Method for preparing peptide containing sample

Abstract

To suppress the loss of peptide when preparing a peptide containing sample.SOLUTION: The adding of 2,2-didecyl propane-1,3-bis-β-D-malto pyranoside(LMNG) to a peptide aqueous solution suppresses the adsorption of peptide to a vessel, etc.; the LMNG can easily remove from the peptide by a reverse phase column; the adding of at least one kind of sugar based nonionic surfactant to an aqueous solvent dissolving dry peptide substantially promotes the dissolution of especially long peptide; and the combination of two kinds of sugar based nonionic surfactant, for example, the combination of n-dodecyl-β-D-maltoside(DDM) and α-D-glucopyranosyl-α-D-glucopyranoside mono-dodecanoate (trehalose C12) enhances the dry peptide dissolution promotion effect.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a technique for minimizing peptide loss in the preparation of a trace peptide-containing sample for use in a wide range of liquid chromatography mass spectrometry (LC-MS) analyses, etc. Specifically, the present invention relates to a method for preparing a peptide-containing sample, a method for dissolving dried peptides generated during the pretreatment of samples for proteome analysis, the use of a surfactant to promote dissolution of the dried peptides, etc. The present invention also relates to a stable aqueous peptide solution. [Background technology]

[0002] Proteome analysis is now in an era where more than 10,000 proteins can be observed in a single shot measurement, and further contributions to the fields of biology and medicine are expected. One way to further improve proteome analysis technology is to reduce peptide loss during sample preparation. The main pretreatment steps in proteome analysis include 1) protein extraction, 2) reduction and alkylation, 3) enzymatic digestion, 4) desalting using a reversed-phase spin column (mainly STAGE-Tip), 5) drying, and 6) resolubilization (Figure 1). Efforts are being made to improve the recovery rate and throughput in various steps. In recent years, with the emergence of the SP3 method and S-Trap method, it has become common to dissolve proteins from cells and tissues with a strong detergent, then reduce and alkylate them to remove the detergent and salt, and replace them with a solvent suitable for digestive enzymes before digesting the proteins. However, in trace samples, peptides are lost due to nonspecific adsorption to tubes and tips during long-term incubation such as protein digestion, or when digested peptides are transferred to another tube.

[0003] To prevent this loss, it is desirable to use surfactants, but the problem is that the surfactants are trapped in the reversed-phase column and eluted together with the peptides. In the case of a small amount of sample, it is common to dry the extract from the reversed-phase column, redissolve the peptides in a small amount of dissolving solution, and analyze the concentrated peptides by LC-MS. Naturally, this means that the concentrated surfactant is also analyzed at the same time, increasing the risk of reduced sensitivity due to MS contamination. There are methods to remove the surfactant after digestion by precipitating or decomposing the surfactant, but such treatments have various disadvantages, such as an increase in the number of work steps, which can result in peptide loss, reduced reproducibility, and labor. For this reason, it would be ideal to be able to remove the surfactant during general-purpose processing without special operations.

[0004] Drying of peptides also promotes non-specific adsorption to tubes, etc., which poses a risk of peptide loss. When the sample is small, the risk of peptide loss is particularly high. Considering that the analysis will be performed by LC-MS, the solution used to dissolve the dried peptide fragments is required to not interfere with the peptide analysis in LC-MS and to cause little contamination of the mass spectrometer. Therefore, to dissolve the dried peptide fragments, a solution containing about 2-5% (v / v) acetonitrile (ACN), which is generally volatile and does not affect the background of MS measurements, and about 0.1% (v / v) trifluoroacetic acid (TFA) or formic acid is often used. However, it is difficult to recover the dried peptide fragments non-specifically adsorbed to the container with a high yield.

[0005] One method to reduce the effect of nonspecific peptide adsorption is to add a carrier protein such as albumin to the sample, but this adds complexity to the sample preparation, affects the background of MS measurements, and has the potential to contaminate the mass spectrometer.

[0006] One method for reducing the effect of nonspecific adsorption of peptides is to add high concentrations of NaCl to the solution in which the peptides are dissolved. However, although high concentrations of NaCl can suppress adsorption due to ionic interactions to some extent, the suppression effect on adsorption due to hydrophobic interactions is limited.

[0007] Such peptide loss can be reduced by dissolving the dried peptide in a solvent containing a surfactant, but the surfactant may hinder peptide analysis by LC-MS due to reduced LC separation ability and suppression of ionization. Examples of surfactants suitable for LC-MS include those that can be decomposed, such as RapiGest SF (trademark) (Non-Patent Document 1) and MaSDeS (Non-Patent Document 2); those that can be removed, such as sodium deoxycholate (Non-Patent Documents 3 and 4) and sodium dodecanoate (Non-Patent Document 5); and those whose retention time of peptides in LC does not overlap with that of the surfactant, such as Invitrosol (trademark) (Non-Patent Document 6). However, decomposing or removing the surfactant requires a time-consuming process, and since the surfactant is no longer present in the solution after the process, there is a risk that the peptides will be nonspecifically adsorbed to tubes or vials. Surfactants that have LC retention times different from those of peptides can be directly measured by LC-MS without much effort, and are thought to have high stability of peptides after dissolution, but Invitrosol is an expensive reagent.

[0008] On the other hand, n-dodecyl-β-D-maltoside (DDM) is a nonionic surfactant with a sugar group in the hydrophilic part, and is mainly used to solubilize membrane proteins. DDM is inexpensive and has been used for pretreatment of single-cell proteome analysis, and has been shown to be effective in preventing loss of proteins and peptides (Non-Patent Documents 7, 8). However, its effect on dissolving dried peptides has not yet been examined.

[0009] Lauryl maltose neopentyl glycol (LMNG) (2,2-didecylpropane-1,3-bis-β-D-maltopyranoside), a nonionic detergent with two linked hydrophobic chains of equal length and two hydrophilic maltoside groups, has been shown to solubilize and stabilize membrane proteins better than DDM. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Mosen, PR et al., Proteomics 2021, 21(20), e2100129 [Non-Patent Document 2] Chang, YH et al., J Proteome Res 2015, 14(3), 1587-1599 [Non-Patent Document 3] Masuda, T. et al., J Proteome Res 2008, 7(2), 731-740 [Non-Patent Document 4] Sialana, FJ et al., J Proteome Res 2022, 21(8), 1842-1856 [Non-Patent Document 5] Lin, Y. et al., PLoS One 2013, 8 (3), e59779 [Non-Patent Document 6] Kawashima, Y. et al., Proteomics 2013, 13(5), 751-755 [Non-Patent Document 7] Liu, J. et al., Anal Chem 2015, 87 (4), 2054-2057 [Non-Patent Document 8] Tsai, CF et al., Commun Biol 2021, 4 (1), 265 Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention aims to provide a technique for suppressing peptide loss while avoiding contamination of LC-MS in the preparation of a sample containing a trace amount of peptide for use in proteome analysis, etc. Another aim of the present invention is to provide a technique for improving dissolution of dried peptides adsorbed on the wall of a tube during the pretreatment process of proteome analysis, etc., while at the same time preventing the dissolved peptides from nonspecifically re-adsorbing to a tube or chip. [Means for solving the problem]

[0012] The present inventors conducted extensive research to solve the above problems, and found that adding LMNG to a protein-containing sample when digesting the sample with a protease reduced the loss of peptides from the sample and increased the number of peptides and proteins identified in LC-MS. Adding LMNG during affinity purification of peptides also reduced the loss of peptides from the sample and increased the number of peptides and proteins identified in LC-MS. LMNG has a strong affinity to a reversed-phase column, and is retained in the column even under elution conditions that would elute most peptides from the reversed-phase column. As a result, LMNG was removed from peptides while avoiding the loss of peptides in the desalting step using the reversed-phase column, and the possibility of contamination of the LC-MS instrument was successfully minimized.

[0013] In addition, by adding a sugar-based nonionic surfactant such as DDM to the solution for recovering dried peptides, the solubility of the dried peptides was improved, and the recovery amount of the peptides was successfully improved. In particular, the recovery amount of long peptide fragments of 11 amino acids or more was significantly improved. Even with a concentration of DDM below the CMC, the effect of improving the recovery amount of peptide fragments was observed, and this effect was achieved even at a low concentration of 0.005% (w / w). Surprisingly, by adding two or more sugar-based nonionic surfactants in combination, the solubility of the dried peptides was improved beyond the additive effect. Based on these findings, the present inventors further investigated and completed the present invention.

[0014] That is, the present invention relates to the following. [1] A method for preparing a peptide-containing sample, comprising the steps of: 1) providing an aqueous peptide solution containing a peptide and 2,2-didecylpropane-1,3-bis-β-D-maltopyranoside (hereinafter referred to as "LMNG"); 2) subjecting the resulting peptide aqueous solution to a reverse phase column to adsorb the peptide and LMNG onto the reverse phase column; and 3) Removal of LMNG from the peptides by eluting the adsorbed peptides from the reversed-phase column while maintaining the adsorption of LMNG to the reversed-phase column. [2] The preparation method of [1], wherein the reverse phase column is a C18 column or a styrene-divinylbenzene copolymer column. [3] A preparation method according to [1] or [2], in which peptides are eluted from a reversed-phase column with an eluent having a polarity equivalent or greater to a mixture of acetonitrile / 0.1% (v / v) aqueous trifluoroacetic acid = 50 / 50 (v / v). [4] Any of the preparation methods [1] to [3], wherein the polarity of the eluent is equivalent to or less than a mixture of acetonitrile / 0.1% (v / v) aqueous trifluoroacetic acid = 30 / 70 (v / v). [5] Any of the preparation methods [1] to [4], in which the LMNG concentration in the aqueous peptide solution is below the critical micelle concentration. [6] The method according to any one of [1] to [5], wherein the aqueous peptide solution is provided in a container having a plastic surface. [7] The method for preparing any one of [1] to [6], further comprising the step of subjecting a protein dissolved in an aqueous solvent containing LMNG to limited hydrolysis with a protease to obtain an aqueous peptide solution containing the peptide and LMNG. [8] The preparation method according to any one of [1] to [7], further comprising subjecting the aqueous peptide solution provided in step 1) to affinity chromatography to obtain an aqueous peptide solution containing affinity-enriched peptides and LMNG. [9] The preparation method according to [8], wherein the affinity chromatography is immobilized metal affinity chromatography or avidin or streptavidin affinity chromatography.

[10] Any of the preparation methods [1] to [9], wherein the peptide-containing sample is for LC-MS analysis.

[0015]

[11] A method for producing an aqueous peptide solution, comprising dissolving a dry peptide in an aqueous solvent containing at least one sugar-based nonionic surfactant.

[12] The sugar-based nonionic surfactant is represented by the formula (I);

[0016] [ka]

[0017] (G1 is a monosaccharide or disaccharide group, and L1 is O, S, -O-(CH2) n -O- or -O-CO-, R1 is a linear alkyl group having 6 to 12 carbon atoms, and n is an integer of 1 to 3), or formula (II);

[0018] [ka]

[0019] (G2 and G3 are the same or different and are monosaccharide or disaccharide groups, and R2 and R3 are the same or different and are linear alkyl groups having 6 to 12 carbon atoms.) A method for producing a compound represented by the formula

[11] .

[13] The method for producing

[12] , wherein the sugar-based nonionic surfactant is a compound represented by formula (I), and G1 is any one selected from the group consisting of a glucose group, a mannose group, a maltose group, or a trehalose group.

[14] The method according to

[13] , wherein the sugar-based nonionic surfactant is n-dodecyl-β-D-maltoside, n-undecyl-β-D-maltoside, n-decyl-β-D-maltoside, n-nonyl-β-D-thiomaltoside, n-octyl-β-D-thioglucoside, 3-oxatridecyl-α-D-mannoside or α-D-glucopyranosyl-α-D-glucopyranoside monododecanoate.

[15] The method for producing

[12] , wherein the sugar-based nonionic surfactant is a compound represented by formula (II), and G2 and G3 are the same and are a glucose group or a maltose group.

[16] The method for producing

[15] , wherein the sugar-based nonionic surfactant is 2,2-dioctylpropane-1,3-bis-β-D-maltopyranoside, 2,2-didecylpropane-1,3-bis-β-D-maltopyranoside or 2,2-dihexylpropane-1,3-bis-β-D-glucopyranoside.

[17] The method according to any one of

[11] to

[16] , wherein the aqueous solvent contains a combination of at least two sugar-based nonionic surfactants.

[18] The method for producing the composition according to

[17] , wherein the combination of sugar-based nonionic surfactants is any one selected from the following group: n-Dodecyl-β-D-maltoside and α-D-glucopyranosyl-α-D-glucopyranoside monododecanoate; 2,2-Dihexylpropane-1,3-bis-β-D-glucopyranoside and α-D-glucopyranosyl-α-D-glucopyranoside monododecanoate; 2,2-Dioctylpropane-1,3-bis-β-D-maltopyranoside and 2,2-dihexylpropane-1,3-bis-β-D-glucopyranoside; and 3-Oxatridecyl-α-D-mannoside and 2,2-dioctylpropane-1,3-bis-β-D-maltopyranoside.

[19] The manufacturing method according to any one of

[11] to

[18] , wherein the dried peptide is contained in a container having a plastic surface.

[20] A dry peptide dissolution enhancer comprising at least one sugar-based nonionic surfactant.

[21] The dry peptide dissolution enhancer according to

[20] , which comprises a combination of at least two sugar-based nonionic surfactants.

[22] The dry peptide dissolution enhancer according to

[21] , wherein the combination of the sugar-based nonionic surfactant is any one selected from the following group: n-Dodecyl-β-D-maltoside and α-D-glucopyranosyl-α-D-glucopyranoside monododecanoate; 2,2-Dihexylpropane-1,3-bis-β-D-glucopyranoside and α-D-glucopyranosyl-α-D-glucopyranoside monododecanoate; 2,2-Dioctylpropane-1,3-bis-β-D-maltopyranoside and 2,2-dihexylpropane-1,3-bis-β-D-glucopyranoside; and 3-Oxatridecyl-α-D-mannoside and 2,2-dioctylpropane-1,3-bis-β-D-maltopyranoside.

[23] An aqueous peptide solution comprising a peptide and a combination of at least two sugar-based nonionic surfactants.

[24] The aqueous peptide solution according to

[23] , wherein the combination of the sugar-based nonionic surfactant is any one selected from the following group: n-Dodecyl-β-D-maltoside and α-D-glucopyranosyl-α-D-glucopyranoside monododecanoate; 2,2-Dihexylpropane-1,3-bis-β-D-glucopyranoside and α-D-glucopyranosyl-α-D-glucopyranoside monododecanoate; 2,2-Dioctylpropane-1,3-bis-β-D-maltopyranoside and 2,2-dihexylpropane-1,3-bis-β-D-glucopyranoside; and 3-Oxatridecyl-α-D-mannoside and 2,2-dioctylpropane-1,3-bis-β-D-maltopyranoside.

[0020]

[25] The method for preparing any one of [1] to

[10] , further comprising the steps of: 4) drying the eluted peptides to obtain dry peptides; and 5) dissolving the obtained dry peptide in an aqueous solvent containing at least one sugar-based nonionic surfactant to obtain an aqueous peptide solution.

[26] The sugar-based nonionic surfactant is represented by the formula (I);

[0021] [ka]

[0022] (G1 is a monosaccharide or disaccharide group, and L1 is O, S, -O-(CH2) n -O- or -O-CO-, R1 is a linear alkyl group having 6 to 12 carbon atoms, and n is an integer of 1 to 3), or formula (II);

[0023] [ka]

[0024] (G2 and G3 are the same or different and are monosaccharide or disaccharide groups, and R2 and R3 are the same or different and are linear alkyl groups having 6 to 12 carbon atoms.) A method for preparing the compound represented by the formula

[25] .

[27] The preparation method of

[26] , wherein the sugar-based nonionic surfactant is a compound represented by formula (I), and G1 is any one selected from the group consisting of a glucose group, a mannose group, a maltose group, or a trehalose group.

[28] The method according to

[27] , wherein the sugar-based nonionic surfactant is n-dodecyl-β-D-maltoside, n-undecyl-β-D-maltoside, n-decyl-β-D-maltoside, n-nonyl-β-D-thiomaltoside, n-octyl-β-D-thioglucoside, 3-oxatridecyl-α-D-mannoside or α-D-glucopyranosyl-α-D-glucopyranoside monododecanoate.

[29] The preparation method of

[26] , wherein the sugar-based nonionic surfactant is a compound represented by formula (II), and G2 and G3 are the same and are a glucose group or a maltose group.

[30] The method for preparing the sugar-based nonionic surfactant according to

[29] , wherein the sugar-based nonionic surfactant is 2,2-dioctylpropane-1,3-bis-β-D-maltopyranoside, 2,2-didecylpropane-1,3-bis-β-D-maltopyranoside or 2,2-dihexylpropane-1,3-bis-β-D-glucopyranoside.

[31] The preparation method of any of

[25] to

[30] , wherein the aqueous solvent contains a combination of at least two sugar-based nonionic surfactants.

[32] The method according to

[31] , wherein the combination of sugar-based nonionic surfactants is any one selected from the following group: n-Dodecyl-β-D-maltoside and α-D-glucopyranosyl-α-D-glucopyranoside monododecanoate; 2,2-Dihexylpropane-1,3-bis-β-D-glucopyranoside and α-D-glucopyranosyl-α-D-glucopyranoside monododecanoate; 2,2-Dioctylpropane-1,3-bis-β-D-maltopyranoside and 2,2-dihexylpropane-1,3-bis-β-D-glucopyranoside; and 3-Oxatridecyl-α-D-mannoside and 2,2-dioctylpropane-1,3-bis-β-D-maltopyranoside.

[33] Any of the preparation methods

[25] to

[32] , wherein the dried peptide is contained in a container having a plastic surface. Effect of the Invention

[0025] According to the present invention, in the preparation of a sample containing a small amount of peptide for use in proteome analysis, etc., the loss of peptides can be minimized while avoiding contamination of LC-MS. By treating a sample for LC-MS analysis with the method of the present invention, the loss of peptides from a small amount of sample is suppressed, the number of peptide identifications and protein identifications in mass spectrometry increases, and the reproducibility of the analysis is improved. In addition, according to the present invention, the recovery amount of peptide fragments adsorbed to the wall of a tube by drying during the pretreatment process of proteome analysis is improved. In particular, since the recovery amount of long peptides of 11 amino acids or more is significantly improved, a mass spectrometry sample rich in long peptides can be prepared, and the number of peptide identifications and protein identifications in mass spectrometry increases, contributing to improving the accuracy of proteome analysis. Since the effect of improving the recovery amount of peptide fragments is observed with a very low concentration of sugar-based nonionic surfactant, the contamination of the mass spectrometry device can also be minimized. In addition, according to the present invention, a stable peptide aqueous solution in which the adsorption of peptides to a container is suppressed is provided. [Brief description of the drawings]

[0026] [Figure 1] FIG. 1 shows the flow of sample pretreatment in proteome analysis. [Figure 2A] Figures 2A-D show the evaluation of the recovery rate of dried tryptic peptides by DDA-LC-MS / MS. Figure 2A shows the TIC chromatogram of dried tryptic peptides from K562 cells dissolved in 2% ACN-0.1% TFA or 0.005-0.04% DDM. 500 ng of dried peptides in a normal polypropylene (PP) tube were dissolved in 25 μL of each solvent, transferred to a PP LC vial, and 2.5 μL was injected into the LC-MS / MS. [Figure 2B] FIG. 2B shows the number of protein groups and peptides identified by DDA-LC-MS / MS. [Figure 2C] FIG. 2C shows the retention time distribution of the identified peptides. [Figure 2D] FIG. 2D shows the amino acid length distribution of the identified peptides. [Figure 3A] Figures 3A-D show the evaluation of the recovery of dried peptides in different vials and dissolving solutions by DIA-LC-MS / MS. Figure 3A shows the number of peptides identified from dried peptides dissolved in 2% ACN-0.1% TFA or 0.02% DDM in a normal PP vial or a hydrophilic vial. 500 ng of dried peptide in each vial was dissolved in 25 μL of each solvent and 2.5 μL was injected into the LC-MS / MS. [Figure 3B] FIG. 3B shows a comparison of total peptide intensities at each amino acid length. [Figure 3C] FIG. 3C shows a Pearson correlation coefficient heat map from hierarchical clustering of DIA protein intensities. [Figure 3D] FIG. 3D shows principal component analysis of DIA protein intensities. [Figure 4A] Figures 4A-D show the effect of DDM on peptide recovery in IP-MS: Figure 4A shows TIC chromatograms of dried tryptic peptides in IP of RELA dissolved in 2% ACN-0.1% TFA or 0.02% DDM. [Figure 4B] FIG. 4B shows the number of protein groups and peptides identified by DIA-LC-MS / MS. [Figure 4C] Figure 4C shows the number of known RELA interactors among the identified proteins. Known RELA interactors were extracted from IntAct (https: / / www.ebi.ac.uk / intact / home). [Figure 4D] Figure 4D shows the DIA protein intensity of RELA and major RELA interactors among identified proteins. A DIA protein intensity of zero means missing value. * p<0.05; ** p<0.01; *** p<0.005. [Diagram 5] 5 shows the number of peptides that could be identified by LC-MS / MS. A to J on the horizontal axis correspond to the sugar-based nonionic surfactants listed in Table 1. [Figure 6] FIG. 6 shows the elution times of sugar-type surfactants in reversed-phase (C18) column separation. [Figure 7] FIG. 7 shows the elution patterns of DDM, DMNG, and LMNG trapped on a reversed-phase (SDB) column and successively eluted with increasing organic solvent concentration in the eluent. [Figure 8] FIG. 8 shows a detailed examination of the elution conditions of LMNG from a reversed-phase (SDB) column. [Figure 9] FIG. 9 is a graph showing that there was no change in the number of peptide identifications between 50% ACN-0.1% TFA elution and 36% ACN-0.1% TFA elution. [Figure 10] FIG. 10 is a graph showing that the loss of proteins and peptides during enzymatic digestion of a trace sample was suppressed by adding LMNG. [Figure 11A] FIG. 11A shows the results of an evaluation demonstrating that the addition of LMNG during enzymatic digestion and DMNG during dissolution of dried peptides improves the number of protein and precursor (peptide) identifications in LC-MS. [Figure 11B] FIG. 11B shows the evaluation results indicating that the addition of LMNG during enzymatic digestion and the addition of DMNG during dissolution of dried peptides improve the reproducibility of LC-MS analysis. [Figure 12A] FIG. 12A shows the results of an evaluation demonstrating that the addition of LMNG during enzymatic digestion and DMNG during dissolution of dried peptides improves the number of protein and precursor (peptide) identifications in Co-IP-LC-MS. [Figure 12B] Figure 12B shows the total number of peptide fragments derived from RELA, NJKB1, IKBKB, NFKBIA, NFKBIB, and NFKBIE identified by Co-IP-LC-MS analysis using anti-RELA (NF-κB p65) antibody. The total number of peptide fragments increased by adding LMNG during enzyme digestion and by adding DMNG during dissolution of dried peptides. [Figure 13] FIG. 13 shows the flow of pretreatment for phosphoproteome analysis. [Figure 14]FIG. 14 is a graph showing that the addition of LMNG to the eluate in immobilized metal affinity chromatography increased the recovery amount of phosphorylated peptides and increased the number of identified phosphorylated peptides. [Figure 15] FIG. 15 shows the flow of sample pretreatment in proteomic analysis of biotinylated proteins. [Figure 16] FIG. 16 is a graph showing that the addition of LMNG to the eluate in streptavidin affinity chromatography increased the recovery amount of biotinylated peptides and increased the number of identified biotinylated peptides. [Figure 17] FIG. 17 is a graph showing the dissolution patterns of DDM, DMNG, and LMNG in EVOSEP ONE. [Figure 18A] Figure 18A shows the results of an evaluation using LC-MS with EVOSEP ONE, which shows that the addition of LMNG during enzymatic digestion of HEK293 cell lysate improves the number of protein and precursor (peptide) identifications. To perform quantitative evaluation, data was also acquired using DIA-MS. [Figure 18B] Figure 18B shows the results of an evaluation showing that the addition of LMNG during enzymatic digestion of cell lysate improved the reproducibility of LC-MS analysis results using EVOSEP ONE. [Figure 19A] Figure 19A shows the results of an evaluation using LC-MS with EVOSEP ONE, which shows that the addition of LMNG during serum exosome enzymatic digestion improves the number of protein and precursor (peptide) identifications. Data was also obtained using DIA-MS for quantitative evaluation. [Figure 19B] Figure 19B shows the results of the evaluation of serum exosome proteome analysis using EVOSEP ONE, showing that the addition of LMNG during serum exosome enzymatic digestion improved the number of peptides identified that were derived from exosome-specific proteins CD9, CD63, and CD81. Data was also obtained by DIA-MS for quantitative evaluation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] 1. Method for producing aqueous peptide solution The present invention provides a method for producing an aqueous peptide solution (hereinafter referred to as the "production method of the present invention"), which comprises dissolving a dry peptide in an aqueous solvent containing at least one sugar-based nonionic surfactant. In producing an aqueous peptide solution, when dissolving a dry peptide in an aqueous solvent, the aqueous solvent contains at least one sugar-based nonionic surfactant, which promotes dissolution of the dry peptide adsorbed to the container in the aqueous solvent, prevents the dry peptide from remaining undissolved, and inhibits re-adsorption of the peptide once dissolved to the container, etc. By adding an aqueous solvent containing at least one sugar-based nonionic surfactant to the dry peptide in the container and mixing, the dry peptide is dissolved in the aqueous solvent, and the desired aqueous peptide solution can be obtained.

[0028] In this specification, "peptide" refers to a compound composed of a chain of amino acid residues linked by peptide bonds, and may be used interchangeably with "polypeptide" and "protein". The length of the peptide used in the production method of the present invention is not particularly limited, but since sugar-based nonionic surfactants are excellent in the effect of promoting the dissolution of long dry peptides, the peptides dissolved in aqueous solvents contain at least a part of a long-chain peptide (e.g., a peptide having a length of 11 amino acids or more, 12 amino acids or more, 13 amino acids or more, 14 amino acids or more, 15 amino acids or more, or 20 amino acids or more). The upper limit of the length of the long-chain peptide is not particularly limited, but is, for example, 100 amino acids or less, 80 amino acids or less, 60 amino acids or less, 50 amino acids or less, 40 amino acids or less, or 30 amino acids or less.

[0029] In the production method of the present invention, the peptide dissolved in the aqueous solvent may be a single type of peptide or a mixture of multiple types of peptides. In one embodiment, the peptide dissolved in the aqueous solvent in the production method of the present invention is a mixture of multiple types of peptides, at least a part of which contains a long-chain peptide (e.g., a peptide having a length of 11 amino acids or more, 12 amino acids or more, 13 amino acids or more, 14 amino acids or more, 15 amino acids or more, or 20 amino acids or more). The upper limit of the length of the long-chain peptide may be, for example, 100 amino acids or less, 80 amino acids or less, 60 amino acids or less, 50 amino acids or less, 40 amino acids or less, or 30 amino acids or less. The proportion of long-chain peptides in the peptide mixture (proportion of the number of molecules) is not particularly limited, and may be, for example, 0.1% or more, 1% or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more.

[0030] The origin of the peptide dissolved in the aqueous solvent in the production method of the present invention is not particularly limited, and the peptide may be isolated from a biological material, chemically synthesized, or a recombinant peptide expressed by genetic recombination technology. In one embodiment, the peptide dissolved in the aqueous solvent in the production method of the present invention is a peptide mixture obtained by limited digestion of a biological sample containing a protein with a protease (e.g., trypsin).

[0031] In the production method of the present invention, a "dried" peptide is dissolved in an aqueous solvent. "Dry" means that the water content is less than 15% (w / w) (e.g., less than 10% (w / w), less than 5% (w / w), less than 1% (w / w)) as measured chemically by at least one method selected from the drying method and the Karl Fischer method. Methods for drying the peptide include, but are not limited to, vacuum drying, heat drying, air drying, and freeze drying.

[0032] In one embodiment, in the manufacturing method of the present invention, the dried peptide contained in a container having a plastic surface is dissolved in an aqueous solvent. The dissolution of the dried peptide attached to the plastic surface in the container is promoted by the sugar-based nonionic surfactant, and the re-adsorption of the peptide dissolved in the aqueous solvent to the plastic surface can be suppressed by the sugar-based nonionic surfactant. The types of plastics include, but are not limited to, polypropylene, polystyrene, polymethyl methacrylate, polyvinyl chloride, polyethylene, polycarbonate, polysulfone, fluoropolymer, polyamide, polydimethylsiloxane, polyurethane, polysulfone, polytetrafluoroethylene, elastomer, etc. The plastic surface in the container may or may not be hydrophilically treated. The hydrophilic treatment means a treatment to add hydrophilic groups such as hydroxyl groups and carboxyl groups to the plastic surface in the container.

[0033] In one embodiment, in the production method of the present invention, the dried peptide attached to the plastic surface in the container is dissolved in an aqueous solvent. The dried peptide may be attached to the plastic surface by a drying treatment (vacuum drying, etc.).

[0034] One of the features of the production method of the present invention is that at least one type of sugar-based nonionic surfactant is contained in the aqueous solvent in which the dry peptide is dissolved. In this specification, the sugar-based nonionic surfactant refers to a nonionic surfactant having a sugar group as a hydrophilic group unit. In this specification, the sugar group is a monovalent group consisting of a structure derived from a sugar, and specifically means a residue obtained by removing one hydroxyl group from a sugar. The sugar group is preferably a hydroxyl group added to the anomeric carbon of the reducing end or a residue obtained by removing one hydroxyl group in a methylol group from a sugar. The sugar-based nonionic surfactant used in the production method of the present invention is preferably a surfactant represented by formula (I);

[0035] [ka]

[0036] (G1 is a monosaccharide or disaccharide group, and L1 is O, S, -O-(CH2) n -O- or -O-CO-, R1 is a linear alkyl group having 6 to 12 carbon atoms, and n is an integer of 1 to 3), or formula (II);

[0037] [ka]

[0038] (G2 and G3 are the same or different and are monosaccharide or disaccharide groups, and R2 and R3 are the same or different and are linear alkyl groups having 6 to 12 carbon atoms.) It is a compound represented by the formula:

[0039] The monosaccharide is preferably a pentose or a hexose. Examples of the pentose include ribose, deoxyribose, and fructose. Examples of the hexose include glucose, mannose, and galactose. Examples of the disaccharide include maltose, trehalose, lactose, isomaltose, sucrose, and cellobiose.

[0040] Examples of the linear alkyl group having 6 to 12 carbon atoms include a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, and a dodecyl group.

[0041] In formula (I), n is 1, 2 or 3, preferably 1 or 2, and more preferably 2.

[0042] In formula (I), when L1 is bonded to the anomeric carbon at the reducing end of G1, the bonding mode may be an α-bond or a β-bond.

[0043] In formula (I), G1 is preferably a maltose group, a trehalose group, a glucose group or a mannose group.

[0044] In one embodiment of formula (I), G1 is a maltose group, a glucose group or a mannose group and L1 is O, S, or -O-(CH2) n It is -O-, R1 is a linear alkyl group having 8 to 12 carbon atoms, n is 2, and L1 is bonded to the anomeric carbon of G1.

[0045] In one embodiment of formula (I), G1 is a maltose group or a glucose group, L1 is O or S, R1 is a linear alkyl group having 8 to 12 carbon atoms, and L1 is β-bonded to the anomeric carbon of G1.

[0046] In another embodiment of formula (I), G1 is a trehalose group, L1 is -O-CO-, and R1 is a linear alkyl group having 8 to 12 carbon atoms.

[0047] The compound represented by formula (I) is n-Dodecyl-β-D-maltoside (DDM), n-Undecyl-β-D-maltoside, n-Decyl-β-D-maltoside, n-nonyl-β-D-thiomaltoside, n-Octyl-β-D-thioglucoside, 3-oxatridecyl-α-D-mannoside, α-D-Glucopyranosyl-α-D-glucopyranoside monododecanoate (Trehalose C12) The following can be mentioned:

[0048] In formula (II), when the oxygen atom is bonded to the anomeric carbon at the reducing end of G2 or G3, the bonding mode may be an α-bond or a β-bond.

[0049] In formula (II), G2 and G3 are preferably the same and are a glucose group or a maltose group.

[0050] In one embodiment of formula (II), G2 and G3 are the same and are a glucose group or a maltose group, R2 and R3 are the same and are a linear alkyl group having 6 to 10 carbon atoms, and the reducing end anomeric carbons of G2 and G3 are β-bonded to an oxygen atom.

[0051] The compound represented by formula (II) is 2,2-Dioctylpropane-1,3-bis-β-D-maltopyranoside (Decylmaltose neopentyl glycol), 2,2-Didecylpropane-1,3-bis-β-D-maltopyranoside (Lauryl Maltose Neopentyl Glycol), 2,2-Dihexylpropane-1,3-bis-β-D-glucopyranoside (Octylglucose neopentyl glycol) The following can be mentioned:

[0052] The sugar-based nonionic surfactant is preferably any one selected from the following group: n-Dodecyl-β-D-maltoside (DDM), n-Undecyl-β-D-maltoside, 3-oxatridecyl-α-D-mannoside, α-D-Glucopyranosyl-α-D-glucopyranoside monododecanoate (Trehalose C12) 2,2-Dioctylpropane-1,3-bis-β-D-maltopyranoside (Decylmaltose neopentyl glycol), 2,2-Didecylpropane-1,3-bis-β-D-maltopyranoside (lauryl maltose neopentyl glycol), and 2,2-Dihexylpropane-1,3-bis-β-D-glucopyranoside (Octylglucose neopentyl glycol).

[0053] In the production method of the present invention, one type of sugar-based nonionic surfactant may be contained alone in the aqueous solvent, or two or more types of sugar-based nonionic surfactants may be combined and contained in the aqueous solvent. By combining two or more types of sugar-based nonionic surfactants, it is expected that the effect of promoting the dissolution of dried peptides will be enhanced compared to when one type of sugar-based nonionic surfactant is used alone. In one embodiment, the aqueous solvent used in the production method of the present invention contains a combination of at least two types of sugar-based nonionic surfactants.

[0054] The type of sugar-based nonionic surfactant to be combined is not particularly limited, but it is preferable to combine at least two compounds selected from the compounds represented by the above formula (I) and the compounds represented by the above formula (II).

[0055] In one embodiment, the aqueous solvent used in the production method of the present invention contains a combination of at least two sugar-based nonionic surfactants selected from the following group: n-Dodecyl-β-D-maltoside (DDM), 3-oxatridecyl-α-D-mannoside, α-D-Glucopyranosyl-α-D-glucopyranoside monododecanoate (Trehalose C12) 2,2-Dioctylpropane-1,3-bis-β-D-maltopyranoside (Decylmaltose neopentyl glycol), 2,2-Didecylpropane-1,3-bis-β-D-maltopyranoside (lauryl maltose neopentyl glycol), and 2,2-Dihexylpropane-1,3-bis-β-D-glucopyranoside (Octylglucose neopentyl glycol).

[0056] Preferred combinations of sugar-based nonionic surfactants include the following: n-Dodecyl-β-D-maltoside and α-D-glucopyranosyl-α-D-glucopyranoside monododecanoate; 2,2-Dihexylpropane-1,3-bis-β-D-glucopyranoside and α-D-glucopyranosyl-α-D-glucopyranoside monododecanoate; 2,2-Dioctylpropane-1,3-bis-β-D-maltopyranoside and 2,2-dihexylpropane-1,3-bis-β-D-glucopyranoside; and 3-Oxatridecyl-α-D-mannoside and 2,2-dioctylpropane-1,3-bis-β-D-maltopyranoside.

[0057] The aqueous solvent may contain additional components other than water and the sugar-based nonionic surfactant. For example, the aqueous solvent may contain surfactants other than the sugar-based nonionic surfactant, buffers, hydrophilic organic solvents, etc. When producing an aqueous peptide solution for analysis by LC-MS or the like, it is preferable to avoid the inclusion of components that may inhibit the analysis or contaminate the analysis device. In one embodiment, the aqueous solvent consists of water and the sugar-based nonionic surfactant.

[0058] The total concentration of the sugar-based nonionic surfactants contained in the aqueous solvent (when the aqueous solvent contains two or more types of sugar-based nonionic surfactants, the total concentration of all types of sugar-based nonionic surfactants contained in the aqueous solvent) can be appropriately adjusted taking into consideration the type of sugar-based nonionic surfactant and the amount of dry peptide to be dissolved, and is, for example, 0.00125% (w / w) or more, 0.0025% (w / w) or more, 0.005% (w / w) or more, or 0.01% (w / w) or more.

[0059] Table 1 shows an example of the minimum concentration of a sugar-based nonionic surfactant in an aqueous solvent when one type of sugar-based nonionic surfactant is used alone.

[0060] [Table 1]

[0061] When each sugar-based nonionic surfactant is used alone, it is preferable to contain each sugar-based nonionic surfactant in the aqueous solvent at a concentration equal to or higher than the minimum concentration listed in Table 1, in order to ensure promotion of dissolution of the dried peptide.

[0062] By combining two or more sugar-based nonionic surfactants, it is expected that the effect of promoting the dissolution of dry peptides will be enhanced, and therefore, when combining two or more sugar-based nonionic surfactants, a certain degree of promotion of the dissolution of dry peptides can be expected even if the concentration of each sugar-based nonionic surfactant in the aqueous solvent is less than the minimum concentration listed in Table 1. However, in order to more reliably promote the dissolution of dry peptides, when combining two or more sugar-based nonionic surfactants, it is preferable to adjust the concentration of each sugar-based nonionic surfactant in the aqueous solvent to be equal to or greater than the minimum concentration listed in Table 1.

[0063] In one embodiment, when two or more sugar-based nonionic surfactants are combined, the concentration of each sugar-based nonionic surfactant in the aqueous solvent is adjusted to 0.005% or more (w / w).

[0064] Although it is not possible to univocally set an upper limit for the total concentration of the sugar-based nonionic surfactant contained in the aqueous solvent, since the promotion of dissolution of the dry peptide does not require the formation of micelles by the sugar-based nonionic surfactant, even a sugar-based nonionic surfactant with a concentration below the critical micelle concentration (CMC) can promote the dissolution of the dry peptide. In one embodiment, the total concentration of the sugar-based nonionic surfactant contained in the aqueous solvent is below the CMC of the added sugar-based nonionic surfactant. When producing an aqueous peptide solution for analysis by LC-MS or the like, in order to avoid contamination of the analysis device, it is preferable that the sugar-based nonionic surfactant concentration is as low as possible within the range where an appropriate dry peptide dissolution promotion effect is achieved. In one embodiment, the total concentration of the sugar-based nonionic surfactant contained in the aqueous solvent is 0.04% (w / w) or less.

[0065] 2. Dry peptide dissolution enhancer The present invention provides a dry peptide dissolution enhancer (hereinafter referred to as "the dry peptide dissolution enhancer of the present invention") containing at least one sugar-based nonionic surfactant. In the above-mentioned production method of the present invention, an aqueous peptide solution can be easily produced by adding the dry peptide dissolution enhancer of the present invention to an aqueous solvent. The dry peptide dissolution enhancer of the present invention may be used as an aqueous solvent for dissolving the dry peptide.

[0066] The definition of "sugar-based nonionic surfactant" is the same as that described in the above item 1. The sugar-based nonionic surfactant used in the dry peptide dissolution enhancer of the present invention is preferably represented by the formula (I);

[0067] [ka]

[0068] Or formula (II);

[0069] [ka]

[0070] (The definitions of the respective substituents in formula (I) and formula (II) are the same as those described in the above item 1.)

[0071] The compound represented by formula (I) is n-Dodecyl-β-D-maltoside (DDM), n-Undecyl-β-D-maltoside, n-Decyl-β-D-maltoside, n-nonyl-β-D-thiomaltoside, n-Octyl-β-D-thioglucoside, 3-oxatridecyl-α-D-mannoside, α-D-Glucopyranosyl-α-D-glucopyranoside monododecanoate (Trehalose C12) The following can be mentioned:

[0072] The compound represented by formula (II) is 2,2-Dioctylpropane-1,3-bis-β-D-maltopyranoside (Decylmaltose neopentyl glycol), 2,2-Didecylpropane-1,3-bis-β-D-maltopyranoside (Lauryl Maltose Neopentyl Glycol), 2,2-Dihexylpropane-1,3-bis-β-D-glucopyranoside (Octylglucose neopentyl glycol) The following can be mentioned:

[0073] The sugar-based nonionic surfactant is preferably any one selected from the following group: n-Dodecyl-β-D-maltoside (DDM), n-Undecyl-β-D-maltoside, 3-oxatridecyl-α-D-mannoside, α-D-Glucopyranosyl-α-D-glucopyranoside monododecanoate (Trehalose C12) 2,2-Dioctylpropane-1,3-bis-β-D-maltopyranoside (Decylmaltose neopentyl glycol), 2,2-Didecylpropane-1,3-bis-β-D-maltopyranoside (lauryl maltose neopentyl glycol), and 2,2-Dihexylpropane-1,3-bis-β-D-glucopyranoside (Octylglucose neopentyl glycol).

[0074] The dry peptide dissolution enhancer of the present invention may contain one type of sugar-based nonionic surfactant alone, or may contain two or more types of sugar-based nonionic surfactants in combination. In one embodiment, the dry peptide dissolution enhancer of the present invention contains a combination of at least two types of sugar-based nonionic surfactants.

[0075] The type of sugar-based nonionic surfactant to be combined is not particularly limited, but it is preferable to combine at least two compounds selected from the compounds represented by the above formula (I) and the compounds represented by the above formula (II).

[0076] In one embodiment, the dry peptide solubility enhancer of the present invention contains a combination of at least two sugar-based nonionic surfactants selected from the following group: n-Dodecyl-β-D-maltoside (DDM), 3-oxatridecyl-α-D-mannoside, α-D-Glucopyranosyl-α-D-glucopyranoside monododecanoate (Trehalose C12) 2,2-Dioctylpropane-1,3-bis-β-D-maltopyranoside (Decylmaltose neopentyl glycol), 2,2-Didecylpropane-1,3-bis-β-D-maltopyranoside (lauryl maltose neopentyl glycol), and 2,2-Dihexylpropane-1,3-bis-β-D-glucopyranoside (Octylglucose neopentyl glycol).

[0077] Preferred combinations of sugar-based nonionic surfactants include the following: n-Dodecyl-β-D-maltoside and α-D-glucopyranosyl-α-D-glucopyranoside monododecanoate; 2,2-Dihexylpropane-1,3-bis-β-D-glucopyranoside and α-D-glucopyranosyl-α-D-glucopyranoside monododecanoate; 2,2-Dioctylpropane-1,3-bis-β-D-maltopyranoside and 2,2-dihexylpropane-1,3-bis-β-D-glucopyranoside; and 3-Oxatridecyl-α-D-mannoside and 2,2-dioctylpropane-1,3-bis-β-D-maltopyranoside.

[0078] The dried peptide dissolution promoter of the present invention may be provided in the form of a powder of a sugar-based nonionic surfactant (e.g., a lyophilized product), or in the form of an aqueous solvent containing a sugar-based nonionic surfactant (e.g., a solution of a sugar-based nonionic surfactant in an aqueous solvent, a suspension of a sugar-based nonionic surfactant in an aqueous solvent, etc.).

[0079] The aqueous solvent may contain additional components other than water and the sugar-based nonionic surfactant. For example, the aqueous solvent may contain surfactants other than the sugar-based nonionic surfactant, buffers, hydrophilic organic solvents, etc. When producing an aqueous peptide solution for analysis by LC-MS or the like, it is preferable to avoid the inclusion of components that may inhibit the analysis or contaminate the analysis device. In one embodiment, the aqueous solvent consists of water and the sugar-based nonionic surfactant.

[0080] In one aspect, the dry peptide dissolution promoter of the present invention is an aqueous solvent containing a sugar-based nonionic surfactant, and can be used as is as the aqueous solvent for dissolving the dry peptide in the above-mentioned production method of the present invention without dilution (non-concentrated type).

[0081] In the non-concentrated type, the total concentration of the sugar-based nonionic surfactant contained in the aqueous solvent can be appropriately adjusted taking into consideration the type of sugar-based nonionic surfactant and the amount of dry peptide to be dissolved, and is, for example, 0.00125% (w / w) or more, 0.0025% (w / w) or more, 0.005% (w / w) or more, or 0.01% (w / w) or more.

[0082] In the case of non-concentrated type, examples of the minimum concentration of the sugar-based nonionic surfactant in the aqueous solvent when one type of sugar-based nonionic surfactant is used alone are shown in Table 1 above.

[0083] When each sugar-based nonionic surfactant is used alone, it is preferable to contain (dissolve) each sugar-based nonionic surfactant in an aqueous solvent at a concentration equal to or higher than the minimum concentration listed in Table 1, in order to ensure promotion of dissolution of the dried peptide.

[0084] By combining two or more sugar-based nonionic surfactants, it is expected that the effect of promoting the dissolution of dry peptides will be enhanced, and therefore, when combining two or more sugar-based nonionic surfactants, good promotion of the dissolution of dry peptides can be expected even if the concentration of each sugar-based nonionic surfactant in the aqueous solvent is less than the minimum concentration listed in Table 1. However, in order to more reliably promote the dissolution of dry peptides, in the case of combining two or more sugar-based nonionic surfactants in a non-concentrated embodiment, it is preferable to adjust the concentration of each sugar-based nonionic surfactant in the aqueous solvent to be equal to or greater than the minimum concentration listed in Table 1.

[0085] In one embodiment of the non-concentrated type, two or more sugar-based nonionic surfactants are combined, and the concentration of each sugar-based nonionic surfactant in the aqueous solvent is adjusted to 0.005% (w / w) or more.

[0086] In the non-concentrated type, the upper limit of the total concentration of the sugar-based nonionic surfactant contained in the aqueous solvent cannot be set uniquely, but since the promotion of dissolution of the dry peptide does not require the formation of micelles by the sugar-based nonionic surfactant, even a sugar-based nonionic surfactant with a concentration below the critical micelle concentration (CMC) can promote the dissolution of the dry peptide. In one embodiment, the total concentration of the sugar-based nonionic surfactant contained in the aqueous solvent is below the CMC of the added sugar-based nonionic surfactant. When producing an aqueous peptide solution for analysis by LC-MS or the like, in order to avoid contamination of the analysis device, it is preferable that the sugar-based nonionic surfactant concentration is as low as possible within the range where an appropriate dry peptide dissolution promotion effect is achieved. In one embodiment, the total concentration of the sugar-based nonionic surfactant contained in the aqueous solvent is 0.04% (w / w) or less.

[0087] In one embodiment, the dry peptide dissolution promoter of the present invention is an aqueous solvent containing a sugar-based nonionic surfactant, and can be diluted with an appropriate aqueous solvent (e.g., water) at an appropriate dilution ratio to obtain an aqueous solvent for dissolving the dry peptide (concentrated type). The dilution ratio is not particularly limited, but is, for example, 2-100 times (e.g., ×2, ×5, ×10, ×20, ×100) in consideration of the convenience of the experimenter.

[0088] In the concentrated type, the total concentration of the sugar-based nonionic surfactant contained in the aqueous solvent can be appropriately adjusted taking into consideration the type of sugar-based nonionic surfactant and the amount of dry peptide to be dissolved, etc., but for example, the total concentration of the sugar-based nonionic surfactant when diluted 2 to 100 times (e.g., x2, x5, x10, x20, x100) with an aqueous solvent (e.g., water) is 0.00125% (w / w) or more, 0.0025% (w / w) or more, 0.005% (w / w) or more, or 0.01% (w / w) or more.

[0089] In the case of using one type of sugar-based nonionic surfactant alone in the concentrated type, examples of the minimum concentration of the sugar-based nonionic surfactant in the aqueous solvent when the dry peptide dissolution promoter of the present invention is diluted 2 to 100 times (e.g., ×2, ×5, ×10, ×20, ×100) with the aqueous solvent are shown in Table 1 above.

[0090] In the case of using each sugar-based nonionic surfactant listed in Table 1 alone in the concentrated type, from the viewpoint of ensuring promotion of dissolution of the dry peptide, it is preferable to prepare the dry peptide dissolution promoter of the present invention so that the concentration of each sugar-based nonionic surfactant when diluted 2 to 100 times (e.g., x2, x5, x10, x20, x100) with an aqueous solvent (e.g., water) is equal to or higher than the minimum concentration listed in Table 1.

[0091] When two or more sugar-based nonionic surfactants are combined in the concentrated type dry peptide dissolution promoter of the present invention, even if the concentration of each sugar-based nonionic surfactant when diluted with an aqueous solvent to an appropriate ratio (e.g., 2-fold to 100-fold (e.g., ×2, ×5, ×10, ×20, ×100)) is less than the minimum concentration listed in Table 1, good promotion of dry peptide dissolution can be expected. However, in order to more reliably promote the dissolution of dry peptides, when two or more sugar-based nonionic surfactants are combined, it is preferable to prepare the dry peptide dissolution promoter of the present invention so that the concentration of each sugar-based nonionic surfactant in the aqueous solvent when diluted with an appropriate ratio (e.g., 2-fold to 100-fold (e.g., ×2, ×5, ×10, ×20, ×100)) is equal to or greater than the minimum concentration listed in Table 1.

[0092] In one embodiment of the concentrated type, when two or more sugar-based nonionic surfactants are combined, the dried peptide dissolution promoter of the present invention is prepared so that the concentration of each sugar-based nonionic surfactant in the aqueous solvent is 0.005% (w / w) or more when diluted 2 to 100 times (e.g., x2, x5, x10, x20, x100) with the aqueous solvent.

[0093] For the concentrated type, the upper limit of the total concentration of the sugar-based nonionic surfactant contained in the aqueous solvent cannot be set univocally. In one embodiment, the final total concentration of the sugar-based nonionic surfactant when diluted with an aqueous solvent at an appropriate ratio (e.g., 2-fold to 100-fold (e.g., ×2, ×5, ×10, ×20, ×100)) is lower than the CMC of the added sugar-based nonionic surfactant. When producing an aqueous peptide solution for analysis by LC-MS or the like, it is preferable that the sugar-based nonionic surfactant concentration is as low as possible within a range where an appropriate dry peptide dissolution promoting effect is achieved in order to avoid contamination of the analysis device. In one embodiment, the dry peptide dissolution promoter of the present invention is prepared so that the final total concentration of the sugar-based nonionic surfactant when diluted with an aqueous solvent at an appropriate ratio (e.g., 2-fold to 100-fold (e.g., ×2, ×5, ×10, ×20, ×100)) is 0.04% (w / w) or less.

[0094] The definitions of the terms relating to the dissolution promoter of the present invention are the same as those described for the production method of the present invention, unless otherwise specified.

[0095] 3. Peptide aqueous solution The present invention provides an aqueous peptide solution (hereinafter referred to as "aqueous solution of the present invention") containing a peptide and at least one sugar-based nonionic surfactant. The aqueous solution of the present invention contains at least one sugar-based nonionic surfactant, which suppresses adsorption of the peptide to a container or the like, and is stable. The aqueous solution of the present invention can be obtained by dissolving a dried peptide in an aqueous solvent containing at least one sugar-based nonionic surfactant according to the above-mentioned production method of the present invention.

[0096] The length of the peptide contained in the aqueous solution of the present invention is not particularly limited, but since the sugar-based nonionic surfactant has an excellent effect of suppressing the adsorption of long peptides to a container, the peptide contained in the aqueous solution of the present invention contains at least a part of a long-chain peptide (e.g., a peptide having a length of 11 amino acids or more, 12 amino acids or more, 13 amino acids or more, 14 amino acids or more, 15 amino acids or more, or 20 amino acids or more). The upper limit of the length of the long-chain peptide is not particularly limited, but is, for example, 100 amino acids or less, 80 amino acids or less, 60 amino acids or less, 50 amino acids or less, 40 amino acids or less, or 30 amino acids or less.

[0097] The peptide contained in the aqueous solution of the present invention may be a single type of peptide or a mixture of multiple types of peptides. In one embodiment, the peptide contained in the aqueous solution of the present invention is a mixture of multiple types of peptides, at least a part of which contains a long-chain peptide (e.g., a peptide having a length of 11 amino acids or more, 12 amino acids or more, 13 amino acids or more, 14 amino acids or more, 15 amino acids or more, or 20 amino acids or more). The upper limit of the length of the long-chain peptide may be, for example, 100 amino acids or less, 80 amino acids or less, 60 amino acids or less, 50 amino acids or less, 40 amino acids or less, or 30 amino acids or less. The proportion of long-chain peptides in the peptide mixture (proportion of the number of molecules) is not particularly limited, and may be, for example, 0.1% or more, 1% or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more.

[0098] In one aspect, the proportion (proportion by number of molecules) of peptides contained in the aqueous solution of the present invention that are 100 amino acids or less in length (e.g., 80 amino acids or less, 60 amino acids or less, 50 amino acids or less, 40 amino acids or less, or 30 amino acids or less) can be, for example, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more.

[0099] The origin of the peptide contained in the aqueous solution of the present invention is not particularly limited, and the peptide may be isolated from a biological material, chemically synthesized, or a recombinant peptide expressed by a gene recombination technique. In one embodiment, the peptide contained in the aqueous solution of the present invention is a peptide mixture obtained by limited digestion of a protein derived from a biological material with a protease (e.g., trypsin).

[0100] The concentration of the peptide contained in the aqueous solution of the present invention is not particularly limited, and may be, for example, 1 fg / ml or more, 10 fg / ml or more, 100 fg / ml or more, 1 pg / ml or more, 10 pg / ml or more, or 100 pg / ml or more. The aqueous solution of the present invention contains the peptide at a concentration equal to or less than the solubility, and the concentration is, for example, 100 mg / ml or less, 10 mg / ml or less, 1 mg / ml or less, or 100 μg / ml or less. In a low-concentration aqueous solution of peptide, loss due to adsorption of the peptide to a container or the like becomes a problem, but the aqueous solution of the present invention contains at least one sugar-based nonionic surfactant, which suppresses the adsorption of the peptide to a container or the like, making it possible to provide a stable aqueous solution of peptide at a low concentration. In one embodiment, the concentration of the peptide contained in the aqueous solution of the present invention is, for example, 100 μg / ml or less, 10 μg / ml or less, 1 μg / ml or less, 100 ng / ml or less, 10 ng / ml or less, or 1 ng / ml or less.

[0101] The aqueous solution of the present invention is characterized in that it contains at least one sugar-based nonionic surfactant. The definition of "sugar-based nonionic surfactant" is the same as that described in the above item 1. The sugar-based nonionic surfactant contained in the aqueous solution of the present invention is preferably represented by the formula (I);

[0102] [ka]

[0103] Or formula (II);

[0104] [ka]

[0105] (The definitions of the respective substituents in formula (I) and formula (II) are the same as those described in the above item 1.)

[0106] The compound represented by formula (I) is n-Dodecyl-β-D-maltoside (DDM), n-Undecyl-β-D-maltoside, n-Decyl-β-D-maltoside, n-nonyl-β-D-thiomaltoside, n-Octyl-β-D-thioglucoside, 3-oxatridecyl-α-D-mannoside, α-D-Glucopyranosyl-α-D-glucopyranoside monododecanoate (Trehalose C12) The following can be mentioned:

[0107] The compound represented by formula (II) is 2,2-Dioctylpropane-1,3-bis-β-D-maltopyranoside (Decylmaltose neopentyl glycol), 2,2-Didecylpropane-1,3-bis-β-D-maltopyranoside (Lauryl Maltose Neopentyl Glycol), 2,2-Dihexylpropane-1,3-bis-β-D-glucopyranoside (Octylglucose neopentyl glycol) The following can be mentioned:

[0108] The sugar-based nonionic surfactant is preferably any one selected from the following group: n-Dodecyl-β-D-maltoside (DDM), n-Undecyl-β-D-maltoside, 3-oxatridecyl-α-D-mannoside, α-D-Glucopyranosyl-α-D-glucopyranoside monododecanoate (Trehalose C12) 2,2-Dioctylpropane-1,3-bis-β-D-maltopyranoside (Decylmaltose neopentyl glycol), 2,2-Didecylpropane-1,3-bis-β-D-maltopyranoside (lauryl maltose neopentyl glycol), and 2,2-Dihexylpropane-1,3-bis-β-D-glucopyranoside (Octylglucose neopentyl glycol).

[0109] The aqueous solution of the present invention may contain one type of sugar-based nonionic surfactant alone, or may contain two or more types of sugar-based nonionic surfactants in combination. In one embodiment, the aqueous solution of the present invention contains a combination of at least two types of sugar-based nonionic surfactants.

[0110] The type of sugar-based nonionic surfactant to be combined is not particularly limited, but it is preferable to combine at least two compounds selected from the compounds represented by the above formula (I) and the compounds represented by the above formula (II).

[0111] In one embodiment, the aqueous solution of the present invention contains a combination of at least two sugar-based nonionic surfactants selected from the following group: n-Dodecyl-β-D-maltoside (DDM), 3-oxatridecyl-α-D-mannoside, α-D-Glucopyranosyl-α-D-glucopyranoside monododecanoate (Trehalose C12) 2,2-Dioctylpropane-1,3-bis-β-D-maltopyranoside (Decylmaltose neopentyl glycol), 2,2-Didecylpropane-1,3-bis-β-D-maltopyranoside (lauryl maltose neopentyl glycol), and 2,2-Dihexylpropane-1,3-bis-β-D-glucopyranoside (Octylglucose neopentyl glycol).

[0112] Preferred combinations of sugar-based nonionic surfactants include the following: n-Dodecyl-β-D-maltoside and α-D-glucopyranosyl-α-D-glucopyranoside monododecanoate; 2,2-Dihexylpropane-1,3-bis-β-D-glucopyranoside and α-D-glucopyranosyl-α-D-glucopyranoside monododecanoate; 2,2-Dioctylpropane-1,3-bis-β-D-maltopyranoside and 2,2-dihexylpropane-1,3-bis-β-D-glucopyranoside; and 3-Oxatridecyl-α-D-mannoside and 2,2-dioctylpropane-1,3-bis-β-D-maltopyranoside.

[0113] In a further aspect, the aqueous solution of the invention contains only 2,2-didecylpropane-1,3-bis-β-D-maltopyranoside as the surfactant alone.

[0114] The aqueous solution of the present invention may contain additional components other than the peptide, the sugar-based nonionic surfactant, and water. For example, the aqueous solution of the present invention may contain surfactants other than the sugar-based nonionic surfactant, buffers, hydrophilic organic solvents, etc. When the aqueous solution of the present invention is used for analysis by LC-MS or the like, it is preferable to avoid the inclusion of components that may inhibit the analysis or contaminate the analysis device. In one embodiment, the aqueous solution of the present invention consists of the peptide, the sugar-based nonionic surfactant, and water.

[0115] The total concentration of the sugar-based nonionic surfactants contained in the aqueous solution of the present invention (when the aqueous solution of the present invention contains two or more types of sugar-based nonionic surfactants, the total concentration of all types of sugar-based nonionic surfactants contained in the aqueous solution) can be appropriately adjusted taking into consideration the type of sugar-based nonionic surfactant, the amount of dried peptide to be dissolved, etc., and is, for example, 0.00125% (w / w) or more, 0.0025% (w / w) or more, 0.005% (w / w) or more, or 0.01% (w / w) or more.

[0116] When the aqueous solution of the present invention contains one type of sugar-based nonionic surfactant alone, examples of the minimum concentration of the sugar-based nonionic surfactant in the aqueous solvent are shown in the above Table 1. When the aqueous solution of the present invention contains each sugar-based nonionic surfactant alone, from the viewpoint of ensuring the effect of suppressing peptide adsorption to the container, the aqueous solution preferably contains each sugar-based nonionic surfactant at a concentration equal to or higher than the minimum concentration shown in Table 1.

[0117] By combining two or more sugar-based nonionic surfactants, it is expected that the effect of suppressing peptide adsorption to the container will be enhanced, and therefore, when combining two or more sugar-based nonionic surfactants, a certain level of peptide adsorption suppression effect can be expected even if the concentration of each sugar-based nonionic surfactant in the aqueous solution is less than the minimum concentration listed in Table 1. However, in order to reliably suppress peptide adsorption, it is preferable to adjust the concentration of each sugar-based nonionic surfactant in the aqueous solution to be equal to or greater than the minimum concentration listed in Table 1, even when combining two or more sugar-based nonionic surfactants.

[0118] In one embodiment, when two or more sugar-based nonionic surfactants are combined, the concentration of each sugar-based nonionic surfactant in the aqueous solution of the present invention is adjusted to 0.005% (w / w) or more.

[0119] Although it is not possible to unequivocally set an upper limit for the total concentration of the sugar-based nonionic surfactant contained in the aqueous solution of the present invention, since suppression of peptide adsorption to a container does not require the formation of micelles by the sugar-based nonionic surfactant, peptide adsorption can be suppressed even with a sugar-based nonionic surfactant at a concentration below the critical micelle concentration (CMC). In one embodiment, the total concentration of the sugar-based nonionic surfactant contained in the aqueous solution of the present invention is below the CMC of the sugar-based nonionic surfactant contained in the aqueous solution. When the aqueous solution of the present invention is used for analysis by LC-MS or the like, it is preferable that the sugar-based nonionic surfactant concentration is as low as possible to avoid contamination of the analysis device. In one embodiment, the total concentration of the sugar-based nonionic surfactant contained in the aqueous solution of the present invention is 0.04% (w / w) or less.

[0120] In one embodiment, the aqueous solution of the present invention is provided as a preparation contained in a container having a plastic surface. Since the sugar-based nonionic surfactant suppresses the adsorption of peptides in the aqueous solution to the plastic surface, a stable aqueous peptide preparation can be provided that is contained in a container having a plastic surface. The types of plastics include, but are not limited to, polypropylene, polystyrene, polymethyl methacrylate, polyvinyl chloride, polyethylene, polycarbonate, polysulfone, fluoropolymer, polyamide, polydimethylsiloxane, polyurethane, polysulfone, polytetrafluoroethylene, elastomer, etc. The plastic surface in the container may or may not be hydrophilically treated.

[0121] The aqueous solution of the present invention is useful as a sample for LC-MS analysis, a peptide standard, a peptide preparation for clinical or non-clinical use, and the like.

[0122] The definitions of each term in the aqueous solution of the present invention are the same as those described in the production method of the present invention and the dissolution promoter of the present invention, unless otherwise specified.

[0123] 4. Method for preparing peptide-containing samples In a further aspect, the present invention provides a method for preparing a peptide-containing sample (hereinafter referred to as "the sample preparation method of the present invention"), comprising the steps of: 1) providing an aqueous peptide solution containing a peptide and 2,2-didecylpropane-1,3-bis-β-D-maltopyranoside (lauryl maltose neopentyl glycol) (LMNG); 2) subjecting the resulting peptide aqueous solution to a reverse phase column to adsorb the peptide and LMNG onto the reverse phase column; and 3) Removal of LMNG from the peptides by eluting the adsorbed peptides from the reversed-phase column while maintaining the adsorption of LMNG to the reversed-phase column. In the process of preparing a peptide-containing sample, when an aqueous peptide solution is provided, the aqueous solution contains LMNG, which suppresses the adsorption of peptides in the solution to a container, thereby avoiding loss of peptides. LMNG also strongly adsorbs to a reversed-phase column, and its elution conditions are significantly different from those of peptides. Even if peptides are eluted from a reversed-phase column under elution conditions that can elute most peptides, including hydrophobic peptides, LMNG is retained in the column, so that LMNG can be easily separated from peptides while avoiding loss of peptides in the desalting step using a reversed-phase column. LMNG effectively suppresses loss of peptides due to adsorption to a container, and can be easily removed from peptides using a reversed-phase column, so it is useful for preparing a trace amount of peptide-containing sample, for example, a peptide-containing sample for LC-MS analysis.

[0124] In the sample preparation method of the present invention, first, an aqueous peptide solution containing LMNG is provided. The peptide may be a single type of peptide or a mixture of multiple types of peptides. In one embodiment, the peptide is a mixture of multiple types of peptides, at least a part of which contains a long-chain peptide (e.g., a peptide having a length of 11 amino acids or more, 12 amino acids or more, 13 amino acids or more, 14 amino acids or more, 15 amino acids or more, or 20 amino acids or more). The upper limit of the length of the long-chain peptide may be, for example, 100 amino acids or less, 80 amino acids or less, 60 amino acids or less, 50 amino acids or less, 40 amino acids or less, or 30 amino acids or less. The proportion of long-chain peptides in the peptide mixture (proportion of the number of molecules) is not particularly limited, and may be, for example, 0.1% or more, 1% or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more.

[0125] The origin of the peptide is not particularly limited, and the peptide may be isolated from a biological material, chemically synthesized, or a recombinant peptide expressed by genetic recombination technology. In one embodiment, the peptide dissolved in the aqueous solvent in the preparation method of the present invention is a peptide mixture obtained by limitedly digesting a biological sample containing a protein with a protease (e.g., trypsin). Such a peptide mixture contains a variety of peptides with different lengths and hydrophobicity, but in the preparation method of the present invention, the adsorption of peptides to the container is suppressed by the addition of LMNG, and most peptides, including long-chain peptides and hydrophobic peptides, can be eluted in reversed-phase column purification while maintaining the adsorption of LMNG to the reversed-phase column, so that the peptide mixture can be purified in a high yield while maintaining the diversity of peptides in the sample.

[0126] The aqueous peptide solution in step 1 may be provided in a state where it is contained in a container having a plastic surface. Adsorption of the peptide in the aqueous solution to the plastic surface can be effectively suppressed by LMNG. The types of plastics include those described in the above item 1. The plastic surface in the container may or may not be hydrophilically treated. The hydrophilic treatment refers to a treatment that adds hydrophilic groups such as hydroxyl groups and carboxyl groups to the plastic surface in the container.

[0127] The aqueous peptide solution may contain only LMNG as a surfactant, or may contain LMNG in combination with one or more other surfactants (e.g., any of the sugar-based nonionic surfactants described above). In order to effectively separate the surfactant from the peptide by the reversed-phase column treatment described below, the aqueous peptide solution preferably contains only LMNG as a surfactant.

[0128] The aqueous peptide solution may contain additional components other than peptide, LMNG, and water. For example, the aqueous peptide solution may contain a buffer, a salt (inorganic salt or organic salt), an amino acid, etc. When preparing a peptide-containing sample for analysis by LC-MS or the like, it is preferable to avoid the inclusion of components that may inhibit the analysis or contaminate the analysis device.

[0129] The concentration of LMNG contained in the aqueous peptide solution may be a concentration that suppresses adsorption of the peptide to the container, for example, 0.00125% (w / w) or more, 0.0025% (w / w) or more, 0.005% (w / w) or more, or 0.01% or more.

[0130] Although it is not possible to unequivocally set an upper limit for the concentration of LMNG contained in the aqueous peptide solution, Suppression of peptide adsorption to a container does not require the formation of micelles by LMNG, and even LMNG at a concentration below the critical micelle concentration (CMC) can suppress peptide adsorption to a container. In one embodiment, the concentration of LMNG contained in the aqueous peptide solution is below the CMC of LMNG. Since LMNG can be removed from peptides by reverse phase column treatment described below, contamination of an analytical device can be avoided when producing a peptide-containing sample for analysis by LC-MS or the like. In one embodiment, the concentration of LMNG contained in the aqueous peptide solution is 0.04% (w / w) or less.

[0131] In one embodiment, the preparation method of the present invention may include obtaining an aqueous peptide solution containing a peptide and LMNG by limited degradation of a protein dissolved in an aqueous solvent containing LMNG with a protease.

[0132] The type of protein is not particularly limited, and for example, a protein-containing sample such as a cell or tissue lysate, serum, or exosome can be appropriately used. The protein may be an affinity purified product such as an immunoprecipitate. The protein may be one in which cysteine ​​residues have been subjected to reduction and alkylation treatment using a reducing reagent such as DTT or TCEP and an alkylating reagent such as iodoacetamide for analysis by LC-MS. The protein may be one that has been modified by phosphorylation, biotinylation, methylation, acetylation, glycosylation, ubiquitination, or the like.

[0133] The proteolytic enzyme is not particularly limited, but for example, trypsin, Glu-C, Lys-N, Lys-C, Asp-N, chymotrypsin, etc. can be used.

[0134] The LMNG concentration in the aqueous protein solution during enzymatic digestion (i.e., the LMNG concentration in the aqueous solvent containing LMNG for dissolving the protein) may be a concentration that suppresses the adsorption of the protein to the container, and is, for example, 0.00125% (w / w) or more, 0.0025% (w / w) or more, 0.005% (w / w) or more, or 0.01% (w / w) or more. The upper limit of the concentration of LMNG contained in the aqueous protein solution cannot be uniquely set, but suppression of the adsorption of the protein to the container does not require the formation of micelles by LMNG. In one embodiment, the concentration of LMNG contained in the aqueous protein solution is lower than the CMC of LMNG. In one embodiment, the concentration of LMNG contained in the aqueous protein solution is 0.04 w / w% or less.

[0135] The protein aqueous solution may contain additional components other than protein, LMNG, protease and water. For example, the protein aqueous solution may contain a buffer suitable for digestion with a protease, a salt (inorganic salt or organic salt), a chaotropic agent, a chelating agent, etc. For example, a buffer suitable for trypsin digestion may include Tris-HCl, ammonium bicarbonate, triethylammonium bicarbonate (TEAB), etc. Salts may include NaCl, etc. Chaotropic agents may include urea, thiourea, etc. When preparing a peptide-containing sample for analysis by LC-MS or the like, it is preferable to avoid the inclusion of components that may inhibit the analysis or contaminate the analysis device.

[0136] Before treating the aqueous peptide solution with the reversed-phase column, the aqueous peptide solution may be subjected to affinity chromatography to concentrate specific peptides, thereby obtaining an aqueous peptide solution containing affinity-enriched peptides and LMNG. Examples of affinity chromatography include, but are not limited to, immobilized metal affinity chromatography, avidin or streptavidin affinity chromatography, and the like. Immobilized metal affinity chromatography includes immobilized metal ion affinity chromatography and metal oxide affinity chromatography. For example, when the peptide contains a phosphorylated peptide, the aqueous peptide solution may be subjected to immobilized metal ion affinity chromatography (IMAC) equipped with a metal ion such as Fe3+ or metal oxide affinity chromatography equipped with a metal oxide such as TiO2 to concentrate the phosphorylated peptide. In addition, when the peptide contains a biotinylated peptide, the aqueous peptide solution may be subjected to avidin or streptavidin affinity chromatography to concentrate the biotinylated peptide. By containing LMNG in the aqueous peptide solution, non-specific adhesion of peptides to the affinity column is suppressed, and the amount of affinity-enriched peptides (e.g., phosphorylated peptides, biotinylated peptides) recovered is increased. When performing this affinity purification, it is preferable to use an eluate containing LMNG and recover affinity-enriched peptides (e.g., concentrated phosphorylated peptides or biotinylated peptides) as an aqueous peptide solution containing LMNG.

[0137] Next, the resulting peptide aqueous solution is applied to a reversed-phase column. This causes the peptide and LMNG to be adsorbed onto the reversed-phase column. The peptide aqueous solution is also desalted by the reversed-phase column treatment, and salts, buffers, chaotropic agents, and the like in the peptide aqueous solution are removed. Examples of reversed-phase columns include, but are not limited to, columns in which hydrophobic groups such as octadecyl groups (C18), octyl groups (C8), butyl groups (C3), phenyl groups, and cyanopropyl groups are bonded to a solid phase carrier (e.g., silica gel), and styrene-divinylbenzene (SDB) copolymer columns. The reversed-phase column is preferably a column in which octadecyl groups (C18) are bonded to a solid phase carrier (e.g., silica gel) (referred to as a C18 column) or an SDB copolymer column. Commercially available reversed-phase columns can be used, and commercially available kits such as EVOSEP ONE and SDB-STAGE chips may also be used.

[0138] Next, the adsorbed peptides are eluted from the reversed-phase column. Since LMNG has a higher affinity for the reversed-phase column than peptides, it is possible to remove LMNG from the peptides by eluting the adsorbed peptides from the reversed-phase column while maintaining the adsorption of LMNG to the reversed-phase column. The solvent used for eluting the peptides is usually a mixture of water and an organic solvent such as acetonitrile, methanol, tetrahydrofuran, isopropanol, or acetone. To promote protonation, it is preferable to add an acid such as trifluoroacetic acid, formic acid, acetic acid, or hydrochloric acid to the water. The concentration of the acid is usually 0.05 to 0.2% (v / v). In order to minimize contamination of the eluted peptides with LMNG, the polarity of the eluent used for peptide elution is preferably equivalent to or greater than a 50 / 50 (v / v) mixture of acetonitrile / 0.1% (v / v) aqueous trifluoroacetic acid, for example, equivalent to or greater than a 40 / 60 (v / v) mixture of acetonitrile / 0.1% (v / v) aqueous trifluoroacetic acid, equivalent to or greater than a 38 / 62 (v / v) mixture of acetonitrile / 0.1% (v / v) aqueous trifluoroacetic acid, or equivalent to or greater than a 37 / 63 (v / v) mixture of acetonitrile / 0.1% (v / v) aqueous trifluoroacetic acid. On the other hand, from the viewpoint of minimizing the amount of peptide remaining in the reverse-phase column and increasing the recovery rate of the peptide, it is preferable that the polarity of the eluent used for eluting the peptide is low, and is usually equivalent to or less than a mixture of acetonitrile / 0.1% (v / v) aqueous trifluoroacetic acid = 30 / 70 (v / v), preferably equivalent to or less than a mixture of acetonitrile / 0.1% (v / v) aqueous trifluoroacetic acid = 32 / 68 (v / v), equivalent to or less than a mixture of acetonitrile / 0.1% (v / v) aqueous trifluoroacetic acid = 34 / 66 (v / v), equivalent to or less than a mixture of acetonitrile / 0.1% (v / v) aqueous trifluoroacetic acid = 35 / 65 (v / v), or equivalent to or less than a mixture of acetonitrile / 0.1% (v / v) aqueous trifluoroacetic acid = 36 / 64 (v / v). In one embodiment, the polarity of the eluent is equivalent to or less than a mixture of acetonitrile / 0.1% (v / v) aqueous trifluoroacetic acid = 30 / 70 (v / v) and equivalent to or more than a mixture of acetonitrile / 0.1% (v / v) aqueous trifluoroacetic acid = 50 / 50 (v / v).In one embodiment, the polarity of the eluent is equivalent to or less than a mixture of acetonitrile / 0.1% (v / v) trifluoroacetic acid aqueous solution = 30 / 70 (v / v), and equivalent to or more than a mixture of acetonitrile / 0.1% (v / v) trifluoroacetic acid aqueous solution = 40 / 60 (v / v). In one embodiment, the polarity of the eluent is equivalent to or less than a mixture of acetonitrile / 0.1% (v / v) trifluoroacetic acid aqueous solution = 30 / 70 (v / v), and equivalent to or more than a mixture of acetonitrile / 0.1% (v / v) trifluoroacetic acid aqueous solution = 38 / 62 (v / v). The standard eluent from the C18 column used in EVOSEP ONE is a mixture of acetonitrile / 0.1% (v / v) formic acid aqueous solution = 35 / 65 (v / v). The polarity of the eluent can be calculated based on the "Snyder polarity parameter" specific to each solvent and the mixing ratio of the solvent. In addition, in this specification, "acetonitrile / 0.1% (v / v) aqueous trifluoroacetic acid solution = 30 / 70 (v / v) mixed solution" means a mixed solution obtained by mixing 30 volumes of acetonitrile and 70 volumes of 0.1% (v / v) aqueous trifluoroacetic acid solution, and other mixed solutions are interpreted similarly.

[0139] This reversed-phase column treatment removes LMNG from the peptides, resulting in a purified peptide-containing sample that is useful for LC-MS analysis, which can be performed by drying the eluted peptides (e.g., in a container with a plastic surface) and redissolving the dried peptides in water.

[0140] Therefore, in one embodiment, the preparation method of the present invention can also be considered as a method for identifying a peptide, comprising the steps of: 1) providing an aqueous peptide solution containing a peptide and 2,2-didecylpropane-1,3-bis-β-D-maltopyranoside (lauryl maltose neopentyl glycol) (LMNG); 2) subjecting the resulting peptide aqueous solution to a reverse phase column to adsorb the peptide and LMNG onto the reverse phase column; 3) removing LMNG from the peptides by eluting the adsorbed peptides from the reversed-phase column while maintaining the adsorption of LMNG to the reversed-phase column; 4) drying the eluted peptides to obtain dry peptides; 5) dissolving the obtained dry peptide in water to obtain an aqueous peptide solution; and 6) The resulting peptide aqueous solution is subjected to LC-MS analysis, and the peptides are identified based on the results of the MS analysis.

[0141] In one embodiment, the preparation method of the present invention can also be considered as a method for identifying a protein in a protein-containing sample (a method for analyzing a protein profile, a method for proteome analysis) comprising the following steps: 1) dissolving proteins in a protein-containing sample in an aqueous solvent containing LMNG and subjecting the proteins to limited hydrolysis with a protease to obtain an aqueous peptide solution containing peptides and LMNG; 2) subjecting the resulting peptide aqueous solution to a reverse phase column to adsorb the peptide and LMNG onto the reverse phase column; 3) removing LMNG from the peptides by eluting the adsorbed peptides from the reversed-phase column while maintaining the adsorption of LMNG to the reversed-phase column; 4) drying the eluted peptides to obtain dry peptides; 5) dissolving the obtained dry peptide in water to obtain an aqueous peptide solution; and 6) subjecting the resulting peptide aqueous solution to LC-MS analysis and identifying the peptides based on the results of the MS analysis; and 7) Identifying proteins in protein-containing samples based on the identified peptides.

[0142] Furthermore, dissolving the dried peptides by the above-mentioned production method of the present invention can promote dissolution of the dried peptides and suppress loss of the peptides during the redissolution step of the dried peptides, thereby further improving the number of peptides and proteins identified in LC-MS. That is, the present invention also provides a method for preparing a peptide-containing sample, comprising the following steps: 1. Providing an aqueous peptide solution containing a peptide and LMNG; 2. The resulting peptide aqueous solution is applied to a reverse phase column to adsorb the peptide and LMNG onto the reverse phase column; 3. Removing LMNG from the peptides by eluting the adsorbed peptides from the reversed-phase column while maintaining the adsorption of LMNG to the reversed-phase column; 4. Drying the eluted peptides to obtain dry peptides; and 5. Dissolving the obtained dry peptide in an aqueous solvent containing at least one sugar-based nonionic surfactant to obtain an aqueous peptide solution.

[0143] In one embodiment, the present invention also provides a method for identifying a peptide comprising the steps of: 1) providing an aqueous peptide solution containing a peptide and 2,2-didecylpropane-1,3-bis-β-D-maltopyranoside (lauryl maltose neopentyl glycol) (LMNG); 2) subjecting the resulting peptide aqueous solution to a reverse phase column to adsorb the peptide and LMNG onto the reverse phase column; 3) removing LMNG from the peptides by eluting the adsorbed peptides from the reversed-phase column while maintaining the adsorption of LMNG to the reversed-phase column; 4) drying the eluted peptides to obtain dry peptides; 5) dissolving the obtained dry peptide in an aqueous solvent containing at least one sugar-based nonionic surfactant to obtain an aqueous peptide solution; and 6) The resulting peptide aqueous solution is subjected to LC-MS analysis, and the peptides are identified based on the results of the MS analysis.

[0144] In one embodiment, the present invention also provides a method for identifying a protein in a protein-containing sample (a method for analyzing a protein profile, a method for proteome analysis) comprising the steps of: 1) dissolving proteins in a protein-containing sample in an aqueous solvent containing LMNG and subjecting the proteins to limited hydrolysis with a protease to obtain an aqueous peptide solution containing peptides and LMNG; 2) subjecting the resulting peptide aqueous solution to a reverse phase column to adsorb the peptide and LMNG onto the reverse phase column; 3) removing LMNG from the peptides by eluting the adsorbed peptides from the reversed-phase column while maintaining the adsorption of LMNG to the reversed-phase column; 4) drying the eluted peptides to obtain dry peptides; 5) dissolving the obtained dry peptide in an aqueous solvent containing at least one sugar-based nonionic surfactant to obtain an aqueous peptide solution; and 6) subjecting the resulting peptide aqueous solution to LC-MS analysis and identifying the peptides based on the results of the MS analysis; and 7) Identifying proteins in protein-containing samples based on the identified peptides.

[0145] The definitions of each term relating to the sample preparation method of the present invention are the same as those described for the production method of the present invention, the dissolution enhancer of the present invention, and the aqueous solution of the present invention, unless otherwise specified.

[0146] All references cited herein, including publications, patent documents, and the like, are hereby incorporated by reference to the same extent as if each was individually and specifically incorporated by reference and the contents of which were specifically set forth in their entirety.

[0147] The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto. EXAMPLES

[0148] [Example 1] Dissolution of dried peptide mixture with DDM solution Materials and Methods Dry peptide mixture Tryptic digests of 100 μg of K562 cells (CAT# V6951, Promega, Madison, WI, USA) were adjusted to 50 ng / μL with 50% acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA) and 10 μL of each were dispensed into standard tubes (1.5 ml safe-lock tubes, CAT# 0030120086, Eppendorf, Hamburg; Germany), standard polypropylene (PP) vials (CAT# C5000-97, Thermo Fisher Scientific, Waltham, MA, USA) or hydrophilic vials (ProteoSave vials, CAT# 11-19-1021-10, AMR Inc, Tokyo, Japan). The samples were dried in a centrifugal evaporator (miVac Duo concentrator, Genevac Ltd., Ipswich, UK) and redissolved in 25 μL of 0.1% TFA in 2% ACN or 0.005–0.04% DDM.

[0149] Co-immunoprecipitation (coIP) and on-bead digestion HeLa cells were mixed with IP lysis buffer (Pierce IP Lysis Buffer: CAT# 87788, Thermo Fisher Scientific, Waltham, MA, USA) containing protease inhibitors (CAT# 5892791001, cOmplete ULTRA Tablets, Sigma-Aldrich, MO, USA) and phosphatase inhibitors (CAT# 4906837001, PhosSTOP Tablets, Sigma-Aldrich) for 30 seconds and incubated on ice for 30 minutes. The cell lysate was then centrifuged at 18,000g for 30 minutes at 4°C, and the supernatant was collected. The protein concentration in the protein extract was measured using a BCA protein assay kit (CAT# 23225, Thermo Fisher Scientific) and adjusted to 2 μg / μL with IP lysis buffer.

[0150] Co-immunoprecipitation (IP) from HeLa cell lysates was performed using an automated TANBead Maelstrom 8 (TANBead, Taipei, Taiwan). Sera-Mag SpeedBeads Protein A / G Magnetic Particles (CAT# 171521040150, Cytiva, Uppsala, Sweden) and anti-RELA (NF-κB p65) antibodies (CAT# ab16502, Abcam, Cambridge, MA, USA) were used as beads and antibodies, respectively. To prepare beads for immunoprecipitation, 1.5 μL of bead slurry was washed once with 500 μL of Tris-buffered saline containing 0.05% Tween 20 (TBST). Anti-RELA antibodies were then captured on the beads by mixing the beads in 200 μL of TBST containing 4 μg of anti-RELA antibodies for 30 min at room temperature and washing twice with 500 μL of TBST to remove unbound antibodies. Then, 200 μL of HeLa cell lysate (2 μg / μL) was added to the beads, incubated for 60 min at room temperature with end-over-end mixing, and washed twice with 500 μL of IP lysis buffer, once with 500 μL of TBST, and once with 500 μL of 50 mM Tris-HCl (pH 8.0). 100 μL of 50 mM Tris-HCl (pH 8.0) was added to the beads, and on-bead digestion was performed using a previously published procedure with minor modifications (Nat Cell Biol 2018, 20 (1), 81-91). Briefly, proteins were digested by adding 500 ng of trypsin / Lys-C Mix (CAT# V5072, Promega, Madison, WI, USA) to the beads and gently mixing overnight at 37 °C. The beads were then aggregated from the digested samples using a magnetic stand (EpiMag HT (96-Well) Magnetic Separator, EpiGentek, Brooklyn, NY, USA), and the supernatant was collected.The collected samples were treated with 20 mM tris(2-carboxyethyl)phosphine at 80°C for 10 min, and alkylated with 35 mM iodoacetamide at room temperature for 30 min in the dark. The alkylated samples were then acidified with 20 μL of 5% trifluoroacetic acid (TFA), desalted using a STAGE tip (CAT# 7820-11200, GL Sciences Inc, Tokyo, Japan) according to the manufacturer's protocol, and dried in a centrifugal evaporator (miVac Duo concentrator, Genevac Ltd., Ipswich, UK). The dried samples were redissolved in 12 μL of 2% ACN or 0.02% DDM containing 0.1% TFA and transferred to regular PP vials (Thermo Fisher Scientific). 2.5 μL of each sample was injected into the LC-MS / MS.

[0151] LC-MS / MS The reconstituted peptides were directly injected into a 75 μm × 12 cm nanoLC nanocapillary column (Nikkyo Technos Co., Ltd., Tokyo, Japan) at 40 °C and separated with a 60 min gradient (A = 0.1% FA in water, B = 0.1% FA in 80% ACN) consisting of 0 min 8% B, 60 min 44% B (for coIP-MS) and 0 min 6% B, 60 min 48% B (for non-cpIP-MS) at a flow rate of 150 nl / min using an UltiMate 3000 RSLCnano LC system (Thermo Fisher Scientific, Waltham, MA, USA). Peptides eluted from the column were analyzed by DDA and overlapping window DIA on an Orbitrap Exploris 480 (Thermo Fisher Scientific) (J Am Soc Mass Spectrom 2019, 30 (4), 669-684). The DDA sets the Auto Gain Control (AGC) target to 3×10 6The maximum injection time was set to 100 msec, and MS1 spectra were collected in the range of 380-1,240 m / z at a resolution of 60,000. 3 The 40 most intense ions with charge states 2+ to 6+ over 10 were dissociated by collision-induced dissociation with a normalized collision energy of 30%. The AGC target value was 1×10 5 For the overlapping window DIA, MS1 spectra were collected in the range of 495-905 m / z at a resolution of 30,000 and an automatic gain control target value of 3 × 10 6 The maximum injection time was set to 55 msec. MS2 spectra were collected in the m / z range of 200-1,800 at a resolution of 30,000 and an automatic gain control target of 3 × 10 6 The MS2 isolation width was set to 8 m / z, and the overlapping window pattern from 500 to 900 m / z used window placement optimized in Xcalibur 4.4 (Thermo Fisher Scientific).

[0152] DDA-MS files were searched against the human UniProt reference proteome (Proteoem ID UP000005640, review, canonical; 20,381 entries). For DDA-MS files, the Proteome Discoverer 2.5 (Thermo Fisher Scientific) search engine was used with Sequest HT and Percolator. The following parameters were set: Enzyme Trypsin Maximum missed cleavage sites 3 Precursor mass tolerance 10 ppm Fragment mass tolerance 0.02 Da Static modification Cysteine ​​carbamidomethylation Dynamic modification Methionine oxidation DIA-MS files were searched against an in silico human spectral library using Scaffold DIA (Proteome Software, Inc., Portland, OR, USA) as previously reported ( J Proteome Res 2022, 21 (5), 1340-1348 ).

[0153] Data analysis Principal component analysis (PCA) and Pearson correlation coefficient heat map analysis with hierarchical clustering were performed using Perseus v1.6.15.0 (Nat Methods 2016, 13 (9), 731-740).

[0154] (Results and Discussion) Dissolving dried peptides with DDM To clarify the change in the recovery rate of dried peptides due to the DDM solution, a small amount (500 ng) of dried tryptic peptides, which are prone to peptide loss, was prepared. The dried peptides were dissolved in 2% ACN-0.1% TFA, a common solution containing no surfactant, and 0.005-0.04% DDM, and subjected to DDA-LC-MS / MS analysis. Compared with 0.005-0.04% DDM, 2% ACN-0.1% TFA had clearly lower peak intensities after a retention time of 40 min (Figure 2A), and the number of identified protein groups and peptides was also smaller (Figure 2B). The main reason for the small number of identified protein groups and peptides in 2% ACN-0.1% TFA was the clear decrease in the number of peptides identified after a retention time of 40 min (Figure 2C). When the length of the peptides identified in each solution was examined, the difference in the number of identified peptides between 2% ACN-0.1% TFA and DDM solutions increased as the peptides became longer (Figure 2D). Thus, it was revealed that the use of DDM mainly improves the recovery rate of long peptides. Although there were some differences in the DDM concentration, 0.02% allowed the identification of the most protein groups and peptides, so in the subsequent tests, a 0.02% DDM solution was used to dissolve the dried peptides.

[0155] Changes in recovery of dried peptides using hydrophilic vials The recovery rate of dried peptides in hydrophilically coated vials was evaluated. 500 ng of tryptic peptides were dried in hydrophilically coated or normal PP vials, dissolved in 2% ACN-0.1% TFA or 0.02% DDM, and quantitatively evaluated by DIA-LC-MS / MS analysis. The number of identified peptides was higher in hydrophilically coated vials when dissolved in 2% ACN-0.1% TFA, and slightly lower when dissolved in 0.02% DDM (Figure 3A). Regardless of the vial, more peptides were identified when dissolved in 0.02% DDM than when dissolved in 2% ACN-0.1% TFA. Regarding peptide intensity, the peak intensity of dissolution with 0.02% DDM in the normal vial was higher than that of dissolution with 0.02% DDM in the hydrophilic vial, followed by dissolution with 2% ACN-0.1% TFA in the hydrophilic vial, and then dissolution with 2% ACN-0.1% TFA in the normal vial, with the difference being more pronounced for longer peptides (Fig. 3B). Next, cluster analysis based on protein intensity level showed that clusters were not separated between vials in DDM, whereas clusters were separated between vials in 2% ACN-0.1% TFA, and that the clusters in the hydrophilic vial were closer to those in the case of dissolution with DDM (Fig. 3C). A similar trend was confirmed by principal component analysis based on protein intensity (Fig. 3D). Both peptide-level and protein-level analyses showed that the recovery rate of dried peptides was improved by using hydrophilic vials for dissolution with 2% ACN-0.1% TFA. On the other hand, when dissolving with 0.02% DDM, the peptide recovery rate was slightly reduced by using hydrophilic vials in peptide-level analysis, but there was no difference between the two vials in protein-level analysis. More than the difference between the vials, the contribution of the use of 0.02% DDM or not to peptide recovery was very large, and it was shown that regular vials are sufficient for use with 0.02% DDM. Vials and tubes with hydrophilic coating are expensive, so dissolving with inexpensive DDM allows high peptide recovery rates to be maintained even with regular PP vials and tubes, which is advantageous in terms of cost.DDM is not a volatile solvent, so there is a risk of contaminating the MS instrument, but even though we have been using DDM for half a year, the frequency of cleaning the MS instrument has not changed compared to when we used 2% ACN-0.1% TFA. It is possible that the use of a low concentration of 0.02% DDM has reduced contamination of the MS instrument.

[0156] Effect of DDM on coIP-MS Since the amount of proteins bound to low-abundance proteins such as transcription factors is even smaller, it has been difficult to search for interactors by coIP-MS for such low-abundance proteins. In this study, in order to observe low-abundance interactors by coIP-MS, peptides were desalted using a reversed-phase spin column, dried in a centrifugal evaporator, and then dissolved in DDM. In coIP, an antibody against the transcription factor RELA (NF-κB p65) was used and reacted with HeLa lysate. Figure 4A shows the TIC chromatograms of the dried tryptic peptides obtained by coIP against RELA, dissolved in 2% ACN-0.1% TFA and 0.02% DDM, respectively, and analyzed by DIA-LC-MS / MS (Figure 4A). As in the results of Figure 2A, the peak intensity was clearly higher in the latter half of the LC retention time when dissolved in 0.02% DDM compared to when dissolved in 2% ACN-0.1% TFA. In addition, the number of identified protein groups and peptides was also higher with 0.02% DDM (Figure 4B). Furthermore, the number of known RELA interactors included in the identified protein groups was also higher with 0.02% DDM (Figure 4C). These results indicate that more interactors can be observed simply by changing the solvent for dissolving dried peptides to 0.02% DDM. In addition, when the protein intensities of RELA and well-known interactors of RELA (NFKB1, NFKB2, NFKBIA, NFKBIB, and CREBBP) were examined, the protein intensities were high for all proteins when dissolved in 0.02% DDM, suggesting that the recovery rate of important proteins can be improved in coIP-MS for RELA (Figure 4D). Thus, dissolving dried peptides in DDM in coIP-MS was very effective in improving the recovery rate of peptides and observing many interactors. DDM may improve the recovery rate of dried peptides in other proteomics approaches, especially when dealing with trace samples.

[0157] (Conclusion) The effect of DDM solution was examined to improve the recovery rate of dried peptides. The use of DDM significantly improved the recovery rate of long peptides. Furthermore, by using DDM, it was possible to recover high peptides in a normal PP vial without using expensive hydrophilic coated vials. This method was applied to RELA coIP-MS analysis, and it was shown that it is possible to improve peptide recovery rate and observe many interacting factors. By simply changing the solvent for dissolving dried peptides to DDM solution, the recovery rate of long peptides in particular is improved, so it can be easily applied to various proteomics approaches.

[0158] [Example 2] Dissolution of dry peptide mixtures with various sugar-based nonionic surfactant solutions As in Example 1, 400 ng of the dry peptide mixture was dissolved in 20 μL of various 0.00125-0.04% sugar-based nonionic surfactant solutions, and 2.5 μL of the resulting peptide solution was analyzed by LC-MS / MS. The sugar-based nonionic surfactants evaluated are shown in Table 2.

[0159] [Table 2]

[0160] The number of peptides that could be identified by LC-MS / MS is shown in Table 3 and FIG.

[0161] [Table 3]

[0162] When the dry peptide mixture was dissolved in a 0.04% solution of each sugar-based nonionic surfactant, the number of identified peptides increased compared to 2% ACN-0.1% TFA regardless of the sugar-based nonionic surfactant used (Figure 5), indicating that the sugar-based nonionic surfactant has the effect of promoting the dissolution of the dry peptide mixture. Even when the surfactant concentration was reduced to 0.0025%, A (n-dodecyl-β-D-maltoside), B (n-undecyl-β-D-maltoside), F (3-oxatridecyl-α-D-mannoside), G (Decyl maltose neopentyl glycol), H (lauryl maltose neopentyl glycol), I (Octyl glucose neopentyl glycol) and J (Trehalose C12) maintained a high number of peptide identifications (Table 3). In particular, surfactant F (3-oxatridecyl-α-D-mannoside) showed a high number of peptide identifications (Table 3).

[0163] [Example 3] Enhancement of solubility of dry peptide mixture by combination of two surfactants In Example 2, the top six surfactants (A, F, G, H, I, J) with the highest number of peptide identifications at a concentration of 0.005% were selected, and the effect of combining two of them on the solubility of the dry peptide mixture was evaluated. An aqueous solution of a mixture of two surfactants was prepared so that the final concentration of each surfactant was 0.005% and the total concentration of the two surfactants was 0.01%. As in Example 2, 400 ng of the dry peptide mixture was dissolved in 20 μL of each surfactant solution, and 2.5 μL of the resulting peptide solution was analyzed by LC-MS / MS to compare the number of peptide identifications. The results are shown in Table 4.

[0164] [Table 4]

[0165] The number of peptide identifications was higher when two surfactants were combined at a total concentration of 0.01% (AG, AH, AI, AJ, FG, FI, FJ, GH, GI, GJ, HI, IJ) than when each surfactant was used alone at a concentration of 0.01% (Table 3), suggesting that the combination of two sugar-based nonionic surfactants synergistically improves the solubility of dried peptides. In particular, the number of peptide identifications was particularly high for the combinations of AJ, JI, GI, and FG. The number of peptide identifications for these four surfactant combinations was compared with the top six surfactants (A, B, F, G, I, J) that had the highest number of peptide identifications at a concentration of 0.01%. The results are shown in Table 5.

[0166] [Table 5]

[0167] As a result, the combination of A and J had the highest number of peptide identifications, suggesting that this is the optimal surfactant combination for dissolving dry peptides.

[0168] [Example 4] Improvement and elimination of peptide loss by LMNG Materials and Methods LC-MS measurement of surfactants The surfactants were directly injected onto a 75 μm × 12 cm nanoLC column (Nikkyo Technos Co., Ltd., Tokyo, Japan) at 50 °C and separated using an UltiMate 3000 RSLCnano LC system (Thermo Fisher Scientific) with a 30 min gradient consisting of 0 min 5% B, 25 min 95% B, and 30 min 95% B (A = 0.1% FA in water, B = 0.1% FA in 80% ACN) at a flow rate of 300 nl / min. Samples eluted from the column were analyzed by DDA on a Q Exactive HF-X (Thermo Fisher Scientific). MS1 spectra were collected in the range of 100–1,500 m / z at a resolution of 60,000 with an automatic gain control (AGC) objective of 1 × 10 6 The maximum injection time was set to 119 msec. MS chromatograms of the monoisotopic mass (single charge) of each surfactant were obtained. FA: Formic acid ACN: Acetonitrile

[0169] Elution conditions for SDB-STAGE chips for LMNG, DMNG and DDM SDB-STAGE chips were washed with 25 μL of 80% ACN in 0.1% TFA and equilibrated with 50 μL of 3% ACN in 0.1% TFA. 10 μL of 0.001% LMNG, DMNG or DDM were then loaded onto the chip, washed with 80 μL of 3% ACN in 0.1% TFA, and stepwise eluted with 50 μL of 30% ACN in 0.1% TFA, 40% ACN in 0.1% TFA, and 50% ACN in 0.1% TFA. Additional DMNG samples were stepwise eluted with 50 μL of 30% ACN in 0.1% TFA, 32% ACN in 0.1% TFA, 34% ACN in 0.1% TFA, 36% ACN in 0.1% TFA, 38% ACN in 0.1% TFA, and 40% ACN in 0.1% TFA. The eluate was dried using a centrifugal evaporator (miVac Duo concentrator, Genevac Ltd., Ipswich, UK). The dried sample was redissolved in 200 μL of H2O and transferred to a regular vial (CAT# C5000-97, Thermo Fisher Scientific, Waltham, MA, USA). TFA: Trifluoroacetic acid

[0170] Protein extraction from HEK293T cells Proteins in HEK293T cells were extracted in 100 mM Tris-HCl (pH 8.0) containing 4% sodium dodecyl sulfate (SDS) and 20 mM NaCl by sonication using a Bioruptor II (Cosmo Bio, Tokyo, Japan) for 10 min. Protein concentrations in the protein extracts were measured using a BCA protein assay kit (CAT# 23225, Thermo Fisher Scientific) and adjusted to 500 ng / μL or 5 ng / μL with 100 mM Tris-HCl (pH 8.0) containing 4% SDS and 20 mM NaCl.

[0171] Purification of extracellular vesicles (EVs) from human serum Human serum EVs were purified by the Tim4-phosphatidylserine (PS) affinity method using MagCapture Exosome Isolation Kit PS Ver.2 (Wako Pure Chemical Industries, Osaka, Japan) combined with a Maelostrom 8 Autostage (TANBeads). Briefly, 250 μl of pooled healthy serum was centrifuged at 3,000 × g for 20 min at 4°C, and 200 μL of the supernatant was collected in a new tube. Then, 300 μL of TBS and 1 μL of Exosome Binding Enhancer (included in the kit) were added to the supernatant and mixed gently. To prepare beads for the Tim4-PS affinity method, 30 μl of Exosome Capture Beads (included in the kit) were washed once with 250 μl of Exosome Immobilizing / Washing Buffer (included in the kit). The beads were then mixed with 10 μl of biotin-labeled Exosome Capture (diluted in 250 μl of Exosome Immobilizing / Washing Buffer) and stirred at 1,000 rpm for 10 min. After washing twice with 250 μl of Exosome Immobilizing / Washing Buffer, the beads were added to the sample. To allow EVs to bind to the beads, the mixture was stirred at 1,000 rpm for 2 h and washed three times with 250 μl of Exosome Immobilizing / Washing Buffer. Finally, the EVs captured by the beads were eluted with 100 mM Tris-HCl (pH 8.0) containing 4% SDS and 20 mM NaCl.

[0172] Protein Digestion Protein lysates and purified EVs were treated with 20 mM tris(2-carboxyethyl)phosphine at 80 °C for 10 min, alkylated with 35 mM iodoacetamide at room temperature for 30 min in the dark, and subjected to cleanup and digestion by single-pot solid-phase enhanced sample preparation (SP3) using a TANBead Maelstrom 8 (TANBead, Taipei, Taiwan). Briefly, two types of Sera-Mag SpeedBead carboxylate-modified magnetic particles (hydrophilic particles, CAT# 45152105050250; hydrophobic particles, CAT# 65152105050250; Cytiva, Marlborough, MA, USA) were used. These beads were combined in a 1:1 (v / v) ratio, washed twice with distilled water, and reconstituted in distilled water at a concentration of 8 μg solids / μL. Then, 20 μL of reconstituted beads were added to the alkylated protein sample, and 99.5% ethyl alcohol was added to a final concentration of 75% (v / v) and stirred for 5 min. The supernatant was discarded, and the pellet was washed twice with 80% ethyl alcohol. The beads were suspended in 100 μL of 50 mM Tris-HCl (pH 8.0) or 50 mM Tris-HCl (pH 8.0) containing 0.02% LMNG, followed by the addition of 500 ng of trypsin / Lys-C Mix (CAT# V5072, Promega, Madison, WI, USA) and gentle mixing overnight at 37 °C to digest the proteins. The digested samples were acidified with 20 μL of 5% trifluoroacetic acid (TFA) and high-level sonicated at room temperature for 5 min using a Bioruptor II (Cosmo Bio). Samples were desalted using SDB-STAGE chips (CAT# 7820-11200, GL Sciences, Tokyo, Japan) or Evotip Pure (CAT# EV2015, EVOSEP, Odense, Denmark). SDB-STAGE chips were washed with 25 μl of 80% ACN in 0.1% TFA, followed by equilibration with 50 μl of 3% ACN in 0.1% TFA. Samples were then loaded onto the chip, washed with 80 μl of 3% ACN in 0.1% TFA, and eluted with 50 μl of 50% ACN in 0.1% TFA or 36% ACN in 0.1% TFA.The eluate was dried using a centrifugal evaporator (miVac Duo concentrator). The dried samples were redissolved in 8 μL of 2% ACN containing 0.1% TFA or 0.01% DMNG and transferred to a regular vial. 4 μL of the sample was injected into the LC-MS / MS. The Evotip Pure was used according to the manufacturer's protocol. Briefly, the tip was washed with 20 μl of ACN in 0.1% formic acid (FA) and then equilibrated with 20 μl of ACN. The sample was then loaded onto the tip and washed with 20 μl of H2O in 0.1% FA. The tip was filled with 0.1% H2O until LC-MS / MS measurement.

[0173] Co-IP and on-beads digestion IP lysis buffer [Pierce IP Lysis Buffer (CAT# 87788, Thermo Fisher Scientific, Waltham, MA, USA)] containing protease inhibitors (CAT# 5892791001, cOmplete ULTRA Tablets, Sigma-Aldrich, MO, USA) and phosphatase inhibitors (CAT# 4906837001, PhosSTOP Tablets, Sigma-Aldrich) was added to HeLa cells and mixed for 30 min at 4°C. The cell lysate was then centrifuged at 18,000g for 30 min at 4°C, and the supernatant was collected. The protein concentration in the protein extract was measured using a BCA protein assay kit (CAT# 23225, Thermo Fisher Scientific) and adjusted to 2 μg / μL with IP lysis buffer. Co-immunoprecipitation (Co-IP) from HeLa cell lysates was performed using an automated TANBead Maelstrom 8 (TANBead). The beads for Co-IP were Sera-Mag SpeedBeads Protein A / G Magnetic Particles (CAT# 171521040150, Cytiva, Uppsala, Sweden) and the antibody was anti-RELA (NF-κB p65) antibody (CAT# ab16502, Abcam, Cambridge, MA, USA). To prepare the beads for immunoprecipitation, 1.5 μL of bead slurry was washed once with 500 μL of Tris-buffered saline (TBST) containing 0.05% Tween 20. Anti-RELA antibody was then captured on the beads by mixing the beads in 200 μL of TBST containing 4 μg of anti-RELA antibody for 30 min at room temperature and washing twice with 500 μL of TBST to remove unbound antibody. Next, 200 μL of HeLa cell lysate (2 μg / μL) was added to the beads and incubated for 60 min at room temperature with end-over-end mixing, followed by washing three times with 500 μL of IP lysis buffer and twice with 500 μL of 50 mM Tris-HCl (pH 8.0).Proteins were digested by adding 100 μL of 50 mM Tris-HCl (pH 8.0) or 50 mM Tris-HCl (pH 8.0) containing 0.02% LMNG to the beads, followed by 500 ng of Trypsin / Lys-C Mix (CAT#V5072, Promega) and gently mixing at 37°C overnight. The beads were then aggregated from the digested samples using a magnetic stand (EpiMag HT (96-Well) Magnetic Separator, EpiGentek, Brooklyn, NY, USA), and the supernatant was collected. The collected samples were treated with 20 mM Tris(2-carboxyethyl)phosphine at 80°C for 10 min and alkylated with 35 mM iodoacetamide at room temperature for 30 min in the dark. The alkylated samples were then acidified with 10 μL of 10% TFA (total volume 120 μL), desalted using SDB-STAGE tips (eluent: 36% ACN in 0.1% TFA), and dried in a centrifugal evaporator (miVac Duo concentrator). The dried samples were redissolved in 10 μL of 2% ACN containing 0.1% TFA or 0.01% DMNG and transferred to regular vials (Thermo Fisher Scientific). 1 μL of the sample was injected into the LC-MS / MS.

[0174] Typical DDA-MS by LC-MS / MS The resolubilized peptides were directly injected onto a 75 μm × 12 cm nanoLC nanocapillary column (Nikkyo Technos Co., Ltd., Tokyo, Japan) at 50 °C using an UltiMate 3000 RSLCnano LC system (Thermo Fisher Scientific, Waltham, MA, USA) and separated with a 30 min gradient consisting of 0 min 5% B, 30 min 45% B (A = 0.1% FA in water, B = 0.1% FA in 80% ACN) at a flow rate of 300 nl / min. Peptides eluted from the column were analyzed by DDA on an Orbitrap Exploris 480 (Thermo Fisher Scientific). MS1 spectra were collected in the range of 380–1,240 m / z at a resolution of 60,000 with an automatic gain control (AGC) objective of 3 × 10 6 The maximum injection time was set to 100 msec. 3 The 50 most intense ions with charge states 2+ to 5+ above 1×10 were fragmented by collision-induced dissociation at a normalized collision energy of 28%. 5 The maximum injection time was set to "Auto" and MS2 spectra were collected over a 200 m / z range with a resolution of 15,000. The dynamic exclusion time was set to 30 seconds.

[0175] Typical DIA-MS by LC-MS / MS The resolubilized peptides were directly injected onto a 75 μm × 12 cm nanoLC column (Nikkyo Technos Co., Ltd., Tokyo, Japan) at 50 °C and separated using an UltiMate 3000 RSLCnano LC system with a 60 min gradient (A = 0.1% FA in water, B = 0.1% FA in 80% ACN) consisting of 0 min 8% B, 50 min 35% B, 57 min 70% B, and 60 min 70% B at a flow rate of 200 nl / min. Peptides eluted from the column were analyzed by DIA using an Orbitrap Exploris 480. MS1 spectra were collected in the range of 495–905 m / z at a resolution of 15,000 with an automatic gain control target of 3 × 10 6The maximum injection time was set to 23 msec. MS2 spectra were collected over a 200 m / z range at a resolution of 30000 and an automatic gain control target of 3 × 10 6 The maximum injection time was set to “auto” and the normalized collision energy was set to 26%. The MS2 isolation width was set to 8 m / z, and the window pattern from 500 to 900 m / z used window placement optimized in Xcalibur 4.4 (Thermo Fisher Scientific).

[0176] EVOSEP ONE LC-MS / MS Evotip Pure samples were analyzed using an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an EVOSEP ONE system (EVOSEP) and an InSpIon system with electrospray ionization XYZ stage and column oven (AMR, Tokyo, Japan). The EVOSEP ONE was acquired using the Whisper 80 SPD method (15 min gradient at a flow rate of 100 nl / min). Digested peptides were separated at 60 °C using an Aurora Rapid 75 C18 capillary column (5 cm × 75 μm id, 1.7 μm particle size; IonOpticks, VIC, Australia). Mobile phases A and B consisted of 0.1% FA in HO and 0.1% FA in ACN, respectively. Peptides eluted from the column were analyzed by DIA using an Orbitrap Exploris 480. Collect MS1 spectra in the range of 645–775 m / z at a resolution of 7,500 with an automatic gain control objective of 1 × 10. 6 The maximum injection time was set to 10 msec. MS2 spectra were collected in the m / z range of 200–1800 with a resolution of 30000 and an automatic gain control target of 3 × 10 6The maximum injection time was set to “auto” and the normalized collision energy was set to 28%. The MS2 isolation width was set to 8 m / z, and the window pattern from 650 to 770 m / z used window placement optimized in Xcalibur 4.4 (Thermo Fisher Scientific).

[0177] Protein identification and quantitative analysis from MS data DDA-MS files were searched against the human protein sequence database (Proteome ID UP000005640, reviewed, canonical, 20,591 entries) using Proteome Discoverer 3.0 with Sequest HT and Percolator (Thermo Fisher Scientific) with the following parameters: Enzyme Trypsin Maximum missed cleavage sites 2 precursor mass tolerance 10 ppm fragment mass tolerance 0.02 Da static modification Cysteine ​​carbamidomethylation dynamic modification Methionine oxidation

[0178] The DIA-MS files were also searched against an in silico human spectral library using DIA-NN (version: 1.8.1, https: / / github.com / vdemichev / DiaNN). First, a spectral library was created from a human protein sequence database using DIA-NN. The parameters for creating the spectral library were as follows: Digestion enzyme Trypsin Missed cleavages 1 Peptide length range 7-45 前体电荷范围2 - 4 前体m / z范围395 - 1005 碎片离子m / z范围200 - 1800 用于无库搜索 / 库生成的FASTA消化功能已启用 基于深度学习的光谱、保留时间和离子淌度预测功能已启用 N端甲硫氨酸切除功能已启用 半胱氨酸的碳酰胺甲基化功能已启用

[0179] DIA-NN的搜索参数如下: 质量精度10 ppm(使用Evosep One时为15 ppm); MS1精度10 ppm(使用Evosep One时为15 ppm) 蛋白质推断基因 神经网络分类单通道模式 定量策略稳健液相色谱(高精度) 跨运行归一化关闭 无关运行已启用 使用同位素异构体已启用 启发式蛋白质推断已启用 无共享光谱已启用

[0180] MBR was turned off when searching for protein and precursor identification and turned on for quantitative analysis. The threshold for protein identification was set at 1% or lower for both precursor and protein FDR.

[0181] Phosphoproteomics 100 μg of peptide digests were dissolved in 80% ACN-0.1 TFA. The dissolved peptides were added to Fe beads washed with 80% ACN-0.1% TFA and mixed for 60 min at room temperature, then washed twice with 1 mL of 80% ACN-0.1% TFA and once with 1 mL of 0.1% TFA. The phosphopeptides were eluted by adding 200 μL of 3% polyphosphoric acid or 3% polyphosphoric acid containing 0.02% LMNG to the beads and mixing for 10 min at room temperature. The eluted phosphopeptides were desalted with an SDB-STAGE tip (eluent: 36% ACN in 0.1% TFA). The dried samples were redissolved in 10 μL of 0.01% DMNG and transferred to regular vials. The reconstituted peptides were directly injected onto a 75 μm × 30 cm nanoLC column (CAT# HEB07503001718IWF, CoAnn Technologies, Richland, WA, USA) at 60 °C and separated using an UltiMate 3000 RSLCnano LC system (Thermo Fisher Scientific, Waltham, MA, USA) with a 100 min gradient (A = 0.1% FA in water, B = 0.1% FA in 80% ACN) at a flow rate of 150 nl / min, consisting of 5% B at 0 min, 35% B at 86 min, 70% B at 93 min, and 70% B at 100 min. Peptides eluted from the column were analyzed by DDA using an Orbitrap Exploris 480 (Thermo Fisher Scientific). Collect MS1 spectra in the range of 400–1,500 m / z at a resolution of 60,000 with an automatic gain control (AGC) objective of 3 × 10. 6 , Maximum injection time was set to “auto”. 2.0×10 4The 50 most intense ions with charge states 2+ to 4+ above 10 were fragmented by collision-induced dissociation with stepwise normalized collision energies of 22, 26, and 30%. MS2 spectra were collected in the m / z range of 200–1,800 at a resolution of 30,000 and an AGC target of 5 × 10 5 The maximum injection time was set to "auto". The dynamic exclusion time was set to 30 seconds. MS files were searched against the human protein sequence database (Proteome ID UP000005640, reviewed, canonical, 20,591 entries) using Proteome Discoverer 3.0 with Sequest HT and Percolator (Thermo Fisher Scientific). The setup parameters were: Enzyme Trypsin Maximum missed cleavage sites 2 Precursor mass tolerance 10 ppm Fragment mass tolerance 0.02 Da Static modification Cysteine ​​carbamidomethylation Dynamic modification Serine, threonine and tyrosine phosphorylation.

[0182] LC-MS / MS analysis of biotinylated BSA A mixture of 10 ng of biotinylated BSA digest and 5 μg of K562 cell digest (Promega) was added to streptavidin beads (10 μl of suspension was used; Cat # 211521040150, Cytiva) that had been washed once with TBST and mixed for 60 min at room temperature, followed by washing three times with 1 mL of 50 mM Tris-HCl (pH 8.0) containing 0.5% SDS and 500 mM NaCl and once with 1 mL of TBS. Biotinylated peptides were eluted by adding 50 μl of 8 M guanidine-HCl (pH 1.5) containing 0.5 mM biotin or 8 M guanidine-HCl (pH 1.5) containing 0.02% LMNG and 0.5 mM biotin to the beads and mixing for 2 h at room temperature. The eluted peptides were desalted on an SDB-STAGE chip (elution: 36% ACN in 0.1% TFA). The dried samples were redissolved in 10 μl of 0.01% DMNG and transferred to regular vials. The redissolved peptides were directly injected into a 75 μm × 12 cm nanoLC nanocapillary column (Nikkyo Technos, Tokyo, Japan) at 50 °C and separated using an UltiMate 3000 RSLCnano LC system (Thermo Fisher Scientific, Waltham, MA, USA) with a flow rate of 300 nl / min and a 30 min gradient consisting of 8% B at 0 min, 45% B at 26 min, 85% B at 29 min, and 85% B at 30 min (A = 0.1% FA in water, B = 0.1% FA in 80% ACN). Peptides eluted from the column were analyzed on a Q Exactive HF-X (Thermo Fisher Scientific) equipped with an InSpIon system. MS1 spectra were collected at a resolution of 120,000 in the range of 450–1,500 m / z with an automatic gain control (AGC) target of 3 × 10 6 The maximum injection time was set to 100 msec. 3 The 20 most intense ions with charge states 2+ to 7+ above 10 were fragmented by collision-induced dissociation at normalized step collision energies of 22, 26, and 30%. MS2 spectra were collected over the 200 m / z range at a resolution of 60,000 and an AGC target of 2 × 10 5, maximum injection time was set to 120 ms. Dynamic exclusion time was set to 30 s. MS files were searched against the human protein sequence UniProt database (proteome ID UP000005640, 20,591 entries, downloaded May 5, 2023) and BSA (UniProt Ac P02769) using PEAKS Studio 11 (Bioinformatics Solution Inc, Waterloo, Canada). MS files were searched against the human protein sequence UniProt database (proteome ID UP000005640, 20,591 entries, downloaded May 5, 2023) and BSA (UniProt Ac P02769) using PEAKS Studio 11 (Bioinformatics Solution Inc, Waterloo, Canada). Setting parameters were as follows: Enzyme Trypsin Maximum missed cleavage sites 4 Precursor mass tolerance 10 ppm Fragment mass tolerance 0.02 Da Static modification Cysteine ​​carbamidomethylation Dynamic modification Lysine biotinylation The threshold for protein identification was set at 1% or less for both peptide and protein FDR.

[0183] 4. Data Analysis Principal component analysis (PCA) and Pearson correlation coefficient heatmap analysis with hierarchical clustering were performed using Perseus v1.6.15.0.

[0184] (Results and Discussion) The surfactants shown in Table 6 were separated on a reversed-phase (C18) column and their elution times were compared. The results are shown in Figure 6.

[0185] [Table 6]

[0186] As a result, it was confirmed that LMNG had a significantly slower elution time than other surfactants and had a strong affinity for reversed-phase columns. Because LMNG has an overwhelmingly strong affinity for reversed-phase columns, it was suggested that if LMNG is added for the purposes of peptide affinity for LC-MS analysis and suppression of adsorption to tubes, when the sample is desalted with a reversed-phase solid-phase column, LMNG will not be eluted from the reversed-phase solid-phase column even if the peptides are eluted, and it may be easily removed.

[0187] Next, the extraction conditions from the reversed-phase solid-phase extraction column were examined for the three surfactants with the slowest elution times: LMNG, DMNG, and DDM. The SDB-based reversed-phase solid-phase extraction column evaluated is often used for desalting digested peptides, and to ensure that peptides are eluted from the column, a 50-80% ACN solution is generally used as the eluent. Therefore, LMNG, DMNG, and DDM were trapped in an SDB solid-phase extraction column and successively eluted with increasing organic solvent concentrations, 30% ACN-0.1% TFA, 40% ACN-0.1% TFA, 50% ACN-0.1% TFA, and 60% ACN-0.1% TFA, and the eluates were analyzed by LC-MS to measure the elution times. As a result, DDM and DMNG were eluted with 30% ACN-0.1% TFA (Figure 7). Therefore, in order to elute digested peptides while keeping these surfactants adsorbed to the reversed-phase solid-phase column, the ACN concentration must be lowered to less than 30%. However, at ACN concentrations lower than 30%, there is a high risk that peptides with high affinity to the reversed-phase support (hydrophobic peptides) will remain adsorbed and not be fully eluted, resulting in their loss. On the other hand, LMNG was not eluted at all with 30% ACN-0.1% TFA, but was slightly eluted with 40% ACN-0.1% TFA (Figure 7). Further detailed investigation of the elution conditions for LMNG revealed that it was not eluted at 38% ACN-0.1% TFA or lower (Figure 8). Since the elution method of the solid-phase extraction column may change slightly depending on the temperature and elution speed, 36% ACN-0.1% TFA can be adopted as a condition in which LMNG is not eluted, with a margin. Next, we evaluated whether 36% ACN-0.1% TFA is sufficient as an elution condition for digested peptides. As a result, the number of peptides identified under the elution conditions of 50% ACN-0.1% TFA and 36% ACN-0.1% TFA hardly changed (Figure 9), and it was determined that the peptide components were almost completely eluted from the reversed-phase solid-phase column with 36% ACN-0.1% TFA. Based on the above, the present inventors have succeeded in establishing a method for removing LMNG without affecting the elution of peptides from a reversed-phase solid-phase extraction column.Since LMNG can now be easily removed, we actually added LMNG to the digested peptide sample solution to test whether peptide loss would be improved. Specifically, the starting sample was 100 ng of HEK293 cell lysate, and it was confirmed that the number of peptides identified by LC-MS increased dramatically by adding LMNG during trypsin digestion compared to the case without LMNG (Figure 10). An increase in the number of identified peptides was confirmed at LMNG concentrations of 0.01% to 0.04%, with the largest number of peptides identified at 0.02%.

[0188] Furthermore, in order to minimize sample loss, the inventors tried adding LMNG to the digested peptide solution, desalting it with a reversed-phase solid-phase extraction column, drying the eluted peptides, and dissolving the dried peptides in a low-concentration DMNG aqueous solution (Figures 11 and 12). In Figure 11, 100 ng of HEK293 cell lysate was used as the starting sample. By adding LMNG to the digested solution, the number of precursor (peptide) and protein identifications in LC-MS was greatly improved, and by dissolving the dried peptides in DMNG, these identification numbers were further improved. In addition, in samples without LMNG addition, the individual protein quantification values ​​were widely dispersed, and the correlation coefficient Pearson r was low. In the case of a trace sample such as 100 ng of HEK293 cell lysate, it was suggested that the addition of LMNG not only improved the number of protein and precursor (peptide) identifications, but also improved the reproducibility of pretreatment. We also investigated the effect of adding LMNG during on-bead digestion in co-immunoprecipitation-mass spectrometry (coIP-MS), and the effect of dissolving dried peptides with DMNG. In Figure 12, coIP-MS was performed using an anti-RELA antibody. As in the test in Figure 6, the addition of LMNG during digestion significantly improved the number of precursor (peptide) and protein identifications, and dissolving dried peptides with DMNG further improved these identifications. The recovery of RELA, the antigen recognized by the antibody, and its known interactors NFKB1, IKBKB, NFKBIA, NFKBIB, and NFKBIE was significantly improved by adding LMNG during digestion, and was also improved by adding DMNG when dissolving dried peptides. This confirmed that the addition of LMNG during digestion and DMNG when dissolving dried peptides have beneficial effects in coIP-MS as well. Furthermore, the number of identified phosphorylated peptides was improved by adding LMNG to the eluate during phosphorylated peptide enrichment by immobilized metal ion affinity chromatography (Figures 13 and 14).In addition, adding LMNG to the eluate during biotinylated peptide enrichment using avidin beads also increased the recovery of biotinylated peptides and improved the number of identified biotinylated peptides (Figures 15 and 16). LMNG can be easily removed using a reverse-phase solid-phase extraction column, so it can be applied not only to peptide loss during digestion but also to peptide affinity purification, and is expected to have a wide range of applications.

[0189] Finally, we investigated the adaptability of LMNG to analysis using EVOSEP ONE, a specialized LC for multi-analyte analysis without carryover developed for clinical proteome analysis. EVOSEP ONE is an LC device used worldwide to perform proteome analysis of hundreds to thousands of samples. With EVOSEP ONE, it is possible to perform LC analysis by trapping a sample on a reversed phase solid-phase extraction column (Evotip pure (C18)) and setting it in EVOSEP ONE. With EVOSEP ONE, the manufacturer has already prepared a method optimized for peptide analysis, and the settings do not allow fine gradient changes. First, we investigated whether LMNG is eluted with EVOSEP ONE. As controls, DDM and DMNG were trapped in Evotip pure, separated with EVOSEP ONE, and measured with MS (Figure 17). Peaks were detected at the end of the elution time for both DDM and DMNG, indicating that they were eluted from Evotip pure, but LMNG elution was not observed. When 100ng of HEK293 cell lysate was digested with or without LMNG and analyzed by LC-MS / MS using EVOSEP ONE, more precursors (peptides) and proteins were identified with LMNG (Figure 18). In addition, when exosomes were purified from serum samples that were the subject of multi-analyte analysis and analyzed by EVOSEP ONE using LC-MS / MS, the addition of LMNG during digestion improved the identification of precursors (peptides) and proteins, and the recovery of representative exosome markers such as CD9, CD63, and CD81 was also high (Figure 19). LMNG also increased the recovery of peptides in EVOSEP ONE and was easily removed by Evotip pure, confirming its high suitability for high-throughput proteome analysis using EVOSEP ONE.

[0190] In the examples of this specification, the concentrations of acetonitrile, trifluoroacetic acid, and formic acid in a solution expressed as a percentage mean volume / volume %, and the concentrations of surfactants in an aqueous solution expressed as a percentage mean weight / weight %, respectively. [Industrial Applicability]

[0191] The present invention provides a surfactant that can suppress peptide loss during pretreatment of a sample for proteome analysis and can easily remove peptides. By treating a sample for proteome analysis with the method of the present invention, peptide loss from a trace amount of sample is suppressed, the number of peptide identifications and protein identifications in mass spectrometry increases, and the reproducibility of analysis is improved. In addition, the present invention improves the recovery amount of peptide fragments adsorbed to the wall of a tube by drying during pretreatment of proteome analysis. In particular, the recovery amount of long peptides of 11 amino acids or more is significantly improved, making it possible to prepare a mass spectrometry sample rich in long peptides, which increases the number of peptide identifications and protein identifications in mass spectrometry, contributing to improving the accuracy of proteome analysis. Since an effect of improving the recovery amount of peptide fragments is observed with a very low concentration of a sugar-based nonionic surfactant, contamination of a mass spectrometry device can also be minimized. In addition, the present invention provides a stable peptide aqueous solution in which peptide adsorption to a container is suppressed.

[0192] This application is based on patent application No. 2022-199022 filed in Japan (filing date: December 13, 2022), the contents of which are incorporated in their entirety herein.

Claims

1. Method for preparing a peptide-containing sample, including the following steps: 1) To provide an aqueous peptide solution containing a peptide and 2,2-didecylpropane-1,3-bis-β-D-maltopyranoside (hereinafter referred to as "LMNG"). 2) The obtained aqueous peptide solution is attached to a reversed-phase column, and the peptide and LMNG are adsorbed onto the reversed-phase column, and 3) Remove LMNG from the peptide by eluting the adsorbed peptide from the reversed-phase column while maintaining the adsorption of LMNG to the reversed-phase column.

2. The preparation method according to claim 1, wherein the reversed-phase column is a C18 column or a styrene-divinylbenzene copolymer column.

3. The preparation method according to claim 1, wherein the peptide is eluted from a reversed-phase column using an eluent having polarity equivalent to or greater than a 50 / 50 (v / v) mixture of acetonitrile / 0.1% (v / v) trifluoroacetic acid aqueous solution.

4. The preparation method according to claim 3, wherein the polarity of the eluate is equivalent to or less than a mixture of acetonitrile / 0.1% (v / v) trifluoroacetic acid aqueous solution = 30 / 70 (v / v).

5. The preparation method according to claim 1, wherein the LMNG concentration in the aqueous peptide solution is below the critical micelle concentration.

6. The preparation method according to claim 1, wherein the aqueous peptide solution is provided in a container having a plastic surface.

7. The preparation method according to claim 1, further comprising obtaining a peptide and an aqueous peptide solution containing LMNG by selectively degrading a protein dissolved in an aqueous solvent containing LMNG with a protease.

8. The preparation method according to claim 1, further comprising subjecting the aqueous peptide solution provided in step 1) to affinity chromatography to obtain an aqueous peptide solution containing affinity-concentrated peptide and LMNG.

9. The preparation method according to claim 8, wherein the affinity chromatography is immobilized metal affinity chromatography or avidin or streptavidin affinity chromatography.

10. The preparation method according to claim 1, wherein the peptide-containing sample is for LC-MS analysis.

11. A method for identifying a peptide, comprising the following steps: 1) To provide an aqueous peptide solution containing peptides and LMNG. 2) The obtained aqueous peptide solution is attached to a reversed-phase column, and the peptide mixture and LMNG are adsorbed onto the reversed-phase column. 3) Remove LMNG from the peptide by eluting the adsorbed peptide from the reversed-phase column while maintaining the adsorption of LMNG to the reversed-phase column. 4) Dry the eluted peptides to obtain a dried peptide mixture. 5) Dissolve the obtained dried peptide in water to obtain an aqueous peptide solution, and 6) The obtained peptide aqueous solution is subjected to LC-MS analysis, and the peptide is identified based on the MS analysis results.

12. A method for identifying proteins in a protein-containing sample, comprising the following steps: 1) Dissolve the protein in the protein-containing sample in an aqueous solvent containing LMNG, and obtain a peptide and an aqueous peptide solution containing LMNG by limited decomposition with a protease. 2) The obtained aqueous peptide solution is attached to a reversed-phase column, and the peptide mixture and LMNG are adsorbed onto the reversed-phase column. 3) Remove LMNG from the peptide by eluting the adsorbed peptide from the reversed-phase column while maintaining the adsorption of LMNG to the reversed-phase column. 4) Dry the eluted peptide to obtain a dried peptide. 5) Dissolve the obtained dried peptide in water to obtain an aqueous peptide solution. 6) The obtained aqueous peptide solution is subjected to LC-MS analysis, and the peptide is identified based on the MS analysis results, and 7) Identify the proteins in the protein-containing sample based on the identified peptides.

13. A method for producing an aqueous peptide solution, comprising dissolving a dried peptide in an aqueous solvent containing at least one sugar-based nonionic surfactant.

14. A sugar-based nonionic surfactant is given by formula (I); 【Chemistry 1】 (G 1 L is a monosaccharide group or a disaccharide group. 1 is O, S, -O-(CH 2 ) n -O- or -O-CO-, R 1 (where n is a linear alkyl group having 6 to 12 carbon atoms, and n is an integer from 1 to 3), or formula (II); 【Chemistry 2】 (G 2 and G 3 are, independently or differently, a monosaccharide group or a disaccharide group, and R 2 and R 3 are, independently or differently, a linear alkyl group having 6 to 12 carbon atoms) The manufacturing method according to claim 13, wherein the compound is represented by the compound.

15. A sugar-based nonionic surfactant is a compound represented by formula (I), and G 1 The manufacturing method according to claim 14, wherein is a glucose group, a mannose group, a maltose group, or a trehalose group.

16. The manufacturing method according to claim 15, wherein the sugar-based nonionic surfactant is n-dodecyl-β-D-maltoside, n-undecyl-β-D-maltoside, n-decyl-β-D-maltoside, n-nonyl-β-D-thiomaltoside, n-octyl-β-D-thioglucoside, 3-oxatridecyl-α-D-mannoside, or α-D-glucopyranosyl-α-D-glucopyranoside monododecanoate.

17. A sugar-based nonionic surfactant is a compound represented by formula (II), G 2 and G 3 The manufacturing method according to claim 14, wherein is the same and is a glucose group or a maltose group.

18. The production method according to claim 17, wherein the sugar-based nonionic surfactant is 2,2-dioctylpropane-1,3-bis-β-D-maltopyranoside, 2,2-didecylpropane-1,3-bis-β-D-maltopyranoside, or 2,2-dihexylpropane-1,3-bis-β-D-glucopyranoside.

19. The manufacturing method according to claim 13, wherein the aqueous solvent comprises a combination of at least two sugar-based nonionic surfactants.

20. The manufacturing method according to claim 19, wherein the combination of sugar-based nonionic surfactants is selected from the following group: n-dodecyl-β-D-maltoside and α-D-glucopyranosyl-α-D-glucopyranosyl monododecanoates; 2,2-dihexylpropane-1,3-bis-β-D-glucopyranoside and α-D-glucopyranosyl-α-D-glucopyranoside monododecanoate; 2,2-dioctylpropane-1,3-bis-β-D-maltopyranoside and 2,2-dihexylpropane-1,3-bis-β-D-glucopyranoside; and 3-Oxatridecyl-α-D-mannoside and 2,2-dioctylpropane-1,3-bis-β-D-maltopyranoside.

21. The method for producing a dried peptide according to claim 13, wherein the dried peptide is contained in a container having a plastic surface.

22. A method for identifying a peptide, comprising the following steps: 1) To obtain an aqueous peptide solution by dissolving the dried peptide in an aqueous solvent containing at least one sugar-based nonionic surfactant, and 2) The obtained peptide aqueous solution is subjected to LC-MS analysis.

23. A dry peptide solubility accelerator comprising at least one sugar-based nonionic surfactant.

24. A dry peptide dissolution accelerator according to claim 23, comprising a combination of at least two sugar-based nonionic surfactants.

25. The combination of sugar-based nonionic surfactants is selected from the following group, the dry peptide dissolution accelerator according to claim 24: n-dodecyl-β-D-maltoside and α-D-glucopyranosyl-α-D-glucopyranosyl monododecanoates; 2,2-dihexylpropane-1,3-bis-β-D-glucopyranoside and α-D-glucopyranosyl-α-D-glucopyranoside monododecanoate; 2,2-dioctylpropane-1,3-bis-β-D-maltopyranoside and 2,2-dihexylpropane-1,3-bis-β-D-glucopyranoside; and 3-Oxatridecyl-α-D-mannoside and 2,2-dioctylpropane-1,3-bis-β-D-maltopyranoside.

26. An aqueous peptide solution comprising a peptide and a combination of at least two sugar-based nonionic surfactants.

27. The aqueous peptide solution according to claim 26, wherein the combination of sugar-based nonionic surfactants is selected from the following group: n-dodecyl-β-D-maltoside and α-D-glucopyranosyl-α-D-glucopyranosyl monododecanoates; 2,2-dihexylpropane-1,3-bis-β-D-glucopyranoside and α-D-glucopyranosyl-α-D-glucopyranoside monododecanoate; 2,2-dioctylpropane-1,3-bis-β-D-maltopyranoside and 2,2-dihexylpropane-1,3-bis-β-D-glucopyranoside; and 3-Oxatridecyl-α-D-mannoside and 2,2-dioctylpropane-1,3-bis-β-D-maltopyranoside.

28. A method for identifying a peptide, comprising subjecting an aqueous peptide solution containing a peptide and a combination of at least two sugar-based nonionic surfactants to LC-MS analysis.

29. A method for preparing a peptide-containing sample according to claim 1, further comprising the following steps: 4) Drying the eluted peptide to obtain a dried peptide, and 5) An aqueous peptide solution is obtained by dissolving the obtained dried peptide in an aqueous solvent containing at least one sugar-based nonionic surfactant.

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