acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, tribromoacetic acid, and triiodoacetic acid, preferably trichloroacetic acid.

[0015] Preferably, the second reagent combination includes methanol, trichloroacetic acid, and 2-hydroxy-3-N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate. Using this second reagent combination yields the highest peptide recovery rate.

[0016] Furthermore, the proteome extraction pretreatment combination also includes a fourth reagent: SDS.

[0017] The compositions of the present invention can use SDS as a lysis buffer for samples and can also efficiently remove SDS. Through the combination of these reagents and reagent combinations, nanogram-level proteome samples can be extracted more effectively.

[0018] Furthermore, the amount of cyclodextrin or cyclodextrin derivative in the first reagent is 5 to 50 times the amount of the fourth reagent.

[0019] Furthermore, the final concentration of SDS in the composition is 0.05%-10% by volume.

[0020] Furthermore, in the second reagent combination, the organic solvent accounts for 0.1% to 90% of the volume / volume concentration of the second reagent combination, preferably 40% to 80%.

[0021] Furthermore, in the second reagent combination, the acid accounts for 0.1% to 20% of the mass / volume concentration of the second reagent combination, preferably 0.5% to 5%.

[0022] Furthermore, in the second reagent combination, the concentration of (2-hydroxypropyl)-α-cyclodextrin and / or (2-hydroxy-3-N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate is 1–300 mM.

[0023] Further, the concentration of the third reagent is 0.01 to 1 μg / μL, preferably 0.1 μg / μL.

[0024] Furthermore, the third reagent may also include one or more of ammonium bicarbonate, chloroacetamide, trypsin, lysine protease, etc.

[0025] Secondly, the present invention provides a use of the above combination for preparing a protein extraction kit.

[0026] Furthermore, the protein is a proteome.

[0027] Furthermore, the protein is a polypeptide.

[0028] Furthermore, the use of the above combination in preparing a nanogram-level protein extraction kit has been improved.

[0029] Thirdly, the present invention provides a protein extraction kit, comprising the proteome extraction pretreatment combination as described above.

[0030] Furthermore, the kit also includes a protein extraction kit.

[0031] 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.

[0032] 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.

[0033] In one specific implementation, the solid-phase extraction column is as follows: Figure 1 The structure is shown.

[0034] Fourthly, the present invention provides a method for extracting proteins, comprising the following steps:

[0035] Protein lysis: A surfactant is added to the sample to induce lysis;

[0036] Protein pre-enrichment and preliminary removal of surfactant: Proteins are enriched and SDS is initially removed using the first reagent as described above;

[0037] Surfactant removal: The surfactant is removed using the second reagent combination as described above;

[0038] Alkylation and enzymatic digestion of proteins: Alkylation and enzymatic digestion of proteins using a third reagent combination; and extraction of proteins.

[0039] 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.

[0040] 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.

[0041] Furthermore, the pH value for protein cleavage is 7–10.

[0042] Furthermore, the protein lysis also includes heating and / or sonication for lysing the sample.

[0043] Furthermore, the protein pre-enrichment and preliminary removal of surfactants also include a protein extraction kit, which may be a solid-phase extraction column.

[0044] Furthermore, the pH value for the protein pre-enrichment and preliminary removal of surfactant is 0.1–3.

[0045] Furthermore, the protein extraction method further includes one or more of the following steps: reduction, transfer of peptides, desalting of peptides, elution of peptides, or drying of peptides. Attached Figure Description

[0046] 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;

[0047] Figure 2 The absorbance values ​​of SDS and samples with concentrations ranging from 0 to 0.005% (w / v) are given for the SDS standard curve at wavelengths of 400-500 nm. Detailed Implementation

[0048] 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.

[0049] Example 1: Protein Extraction Method

[0050] (1) Initial protein extraction: Add 0.4 (w / v)% SDS solution (containing 20 mM 4-hydroxyethylpiperazine ethanesulfonic acid, pH 9, protease inhibitor) to the sample to fully lyse the biological sample and obtain crude protein extract.

[0051] (2) Protein sample pretreatment: Take the crude protein extract from (1) and add 0.4 (w / v)% SDS solution to dilute to a protein concentration of 66.7 ng / μL;

[0052] (3) Protein pre-enrichment and removal:

[0053] ① Add 60 μL of acetonitrile and 60 μL of 3 mol / L ammonia water to the self-made solid phase extraction column in sequence, and activate the solid phase extraction column by centrifugation.

[0054] ② Protein pre-enrichment and preliminary SDS removal:

[0055] 60 μL of a 3 mol / L ammonia solution containing 0.1% (w / v) DDM, 30 μL of the crude protein extract (containing 2 μg of total protein) from (2) above, and 33.3 μL of the first reagent (so that the amount of cyclodextrin or cyclodextrin derivative added is 20 times the amount of SDS in the crude protein extract) were sequentially added to a self-made solid-phase extraction column. The sample solution was alkalized to pH 12, and the protein was enriched onto the mixed packing material of strong anion exchange resin by centrifugation.

[0056] (4) Protein precipitation: 200 μL of ammonia and acetonitrile mixed solution (containing 20% ​​(v / v) acetonitrile and 3 mol / L ammonia) and 200 μL of acetonitrile were passed through a self-made solid phase extraction column to precipitate the protein in the mixed packing of strong anion exchange resin.

[0057] (5) SDS removal: SDS is further removed using a second reagent combination of the washing composition of the present invention.

[0058] (6) Cleaning: Use 60 μL of 80% (v / v) acetonitrile to clean twice to remove residual washing composition of the present invention, etc.

[0059] (7) Reduction: Add 5 μL of reducing agent containing 50 mM dithiothreitol and 20 mM ammonium bicarbonate, and react at 37 °C for 30 min. Then add 20 μL of 20 mM ammonium bicarbonate solution to wash the self-made solid phase extraction column.

[0060] (8) Alkylation and enzymatic hydrolysis: Add 6 μL of alkylation and enzymatic hydrolysis reagent, i.e. the third reagent combination, into the solid phase extraction column. Centrifuge to allow the alkylation and enzymatic hydrolysis reagent to enter the packing material. React at 37°C for 3 h to enzymatically hydrolyze the protein in the packing material into peptides.

[0061] (9) Transfer of peptides: Add 60 μL of high-salt solution (containing 1 mol / L NaCl and 1% (v / v) formic acid) to the solid-phase extraction column, and transfer the peptides in the packing material to the solid-phase extraction membrane by centrifugation.

[0062] (10) Desalting of peptides: Add 60 μL of 1% (v / v) formic acid to the solid phase extraction column and wash the solid phase extraction column to remove residual salt by centrifugation.

[0063] (11) Elution: Add 20 μL of elution buffer (containing 0.5% (v / v) acetic acid and 80% (v / v) acetonitrile) to the solid phase extraction column and collect the peptides into the collection tube by centrifugation.

[0064] (12) Drying: The polypeptide solution collected in the above steps is placed in a freeze concentrator and dried at 35°C for 2 hours to prepare polypeptide powder.

[0065] Example 2: SDS Residue Detection Method

[0066] The peptide powder obtained in Example 1 was reconstituted by adding 20 μL of PBS working solution (containing 14.3 mM phosphate buffer, 21.4 mM sodium chloride, pH 7.2). 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 shown in Table 1 (detection range 0-0.005% (w / v) SDS), and the full spectrum scan was exported. The results are as follows: Figure 2 As shown.

[0067] Table 1

[0068] name Protein sample types SDS concentration (w / v) % 0% SDS / 0 0.001% SDS / 0.001 0.002% SDS / 0.002 0.003% SDS / 0.003 0.004% SDS / 0.004 0.005% SDS / 0.005 Pos-Ctrl / 0.0045 Neg-Ctrl / 0 Sample / To be tested

[0069] like Figure 2 As shown, the absorbance of the 0-0.005% (w / v) SDS standard curve at a wavelength of 453 nm increases linearly; the Pos-Ctrl group is a peptide solution diluted with 0.0045% (w / v) SDS; while the Neg-Ctrl group is a peptide solution without SDS; the Sample group is a peptide solution obtained by pretreatment of the protein cleaved with 0.4% (w / v) SDS according to the present invention.

[0070] The spectra show that the absorbance values ​​of the Pos-Ctrl group at 450-460 nm fall between the absorbance values ​​of the standard curves for 0.004% (w / v) SDS and 0.005% (w / v) SDS at the same 450-460 nm. In contrast, the Neg-Ctrl group and the Sample group almost completely overlap with the curves for 0% (w / v) SDS, and their absorbance values ​​at 450 nm and 455 nm are identical to those for 0% (w / v) SDS at the same 450 nm and 455 nm. This result indicates that the peptides obtained by the protein extraction method of this invention do not contain SDS.

[0071] Example 3: Extraction results of different combinations of the present invention

[0072] To further clarify the superiority of the combination of the present invention, the following system combination was designed and extracted according to the method described in Example 1 (the sample used 293T cell lysis buffer).

[0073] Group A:

[0074] First reagent:

[0075] AD1: 33.3 μL of purified water;

[0076] Second reagent combination:

[0077] Wash B: 60 μL containing 1% (w / v) trichloroacetic acid, 46% (v / v) acetonitrile, and 60 mM (2-hydroxy-3-N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate;

[0078] Third reagent combination: 1 μg / μL sodium 3-[(2-methyl-2-undecyl-1,3-dioxolane-4-yl)methoxy]-1-propanesulfonate;

[0079] Group B:

[0080] First reagent:

[0081] AD2: 33.3 μL of 250 mM (2-hydroxy-3N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate;

[0082] Second reagent combination:

[0083] Wash B: 60 μL containing 1% (w / v) trichloroacetic acid, 46% (v / v) acetonitrile, and 60 mM (2-hydroxy-3-N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate;

[0084] Third reagent combination: 1 μg / μL sodium 3-[(2-methyl-2-undecyl-1,3-dioxolane-4-yl)methoxy]-1-propanesulfonate;

[0085] Group C:

[0086] First reagent:

[0087] AD3: 33.3 μL of 250 mM (2-hydroxypropyl)-α-cyclodextrin;

[0088] Second reagent combination:

[0089] Wash B: 60 μL containing 1% (w / v) trichloroacetic acid, 46% (v / v) acetonitrile, and 60 mM (2-hydroxy-3-N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate;

[0090] Third reagent combination: 1 μg / μL sodium 3-[(2-methyl-2-undecyl-1,3-dioxolane-4-yl)methoxy]-1-propanesulfonate;

[0091] Group D:

[0092] First reagent:

[0093] AD3: 33.3 μL of 250 mM (2-hydroxypropyl)-α-cyclodextrin;

[0094] Second reagent combination:

[0095] Wash D: 60 μL containing 1% (w / v) formic acid, 46% (v / v) acetonitrile, and 60 mM (2-hydroxy-3-N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate;

[0096] Third reagent combination: 1 μg / μL sodium 3-[(2-methyl-2-undecyl-1,3-dioxolane-4-yl)methoxy]-1-propanesulfonate;

[0097] Group E:

[0098] First reagent:

[0099] AD3: 33.3 μL of 250 mM (2-hydroxypropyl)-α-cyclodextrin;

[0100] Second reagent combination:

[0101] Wash E: 60 μL containing 1% (w / v) trichloroacetic acid, 46% (v / v) methanol, and 60 mM (2-hydroxy-3-N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate;

[0102] Third reagent combination: 1 μg / μL sodium 3-[(2-methyl-2-undecyl-1,3-dioxolane-4-yl)methoxy]-1-propanesulfonate;

[0103] Group F:

[0104] First reagent:

[0105] AD3: 33.3 μL of 250 mM (2-hydroxypropyl)-α-cyclodextrin;

[0106] Second reagent combination:

[0107] Wash F: 60 μL containing 1% (w / v) trichloroacetic acid, 46% (v / v) acetonitrile, and 60 mM (2-hydroxypropyl)-α-cyclodextrin;

[0108] Third reagent combination: 1 μg / μL sodium 3-[(2-methyl-2-undecyl-1,3-dioxolane-4-yl)methoxy]-1-propanesulfonate;

[0109] Group G:

[0110] First reagent:

[0111] AD3: 33.3 μL of 250 mM (2-hydroxypropyl)-α-cyclodextrin;

[0112] Second reagent combination:

[0113] Wash G:

[0114] 60 μL of a solution containing 46% (v / v) acetonitrile and 60 mM (2-hydroxy-3-N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate;

[0115] Third reagent combination: 1 μg / μL sodium 3-[(2-methyl-2-undecyl-1,3-dioxolane-4-yl)methoxy]-1-propanesulfonate;

[0116] Group H:

[0117] First reagent:

[0118] AD3: 33.3 μL of 250 mM (2-hydroxypropyl)-α-cyclodextrin;

[0119] Second reagent combination:

[0120] Wash H: 60 μL of hydrate containing 1% (w / v) trichloroacetic acid and 60 mM (2-hydroxy-3-N,N,N-trimethylamino)propylchloro-β-cyclodextrin;

[0121] Third reagent combination: 1 μg / μL sodium 3-[(2-methyl-2-undecyl-1,3-dioxolane-4-yl)methoxy]-1-propanesulfonate;

[0122] Group I:

[0123] First reagent:

[0124] AD3: 33.3 μL of 250 mM (2-hydroxypropyl)-α-cyclodextrin;

[0125] Second reagent combination:

[0126] Wash I: Contains 1% (w / v) trichloroacetic acid and 46% (v / v) acetonitrile;

[0127] Third reagent combination: 1 μg / μL sodium 3-[(2-methyl-2-undecyl-1,3-dioxolane-4-yl)methoxy]-1-propanesulfonate;

[0128] Group J:

[0129] First reagent:

[0130] AD3: 33.3 μL of 250 mM (2-hydroxypropyl)-α-cyclodextrin;

[0131] Second reagent combination:

[0132] Wash J: 60 μL of 1% (w / v) trichloroacetic acid;

[0133] Third reagent combination: 1 μg / μL sodium 3-[(2-methyl-2-undecyl-1,3-dioxolane-4-yl)methoxy]-1-propanesulfonate;

[0134] Group K:

[0135] First reagent:

[0136] AD3: 33.3 μL of 250 mM (2-hydroxypropyl)-α-cyclodextrin;

[0137] Second reagent combination:

[0138] Wash K: 60 μL of 46% (v / v) acetonitrile;

[0139] Third reagent combination: 1 μg / μL sodium 3-[(2-methyl-2-undecyl-1,3-dioxolane-4-yl)methoxy]-1-propanesulfonate;

[0140] Group L:

[0141] First reagent:

[0142] AD3: 33.3 μL of 250 mM (2-hydroxypropyl)-α-cyclodextrin;

[0143] Second reagent combination:

[0144] Wash K: 60 μL of 60 mM (2-hydroxy-3-N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate;

[0145] Third reagent combination: 1 μg / μL sodium 3-[(2-methyl-2-undecyl-1,3-dioxolane-4-yl)methoxy]-1-propanesulfonate;

[0146] The peptide content and SDS content in the peptide solution were measured separately, and the experimental results are shown in Table 2 below.

[0147] Table 2

[0148]

[0149] As can be seen from Table 2, using the combination of the present invention, the peptide recovery rate can reach more than 60%, and the residual SDS is 0.

[0150] Example 4: Results of the combined extraction of nanogram-level samples according to the present invention

[0151] The effect of sodium 3-[(2-methyl-2-undecyl-1,3-dioxolane-4-yl)methoxy]-1-propanesulfonate in the third reagent combination in step (8) of Example 1 above on the experimental results was tested.

[0152] Group A: Alkylation and enzymatic digestion reagents, a mixed solution containing 20 mM ammonium bicarbonate, 50 mM chloroacetamide, 0.1 μg / μL sodium 3-[(2-methyl-2-undecyl-1,3-dioxolane-4-yl)methoxy]-1-propanesulfonate, 20 ng / μL trypsin, 2 ng / μL LysC lysine protease, etc.

[0153] Group B: Alkylation and enzymatic hydrolysis reagents, a mixed solution containing 20 mM ammonium bicarbonate, 50 mM chloroacetamide, 20 ng / μL Trypsin trypsin, 2 ng / μL LysC lysine protease, etc.

[0154] Group C: Alkylation and enzymatic hydrolysis reagents, a mixed solution containing 20mM ammonium bicarbonate, 50mM chloroacetamide, 1mol / L urea, 20ng / μL LTrypsin trypsin, 2ng / μL LysC lysine protease, etc.

[0155] Group D: Alkylation and enzymatic digestion reagents, a mixed solution containing 20 mM ammonium bicarbonate, 50 mM chloroacetamide, 1 μg / μL LDC (sodium deoxycholate), 20 ng / μL Trypsin, 2 ng / μL LysC lysine protease, etc.

[0156] 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 3 below.

[0157] Table 3

[0158]

[0159] The experimental results are shown in Table 4.

[0160] Table 4

[0161]

[0162] As can be seen from the table, the sodium 3-[(2-methyl-2-undecyl-1,3-dioxolane-4-yl)methoxy]-1-propanesulfonate used in the combination of the present invention has a specific effect of increasing the depth of protein identification, and has a better effect on the auxiliary enzymatic hydrolysis compared with other denaturing agents.

[0163] Example 5: Results of sample processing with different combinations according to the present invention

[0164] The following system combination was designed and extracted according to the method described in Example 1 (samples were extracted using 293T cell lysis buffer):

[0165] Group A:

[0166] Protein sample: 30 μL of crude protein extract (0.4% SDS lysis, containing 20 ng of total protein)

[0167] First reagent combination:

[0168] AD1: 33.3 μL of 250 mM (2-hydroxypropyl)-α-cyclodextrin;

[0169] Second reagent combination:

[0170] Wash B: 60 μL containing 1% (w / v) trichloroacetic acid, 46% (v / v) acetonitrile, and 60 mM (2-hydroxy-3-N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate;

[0171] Third reagent combination:

[0172] A mixed solution containing 20 mM ammonium bicarbonate, 50 mM chloroacetamide, 1 μg / μL sodium 3-[(2-methyl-2-undecyl-1,3-dioxolane-4-yl)methoxy]-1-propanesulfonate, 20 ng / μL trypsin, 2 ng / μL LysC lysine protease, etc.

[0173] Group B:

[0174] Protein sample: 30 μL of crude protein extract (0.4% SDS lysis, containing 20 ng of total protein)

[0175] First reagent combination:

[0176] AD1: 33.3 μL of 250 mM (2-hydroxypropyl)-α-cyclodextrin; Second reagent combination:

[0177] Wash C: 60 μL containing 1% (w / v) trichloroacetic acid, 46% (v / v) methanol, and 60 mM (2-hydroxy-3-N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate;

[0178] Third reagent combination:

[0179] A mixed solution containing 20 mM ammonium bicarbonate, 50 mM chloroacetamide, 1 μg / μL sodium 3-[(2-methyl-2-undecyl-1,3-dioxolane-4-yl)methoxy]-1-propanesulfonate, 20 ng / μL trypsin, 2 ng / μL LysC lysine protease, etc.

[0180] Group C:

[0181] Protein sample: 30 μL of crude protein extract (0.4% SDS lysis, containing 20 ng of total protein)

[0182] First reagent combination:

[0183] AD1: 33.3 μL of 250 mM (2-hydroxypropyl)-α-cyclodextrin;

[0184] Second reagent combination:

[0185] Wash D: 60 μL containing 1% (w / v) trichloroacetic acid, 80% (v / v) methanol, and 100 mM (2-hydroxy-3-N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate;

[0186] Third reagent combination:

[0187] A mixed solution containing 20 mM ammonium bicarbonate, 50 mM chloroacetamide, 1 μg / μL sodium 3-[(2-methyl-2-undecyl-1,3-dioxolane-4-yl)methoxy]-1-propanesulfonate, 20 ng / μL trypsin, 2 ng / μL LysC lysine protease, etc.

[0188] The conditions for using the computer are shown in Table 5 below.

[0189] Table 5

[0190]

[0191]

[0192] The results are shown in Table 6.

[0193] Table 6

[0194]

[0195] Example 6: Sample results from different methods

[0196] The control group method is as follows:

[0197] (1) Protein pre-enrichment:

[0198] 15 μL of 140 mM potassium citrate (pH 1) was loaded into a pre-activated solid-phase extraction column; 30 μL of the crude protein extract containing 20 ng of protein was loaded into the solid-phase extraction column; 15 μL of 140 mM potassium citrate (pH 1) was added above the solid-phase extraction column packing; and the protein was enriched by centrifugation.

[0199] (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.

[0200] (4) SDS removal: SDS was removed using a mixed solution of 60 μL containing 1% (w / v) trichloroacetic acid, 46% (v / v) acetonitrile and 60 mM (2-hydroxy-3-N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate.

[0201] (5) Cleaning: Clean twice with 60 μL of acetonitrile;

[0202] (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.

[0203] (7) Alkylation and enzymatic hydrolysis: Add 8 μL of alkylation and enzymatic hydrolysis reagent (containing 50 mM Tris-HCl, 50 mM chloroacetamide, 110 ng / μL Trypsin, and 11 ng / μL LysC lysine protease) 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.

[0204] (8) Transfer of peptides: 60 μL of a high-salt solution containing 1 mol / L NaCl and 10 mM formate was added to the solid-phase extraction column, and the peptides in the packing were transferred to the solid-phase extraction membrane by centrifugation.

[0205] (9) Desalting of peptides: Add 60 μL of 0.1% (v / v) formic acid and wash the solid phase extraction column by centrifugation to remove residual salt. Repeat the washing once.

[0206] (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.

[0207] (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.

[0208] (12) Add 5 μL of 0.1% FA to reconstitute.

[0209] The conditions for using the computer are shown in Table 7 below.

[0210] Table 7

[0211]

[0212] The results are shown in Table 8.

[0213] Table 8

[0214]

[0215]

[0216] Experimental results show that, after preprocessing 20 ng protein samples using the methods of the present invention and the control group, the number of proteins identified by the present invention is 1148 higher than that of the control group, and the number of peptides is also 12,800 more.

[0217] Example 7: Sample loading range of the extraction combination of the present invention

[0218] 293T cells were lysed with 0.4% (w / v) SDS (buffered with 20 mM 4-hydroxyethylpiperazine ethanesulfonic acid, pH 9, and protease inhibitor) to obtain crude protein extracts. Appropriate amounts of the crude protein extract were diluted with 0.4% (w / v) SDS to create sample groups containing 2.5 ng, 10 ng, 20 ng, 40 ng, 2 μg, and 4 μg of protein per 30 μL. The instrumentation conditions are shown in Table 9, and the results are shown in Table 10.

[0219] Experimental results show that the clean peptides prepared using the combination of the present invention for proteomics research can process protein samples as low as 2.5 ng.

[0220] Table 9

[0221]

[0222]

[0223] Table 10

[0224]

[0225] Example 8: Detection results of washing samples with different concentrations of SDS using the combination of the present invention (0.1%-4% (w / v) SDS)

[0226] Furthermore, the SDS residue results of the combined washing of the present invention with SDS samples containing different mass concentrations were investigated. The SDS residue results are shown in Table 11, which lists the different mass concentrations of SDS added. As can be seen from Table 11, even when the mass concentration of SDS used for pyrolysis is as high as 4%, the composition of the present invention can still completely remove SDS residue.

[0227] Table 11

[0228]

[0229] Example 9: Detection results of FFPE slide samples processed using the combined method of the present invention (1% DDM and 0.4% SDS)

[0230] This embodiment aims to clarify the results for difficult-to-process samples. When faced with difficult-to-process samples, 1% (w / v) DDM and 0.4% (w / v) SDS were used to lyse FFPE mouse liver tissue sections. However, in practice, DDM treatment of some difficult-to-process samples was not effective, so SDS was preferred. However, residual SDS could affect identification. Therefore, the composition of this invention was developed. The results of peptide analysis after treatment with the composition of this invention are as follows:

[0231] The experimental results are shown in Table 13. The cleavage area was 0.25 mm using 1% DDM and 0.4% SDS, respectively. 2 After processing liver tissue sections from FFPE mice, the 0.4% SDS group showed 600 more protein identifications and over 10,000 more peptide identifications compared to the 1% DDM group, indicating a higher identification depth.

[0232] Table 12

[0233]

[0234]

[0235] Table 13

[0236]

Claims

1. A proteomics extraction pretreatment assembly, comprising: First reagent: cyclodextrin or cyclodextrin derivative; The second reagent combination consists of an organic solvent, an acid, and (2-hydroxypropyl)-α-cyclodextrin and / or (2-hydroxy-3-N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate. Third reagent: Sodium 3-[(2-methyl-2-undecyl-1,3-dioxolane-4-yl)methoxy]-1-propanesulfonate.

2. The proteomics extraction pretreatment combination according to claim 1, characterized in that, The first reagent, the second reagent combination, and the third reagent are packaged separately.

3. The proteomics extraction pretreatment combination according to claim 1, characterized in that, The cyclodextrin or cyclodextrin derivative in the first reagent is (2-hydroxypropyl)-α-cyclodextrin and / or (2-hydroxy-3-N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate.

4. The proteomics extraction pretreatment combination according to claim 1, characterized in that, In the second reagent combination, the organic solvent is one or more of methanol, ethanol, acetonitrile, and isopropanol, preferably methanol.

5. The proteomics extraction pretreatment combination according to claim 1, characterized in that, In the second reagent combination, the acid is one or more of formic acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, tribromoacetic acid, and triiodoacetic acid, preferably trichloroacetic acid.

6. The proteomics extraction pretreatment combination according to claim 1, characterized in that, The second reagent combination includes methanol, trichloroacetic acid, and 2-hydroxy-3-N,N,N-trimethylamino)propylchloro-β-cyclodextrin hydrate.

7. The proteomics extraction pretreatment combination according to any one of claims 1 to 6, characterized in that, The pretreatment combination for proteome extraction also includes a fourth reagent: SDS.

8. Use of the proteomics extraction pretreatment combination as described in any one of claims 1 to 7 for the preparation of a proteomics extraction kit.

9. A proteomics extraction kit, comprising a proteomics extraction pretreatment combination as described in any one of claims 1 to 6.

10. The proteomics extraction kit according to claim 9, characterized in that, The kit also includes a protein extraction kit.

Images