JP7203407B2 - Methods of preparing peptide samples for mass spectrometry - Google Patents

Description

The present invention relates to a method for preparing a sample for mass spectrometry containing peptide fragments that constitute mammalian cell proteins.

Proteomics is a technique for comprehensively analyzing proteins using a mass spectrometer, and is an important technique for life science, especially biomarker research. When preparing a peptide sample for mass spectrometry, first, proteins are solubilized from tissues or cells, and the solubilized proteins are fragmented with a proteolytic enzyme such as trypsin. After that, the fragmented peptides are measured using a mass spectrometer such as a liquid chromatograph-tandem mass spectrometer (LC-MS/MS), and based on the mass spectrum and mass chromatogram of the obtained peptides, In addition to identification and quantification of proteins, it is possible to identify and quantify various mutants of proteins (Patent Document 1).

Mass spectrometry generally uses a sample containing a large number of cells, so the information obtained therefrom is the average information of a large number of cells. Analysis of protein expression at the single-cell level is performed using, for example, flow cytometry or immunofluorescence staining. However, these methods are intended to measure specific proteins, and cannot comprehensively analyze protein expression. The main problem with single-cell proteomics is the low recovery rate of peptide fragments from a minute amount of protein, and the current situation is that it has not yet been realized.

The present inventors solubilized proteins using a surfactant that can be modified to be fat-soluble, and after proteolytic treatment, modified the surfactant to be fat-soluble and separated in an organic solvent solution, resulting in peptide fragments. reported that the recovery rate of is improved (Patent Document 2).

WO 2017/022562 Pamphlet WO 2009/044903 Pamphlet

An object of the present invention is to provide a mass method that is excellent in convenience and rapidity and that can efficiently recover peptide fragments that constitute the protein even from an amount of mammalian cell protein equivalent to one cell. An object of the present invention is to provide a method for preparing a peptide sample for analysis.

The present inventors are continuing intensive research to solve the above problems. In the process, a mammalian cell protein solubilized with a surfactant is placed in a droplet whose surface is covered with an organic solvent liquid (i.e., a droplet that does not contact the container surface), a peptide-binding solid-phase carrier and Proteolytic treatment in the presence of a proteolytic enzyme to prepare a peptide-binding solid-phase carrier to which peptide fragments constituting the protein are bound, and then separating the peptide-binding solid-phase carrier from the organic solvent solution. Peptide fragments constituting the protein can be efficiently recovered even from mammalian cell proteins in an amount equivalent to cells, and as a result, the number of peptide fragments identified by mass spectrometry and the types of cellular proteins are increased. and completed the present invention.

That is, the present invention is as follows.
[1] A method for preparing a peptide sample for mass spectrometry, comprising the following steps (a) and (b) (hereinafter sometimes referred to as "present preparation method 1").
(a) one or more proteins of mammalian cells solubilized with a surfactant are added to a peptide-binding solid-phase carrier and a proteolytic enzyme in a droplet whose surface is covered with an organic solvent liquid; Preparing a peptide-binding solid-phase carrier bound with a peptide fragment constituting a mammalian cell protein by proteolytic treatment in the presence;
(b) separating the peptide-binding solid-phase carrier to which the peptide fragment constituting the mammalian cell protein is bound, and preparing a peptide sample for mass spectrometry;
[2] A method for preparing a peptide sample for mass spectrometry, comprising the following steps (a-1), (a-2), and (b) (hereinafter referred to as "preparation method 2") be).
(a-1) proteolysis of one or more mammalian cell proteins solubilized with a surfactant in the presence of a proteolytic enzyme in a droplet whose surface is covered with an organic solvent liquid; processing;
(a-2) A peptide-binding solid-phase carrier is added to the droplets obtained in step (a-1) to prepare a peptide-binding solid-phase carrier to which a peptide fragment constituting a mammalian cell protein is bound. the step of
(b) separating the peptide-binding solid-phase carrier to which the peptide fragment constituting the mammalian cell protein is bound, and preparing a peptide sample for mass spectrometry;
[3] A method for preparing a peptide sample for mass spectrometry, comprising the following steps (A) and (b) (hereinafter sometimes referred to as "Preparation Method 3").
(A) Solubilization treatment of one or more mammalian cells in droplets whose surface is covered with an organic solvent liquid in the presence of a surfactant, a protease, and a peptide-binding solid-phase carrier and proteolytic treatment to prepare a peptide-binding solid-phase carrier to which a peptide fragment constituting a mammalian cell protein is bound;
(b) separating the peptide-binding solid-phase carrier to which the peptide fragment constituting the mammalian cell protein is bound, and preparing a peptide sample for mass spectrometry;
[4] The preparation method according to any one of [1] to [3] above, wherein the peptide-binding solid-phase carrier is a magnetic bead whose surface is coated with carboxyl groups.
[5] The preparation method according to any one of [1] to [4] above, wherein the surfactant is a phase transfer solubilizer.
[6] the phase transfer dissolving agent contains sodium deoxycholate, and in step (p), before separating the peptide-binding solid-phase carrier to which the peptide fragment constituting the protein of mammalian cells is bound, an acid is added; The preparation method according to [5] above, which further comprises the step (x) of oil-solubilizing the sodium deoxycholate in an organic solvent liquid in addition to the droplets.
[7] The preparation method according to any one of [1] to [6] above, wherein the organic solvent liquid is a liquid containing ethyl acetate.

By using the present preparation methods 1 to 3 (hereinafter collectively referred to as "the present preparation method"), even from one mammalian cell, the mammalian cell can be prepared relatively easily and rapidly Since the peptide fragments that make up the protein inside can be recovered efficiently, not only can single-cell proteomics be possible, but also the number of mammalian cells required for mass spectrometry can be reduced. In addition, using this preparation method enables proteomics of valuable cell types with a limited number of cells such as cancer stem cells and hematopoietic stem cells. It is expected to make a significant contribution to development.

FIG. 4 is a diagram showing the results of mass spectrometric analysis of four types of peptide fragment samples (Example samples and Comparative Examples 1 to 3 samples) and analysis of signal intensities of all mass spectra. [Comparative Example 2 sample preparation method] and [Example sample preparation method] were performed in the absence (0 ng) or presence (1 ng) of the solubilized cell protein, and the peptide fragments were identified by mass spectrometry. FIG. 10 shows. The vertical axis indicates the average number of identified peptide fragments (n=3). The solubilized cell proteins are bound to Magnosphere (“Magnosphere-COOH” in the figure) or FG beads (“FG beads-COOH” in the figure), collected and purified, and the amount of cell protein is measured by CBB staining. It is a figure which shows the result of having analyzed. Example samples were prepared from cell proteins solubilized with sodium deoxycholate (“SDC” in FIG. 4) or urea (“urea” in FIG. 4), and the recovery rate of peptide fragments was analyzed by mass spectrometry. (n=3); FIG. Example samples and Comparative Example 2 samples were prepared from 0.1 ng of HeLa cell protein extract, and the results of analyzing the number of peptide fragments (Fig. 5A) and the number of cell proteins (Fig. 5B) identified by mass spectrometry are shown. It is a diagram.

In step (a) of Preparation Method 1, proteolytic treatment of one or more proteins of mammalian cells solubilized with a surfactant (hereinafter sometimes simply referred to as "solubilized protein"); , binding reaction of the peptide fragment produced by such decomposition treatment to the peptide-binding solid-phase carrier is performed in a droplet whose surface is covered with an organic solvent liquid, and the peptide fragment constituting the protein (hereinafter referred to as , sometimes simply referred to as "peptide fragment") is bound to a peptide-binding solid phase carrier (hereinafter sometimes referred to as "peptide fragment-peptide-binding solid phase carrier complex"). As a specific method of the step (a), for example, a solubilized protein is dissolved in an organic solvent liquid in a container (for example, a tube made of a material such as glass or plastic, a test tube, etc.; the same shall apply hereinafter). and a droplet containing a peptide-binding solid-phase carrier and a droplet containing a protease are brought into contact to prepare a droplet containing the solubilized protein, the peptide-binding solid-phase carrier, and the protease, and proteolyze treatment; and binding of the peptide fragment to the peptide-binding solid-phase carrier; A droplet containing a phase carrier and a droplet containing a protease are contacted to prepare a droplet containing a solubilized protein, a peptide-binding solid phase carrier, and a protease, and subjected to proteolytic treatment; binding of the fragment to the peptide-binding solid-phase carrier; a method of incubating under appropriate conditions, or a droplet containing a solubilized protein, a peptide-binding solid-phase carrier, and proteolysis in an organic solvent liquid in a container; Contact with a droplet containing an enzyme, prepare a droplet containing a solubilized protein, a peptide-binding solid phase carrier and a proteolytic enzyme, perform proteolytic treatment; a method of incubating under conditions suitable for binding; The number of droplets before contact and the number of droplets obtained after contact should be at least one.

In the present preparation method 2, first, in step (a-1), proteolytic treatment of the solubilized protein is performed in a droplet whose surface is covered with an organic solvent liquid, and then step (a-2) ), the peptide fragments produced by such degradation treatment are bound to a peptide-binding solid-phase carrier. As a specific method of such steps (a-1) and (a-2), for example, droplets containing a solubilized protein and droplets containing a protease are mixed in an organic solvent liquid in a container. Droplets containing the solubilized protein and proteolytic enzyme are prepared by contact, incubated under conditions suitable for proteolytic treatment (step [a-1]), and then containing peptide fragments produced by such hydrolytic treatment. A method of contacting a droplet with a droplet containing a peptide-binding solid-phase carrier and incubating under conditions suitable for binding of the peptide fragment to the peptide-binding solid-phase carrier (step [a-2]). be able to. The number of droplets before contact and the number of droplets obtained after contact should be at least one.

The solubilized proteins in Preparation Methods 1 and 2 can be prepared by solubilizing mammalian cell proteins in a surfactant solution in contact with the surface of a container, and mammalian cell proteins can be prepared by subjecting them to surfactant Solubilization treatment and preparation in droplets containing an agent and the surface of which is covered with an organic solvent liquid (i.e., the surface is not in contact with the container surface due to the surface being covered with the organic solvent liquid) can also

In step (A) of Preparation Method 3, one or more mammalian cells (hereinafter sometimes simply referred to as "cells") are solubilized, proteolytically treated, and Binding of the peptide fragment to the peptide-binding solid-phase carrier is performed in a droplet whose surface is covered with an organic solvent liquid, and the peptide fragment is bound to the peptide-binding solid-phase carrier. As a specific method of such step (A), for example, droplets containing cells, droplets containing a protease, and droplets containing a peptide-binding solid-phase carrier are placed in an organic solvent liquid in a container. or contacting a droplet containing cells with a droplet containing a protease and a peptide-binding solid-phase carrier, or contacting a droplet containing cells and a peptide-binding solid-phase carrier with a protein Contact with a droplet containing a degrading enzyme, prepare a droplet containing cells, a protease, and a peptide-binding solid phase carrier, solubilize; proteolytic treatment; and a peptide-binding solid phase of a peptide fragment binding to a carrier; and incubation under appropriate conditions. The number of droplets before contact and the number of droplets obtained after contact should be at least one.

As the above-mentioned "suitable conditions for proteolytic treatment", most of the solubilized protein (for example, within the range of 70 to 100%, preferably within the range of 90 to 100%) is fragmented specifically with a proteolytic enzyme. The temperature is usually in the range of 20 to 39° C. (preferably in the range of 30 to 37° C.), and the treatment time is usually in the range of 15 seconds to 48 hours (preferably, within 16-36 hours).

The above-mentioned "suitable conditions for binding of peptide fragments to peptide-binding solid-phase carriers" means that most of the peptide fragments (for example, within the range of 70 to 100%, preferably within the range of 90 to 100%) are peptide Any conditions are acceptable as long as they can bind to the binding solid-phase carrier. The temperature is usually in the range of 20 to 39° C., and the treatment time is usually in the range of 15 seconds to 48 hours.

The above "appropriate conditions for solubilization treatment" include most (for example, within the range of 70 to 100%, preferably within the range of 90 to 100%) proteins in mammalian cells (cytoplasm and/or cell nucleus). The temperature is usually in the range of 20 to 39° C., and the treatment time is usually in the range of 15 seconds to 48 hours.

In the preparation method of the present invention, the conditions for simultaneous proteolytic treatment and binding of the peptide fragment to the peptide-binding solid phase carrier, the proteolytic treatment, the solubilization treatment, and the peptide fragment to the peptide-binding solid phase carrier Conditions suitable for proteolytic treatment are preferable as the conditions for simultaneously carrying out the binding of .

In the present preparation method, the method of contacting one droplet with the other droplet may be a method of directly contacting both droplets in an organic solvent liquid, or both droplets may be brought into contact with each other. A method may also be used in which the droplets are dropped into the organic solvent liquid so as not to come into contact with each other, and then the two droplets are brought into indirect contact with each other in the organic solvent liquid using a force such as gravity.

In the present preparation method, in order to further increase the efficiency of solubilizing mammalian cell proteins and to solubilize DNA binding proteins and histone proteins present in the cell nuclei of mammalian cells, mammalian cells are treated with a surfactant. Before or after the solubilization treatment with a surfactant, or simultaneously with the solubilization treatment with a surfactant, a high salt concentration (for example, within the range of 200 to 800 mM, preferably within the range of 300 to 600 mM) treatment; , Benzonase, Micrococcal Nuclease) treatment; ultrasonic treatment; within the range of 20 minutes [preferably within the range of 1 to 10 minutes] heat treatment); These processes may be one type of process or two or more types of processes.

In the preparation method of the present invention, a reducing agent (for example, dithiothreitol; DTT) is added before the proteolytic treatment or at the same time as the proteolytic treatment in order to more effectively fragment the protein with the proteolytic enzyme. ], 2-mercaptoethanol, tris[2-carboxyethyl]phosphine [TCEP]) to reduce disulfide bonds (S—S bonds) in cysteine (S) residues constituting mammalian cell proteins to SH groups. treatment to alkylate the SH group with an alkylating agent (e.g., iodoacetamide [IAM], iodoacetic acid [IAA], 2-bromoacetamide, glutathione bromoacetamide, bromoacetic acid, 1,3-propanesultone). preferably.

In the preparation method of the present invention, the proteolytic treatment is usually performed only in the droplets whose surface is covered with the organic solvent liquid, and not in the liquid in contact with the surface of the container.

In Preparation Methods 1 and 2, the amount of solubilized protein to be analyzed is not particularly limited. A solubilized protein in an amount corresponding to one cell (for example, within the range of 0.01 to 10 ng, preferably within the range of 0.1 to 1 ng) can be preferably exemplified because it can be recovered well. In addition, in the present preparation method 3, the number of mammalian cells to be analyzed is not particularly limited, and may be, for example, in the range of 1 to 1000 cells. Within the range can be preferably exemplified.

In the preparation method of the present invention, the volume of droplets formed in the organic solvent liquid is, for example, 0.3 μL or more, preferably 0.6 μL or more, more preferably 0.9 μL or more. When preparing a peptide sample for mass spectrometry from protein (the amount of solubilized protein derived from one cell), from the viewpoint of increasing the concentration of solubilized protein, the amount is, for example, 100 μL or less, preferably 30 μL or less, more preferably 10 μL or less, More preferably, it is 7.0 μL or less. Therefore, in the present specification, the volume of droplets formed in the organic solvent liquid is usually within the range of 0.3 to 100 μL, preferably within the range of 0.6 to 30 μL, more preferably It is within the range of 0.6 to 10 μL, more preferably within the range of 0.9 to 7.0 μL.

The above surfactant can solubilize proteins present in cells and is usually liquid at normal temperature and normal pressure (surfactant liquid). propanesulfonate), CHAPSO (3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate), CYMAL-5 (5-Cyclohexylpentyl β-D-maltoside), Deoxy-BIGCHAP (N,N-Bis(3 -D-gluconamidopropyl)deoxycholamide), n-Dodecyl-β-D-maltopyranoside, MEGA10 (n-Decanoyl-N-methyl-D-glucamine), Nonylthiomaltoside, NP-40, n-Octyl-・D-glucopyranoside, OTG ( octylthioglucoside), Oxatridicylmannoside, sodium N-lauroylsarcosinate, sodium cholate, sodium glycocholate, sucrose monochoalte, Triton X-100, and Tween 20, and Phase Transfer Surfactant (PTS) (e.g., Sodium deoxycholate (SDC), Sodium lauroyl sarcosinate (SLS), Sodium cholate (SC), Sodium chenodeoxycholate (SCDC), Sodium ursodeoxycholate (Sodium ursodeoxycholate; SUDC], sodium dodecyl sulfate; Since it can be transferred (removed) to the solvent liquid), those containing PTS are preferable, and those containing sodium deoxycholate are particularly preferable. Sodium deoxycholate is a substance that migrates from the aqueous phase to the organic phase as a result of increasing the degree of hydrophobicity (ie, decreasing the degree of hydrophilicity) by lowering the pH. In step (p), the PTS to be added may be one type or multiple types (for example, 2, 3, 4, 5 types).

Examples of the surfactant include physiological aqueous solutions (e.g., phosphate buffered saline (PBS), Tris-HCl, Tris Buffered Saline (TBS), Physiological aqueous solutions with a buffering effect such as HEPES-buffered saline and ammonium bicarbonate buffer; It may be a liquid prepared by adding to the above surfactant. As used herein, “isotonic” means that the osmotic pressure is within the range of 250-380 mOsm/L.

As used herein, the term “phase transfer solubilizer (PTS)” refers to a change in the degree of hydrophobicity or hydrophilicity due to changes in the state of pH, peripheral ions, etc., resulting in a transition from the aqueous phase to the organic phase, or means a solubilizer (a substance that dissolves fat-soluble substances) that migrates from to the aqueous phase.

As used herein, mammals include rodents such as mice, rats, hamsters and guinea pigs, lagomorphs such as rabbits, ungulates such as pigs, cows, goats, horses and sheep, and cats such as dogs and cats. Examples include eyes, humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, primates such as chimpanzees, etc. Among them, mice, pigs, and humans are preferred.

The organic solvent liquid may contain an organic compound that can separate from the aqueous phase to form an organic phase and is liquid at normal temperature and normal pressure. Examples include octanol, ethyl acetate, diethyl ether, chloroform, and hexanol. , heptanol, dichloromethane, dioxane, benzene, toluene, hexane, heptane, octane, and pyridine. Ethyl acetate is a suitable example, since the effect thereof is demonstrated in this example. Further, the organic solvent liquid may be a single substance consisting of one type of organic solvent liquid, or a mixture containing a plurality of types (at least two types) of organic solvent liquids.

The peptide-binding solid-phase carrier is not particularly limited as long as it can bind solubilized proteins and peptides produced after proteolytic treatment and is insoluble in droplets. And the bond with the peptide may be a non-covalent bond (e.g., hydrophobic bond, hydrogen bond) or a covalent bond. preferable. Peptide-binding solid-phase carriers that covalently bind proteins and peptides include, for example, the surface of the solid-phase carrier that has an affinity for functional groups (e.g., amino [NH ₂ ] groups, carboxyl [COOH] groups) in peptides. functional group (e.g., carboxyl group, amino group, tosyl group, epoxy group, N-hydroxysuccinimide [NHS] group, azide [N ₃ ] group, hydroxyl [OH] group). A solid phase carrier having a surface coated with a carboxyl group is preferable because the effect of the solid phase carrier has been demonstrated in the Examples described later.

Examples of base materials for the peptide-binding solid-phase carrier include magnetic materials, polystyrene, polypropylene, polyvinyl chloride, nitrocellulose, nylon, polyacrylamide, polyallomer, polyethylene, glass, and agarose.

Examples of the shape of the peptide-binding solid-phase carrier include beads (particles), plates, tubes, membranes, gels, and the like.

As the substrate and shape of the peptide-binding solid-phase carrier, magnetic beads (magnetic beads) are preferable from the viewpoint of rapidity and convenience.

Commercially available magnetic beads coated with carboxyl groups (e.g., FG beads-COOH [Tamagawa Seiki], Dynabeads-COOH [Thermo Fisher Scientific], Magnosphere MX200/Carboxyl [ JSR Life Science Co., Ltd.]) or commercially available amino group-coated magnetic beads (for example, FG beads-NH ₂ [Tamagawa Seiki Co., Ltd.]) can be used.

The proteolytic enzyme may be any enzyme that can hydrolyze peptide bonds in proteins. Specifically, trypsin, chymotrypsin, lysyl endopeptidase, clostripain. , Endo Arg-C, Endo Lys-C, Endo Asn-C, etc. Among them, trypsin is preferable.

In the step (b) of the present preparation method, the peptide fragment-peptide binding solid phase carrier complex is separated to prepare a peptide sample for mass spectrometry. Any method can be used as long as it is separated from the organic solvent liquid and prepared into a sample containing peptide fragments that can be analyzed by mass spectrometry. - A method of mixing a liquid containing a peptide-binding solid-phase carrier complex with a solvent for mass spectrometry, or a method of collecting a liquid containing a peptide fragment-peptide-binding solid-phase carrier complex with a pipette man or a pipette, and transferred to a new container and mixed with a solvent for mass spectrometry. If necessary, the liquid containing the peptide fragment-peptide-binding solid phase carrier complex may be further subjected to desalting, solubilization, extraction, concentration, drying, etc., and redissolved in a solvent for mass spectrometry. good.

Examples of the solvent for mass spectrometry include acetonitrile, trifluoroacetic acid (TFA), formic acid (FA), heptafluorobutyric acid (HFBA), and pure water.

As used herein, the term “peptide sample for mass spectrometry” refers to converting a peptide sample into gaseous ions (ionization) using an ion source, moving the peptide sample in vacuum in the analysis unit using electromagnetic force, or It means a peptide sample (a sample containing peptide fragments that constitute mammalian cell proteins) that can be detected by a mass spectrometer when the peptide sample ionized by time-of-flight is separated according to the mass-to-charge ratio. Here, the ionization method using the ion source includes electron ionization (EI) method, chemical ionization (CI) method, field desorption ionization (FD) method, fast atom bombardment (FAB) method, matrix-assisted laser desorption. A method such as an ionization (MALDI) method, an electrospray ionization (ESI) method, or the like can be appropriately selected. Separation methods such as ion trap type, time-of-flight (TOF) type, and Fourier transform ion cyclotron resonance type can be appropriately selected. Tandem mass spectrometry (MS/MS), which combines two or more mass spectrometry methods, can also be used. If necessary, the peptide fragment can be separated and purified from contaminants and analyzed by gas chromatography (GC), liquid chromatography (LC), or high performance liquid chromatography (HPLC).

In the step (b) of the present preparation method, when the droplets contain PTS (e.g., sodium deoxycholate) as a surfactant, the peptide-binding solid-phase carrier to which the peptide fragment is bound and the organic solvent liquid A preferred method further includes a step (x) of adding an acid (eg, TFA, FA, hydrochloric acid) to the droplets to oil-solubilize the PTS in the droplets in the organic solvent liquid before separating the and. The pH of the droplets after adding the acid is, for example, 3.0 or less, preferably 2.0 or less.

In the preparation method of the present invention, the peptide-binding solid-phase carrier hardly interferes with mass spectrometry, as demonstrated in the examples below. Therefore, in the present preparation method, it is not necessary to remove the peptide-binding solid phase carrier from the peptide sample for mass spectrometry containing the peptide fragment-peptide-binding solid phase carrier complex.

EXAMPLES The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples. In the following Examples and Comparative Examples 1 and 2, a human embryonic kidney cell-derived cell line (HEK293 cells) (obtained from ATCC [American Type Culture Collection]) was seeded in a 10 cm dish containing 10% FBS. The cells were used after being cultured in a DMEM medium in a _CO2 incubator (at 37°C) until 80% confluent.

1-1 Method [Preparation method of example sample]
After degrading the solubilized cell protein in a droplet whose surface is covered with an organic solvent liquid and in the presence of a peptide-binding solid-phase carrier, The solid phase carrier was collected and a peptide sample (example sample) for mass spectrometry was prepared. Specifically, experiments were conducted according to the following procedures [1] to [7].

[1] HEK293 cells were trypsinized, detached from the culture vessel, collected, and added to 1 mL of surfactant solution (12 mM sodium deoxycholate [SDC], 3 mM N- sodium lauroyl sarcosinate, 50 mM Tris-HCl [pH 9.0]), sonication was performed for 20 minutes, followed by heating at 95° C. for 5 minutes to solubilize cell proteins.
[2] Magnetic beads coated with carboxyl groups (particle diameter: 180 nm ± 30 nm, FG beads-COOH, manufactured by Tamagawa Seiki Co., Ltd., hereinafter referred to as Dilute with 29 μL of a solution (3 mM SDC, 3 mM N-lauroyl sarcosinate sodium, 25 mM Tris-HCl [pH 9.0], and 37.5 mM ammonium bicarbonate buffer) containing 17.2 μg/μL (simply referred to as “FG beads”) - After preparation, 1 μL of it was added to 50 μL of ethyl acetate solution added to a 1.5 mL Eppendorf tube to prepare a droplet containing cell protein-bound FG beads, the surface of which was covered with the ethyl acetate solution. Such droplets consisted of 1 ng of cellular protein-bound FG beads (166.7 ng) added to the droplets (3 mM SDC, 3 mM N-lauroyl sarcosinate sodium, 25 mM Tris-HCl [pH 9.0], and 37.5 mM weight). ammonium carbonate buffer).
[3] In order to reduce and alkylate cysteine side chains in cellular proteins, a solution containing DTT (dithiothreitol) and iodoacetamide (IAA) was added so that the final concentrations of DTT and IAA were 10 mM and 50 mM, respectively. , was mixed with droplets containing cell protein-bound FG beads.
[4] A liquid containing trypsin was mixed with a droplet containing cell protein-bound FG beads so that 1 ng of trypsin was added, and proteolytic treatment was performed overnight at 37 ° C., resulting in Droplets containing FG beads bound with peptide fragments (peptide fragment bound FG beads) were prepared.
[5] In order to transfer the SDC contained in the droplets to the organic phase (ethyl acetate solution), a trifluoroacetic acid (TFA) solution was added to the peptide fragment-bound FG beads so that the final concentration of TFA was 10%. was mixed with a droplet containing In addition, the pH of the said droplet was 2.0 or less. After shaking the Eppendorf tube up and down for 1 minute and incubating at room temperature for 5 minutes, the organic phase (ethyl acetate solution) was aspirated and removed using a Pipetman.
[6] The aqueous phase (liquid containing peptide fragment-bound FG beads) is concentrated and dried under reduced pressure for 3 minutes using a centrifugal concentrator (manufactured by TOMY), followed by a liquid containing 5% acetonitrile and 0.5% TFA. Suspended in 30 μL.
[7] Desalting and concentrating using SDB-XC StageTip (manufactured by GL Sciences) to purify the peptide fragment, and then resuspending in 0.1% TFA solution to prepare the example sample. A chromatography tandem mass spectrometer (manufactured by Sciex) was used to analyze the signal intensity of the entire mass spectrum (see "Example sample" in Fig. 1).

[Comparative Example 1 sample preparation method]
Degradation treatment of the solubilized cell protein is performed in a droplet whose surface is covered with an organic solvent liquid and in the presence of a peptide-binding solid-phase carrier as described in [Example sample preparation method] above. Instead of conducting in a liquid in contact with the surface of the container and in the absence of a peptide-binding solid-phase carrier, peptide fragments were recovered and a peptide sample for mass spectrometry (Comparative Example 1 sample) was prepared. Specifically, experiments were conducted according to the following procedures [1] to [8].

[1] HEK293 cells were trypsinized, detached from the culture vessel, collected, and added to 1 mL of surfactant solution (12 mM SDC, 3 mM N-lauroyl sarcosinate sodium, 50 mM Tris-HCl [pH 9.0]). After sonication for 20 minutes, the mixture was heated at 95° C. for 5 minutes to solubilize cellular proteins.
[2] The solubilized cell protein concentration was adjusted to a final concentration of 1 ng/μL with a solution (3 mM SDC, 3 mM N-lauroyl sarcosinate sodium, 25 mM Tris-HCl [pH 9.0], and 37.5 mM weight After diluting and adjusting with 100 μL of ammonium carbonate buffer), 1 μL thereof was added to a 1.5 mL Eppendorf tube. Such a solution is a solution in which 1 ng of cell protein is in contact with the tube surface (3 mM SDC, 3 mM N-lauroyl sarcosinate sodium, 25 mM Tris-HCl [pH 9.0], and 37.5 mM ammonium bicarbonate buffer). is contained within.
[3] In order to reduce and alkylate cysteine side chains in cellular proteins, a solution containing DTT and IAA was added to a final concentration of DTT and IAA of 10 mM and 50 mM, respectively. mixed with
[4] A solution containing trypsin is mixed with a solution containing cell protein so that 1 ng of trypsin is added, subjected to proteolytic treatment at 37° C. overnight, and peptide fragments obtained by such treatment are included. A liquid was prepared.
[5] To remove SDC contained in the liquid, first, 50 μL of ethyl acetate liquid was added to prepare droplets containing the peptide fragment, the surface of which was covered with the ethyl acetate liquid.
[6] Next, in order to move the SDC contained in the droplets to the organic phase (ethyl acetate solution), the TFA solution is mixed with the peptide fragment-containing droplets so that the final concentration of TFA is 10%. bottom. The entire tube was agitated up and down for 1 minute and incubated at room temperature for 5 minutes, after which the organic phase (ethyl acetate solution) was aspirated and removed using a Pipetman.
[7] The aqueous phase (liquid containing the peptide fragment) is concentrated and dried under reduced pressure for 3 minutes using a centrifugal concentrator (manufactured by TOMY), and then suspended in 30 μL of a liquid containing 5% acetonitrile and 0.5% TFA. muddy.
[8] Desalting and concentrating using SDB-XC StageTip (manufactured by GL Sciences), purifying the peptide, and resuspending in 0.1% TFA solution to prepare Comparative Example 1 sample. Using a chromatography tandem mass spectrometer (manufactured by Sciex), the signal intensity of the entire mass spectrum was analyzed (see "Comparative Example 1 sample" in Fig. 1).

[Comparative Example 2 sample preparation method]
The decomposition treatment of the solubilized cell protein is not performed in a droplet whose surface is covered with an organic solvent liquid as in the above [Example sample preparation method], but in a state in which it is in contact with the container surface. After the measurement in the liquid, the peptide-binding solid-phase carrier to which the peptide fragment was bound was collected, and a peptide sample for mass spectrometry (Comparative Example 2 sample) was prepared. Specifically, experiments were conducted according to the following procedures [1] to [8].

[1] HEK293 cells were trypsinized, detached from the culture vessel, collected, and added to 1 mL of surfactant solution (12 mM SDC, 3 mM N-lauroyl sarcosinate sodium, 50 mM Tris-HCl [pH 9.0]). After sonication for 20 minutes, the mixture was heated at 95° C. for 5 minutes to solubilize cellular proteins.
[2] The solubilized cell protein concentration was adjusted to a final concentration of 1 ng/μL with a solution containing 17.2 μg/μL of FG beads (3 mM SDC, 3 mM N-lauroyl sarcosinate sodium, 25 mM Tris-HCl [pH 9.0] and 37.5 mM ammonium bicarbonate buffer), and 1 μL thereof was added to a 1.5 mL Eppendorf tube. Such a solution is a solution in which 1 ng of cell protein-bound FG beads (166.7 ng) are in contact with the tube surface (3 mM SDC, 3 mM N-lauroyl sodium sarcosinate, 25 mM Tris-HCl [pH 9.0], and 37.5 mM ammonium bicarbonate buffer).
[3] In order to reduce and alkylate cysteine side chains in cell proteins, a solution containing DTT and IAA was added to cell protein-bound FG beads so that the final concentrations of DTT and IAA were 10 mM and 50 mM, respectively. was mixed with a liquid containing
[4] A solution containing trypsin is mixed with a solution containing cell protein-bound FG beads so that 1 ng of trypsin is added, and subjected to proteolytic treatment at 37° C. overnight, and peptides obtained by such treatment A liquid containing FG beads bound with fragments (peptide fragment-bound FG beads) was prepared.
[5] To remove SDC contained in the liquid, first, 50 μL of ethyl acetate liquid was added to prepare droplets containing peptide fragment-bound FG beads, the surface of which was covered with the ethyl acetate liquid.
[6] Next, in order to transfer the SDC contained in the droplets to the organic phase (ethyl acetate solution), a TFA solution was added to a solution containing peptide fragment-bound FG beads so that the final concentration of TFA was 10%. mixed with drops. The entire tube was agitated up and down for 1 minute and incubated at room temperature for 5 minutes, after which the organic phase (ethyl acetate solution) was aspirated and removed using a Pipetman.
[7] The aqueous phase (liquid containing peptide fragment-bound FG beads) is concentrated and dried under reduced pressure for 3 minutes using a centrifugal concentrator (manufactured by TOMY), followed by a liquid containing 5% acetonitrile and 0.5% TFA. Suspended in 30 μL.
[8] Desalting and concentrating using SDB-XC StageTip (manufactured by GL Sciences), purifying the peptide, and resuspending in 0.1% TFA solution to prepare Comparative Example 2 sample. Using a chromatography tandem mass spectrometer (manufactured by Sciex), the signal intensity of the entire mass spectrum was analyzed (see "Comparative Example 2 sample" in Fig. 1).

[Comparative Example 3 sample preparation method]
The solubilized cell protein is degraded in the absence of a peptide-binding solid-phase carrier, instead of being performed in the presence of a peptide-binding solid-phase carrier as in [Example sample preparation method] above. After the measurement, peptide fragments were recovered to prepare a peptide sample for mass spectrometry (Comparative Example 3 sample). Specifically, experiments were conducted according to the following procedures [1] to [7].

[1] HEK293 cells were trypsinized, detached from the culture vessel, collected, and added to 1 mL of surfactant solution (12 mM SDC, 3 mM N-lauroyl sarcosinate sodium, 50 mM Tris-HCl [pH 9.0]). After sonication for 20 minutes, the mixture was heated at 95° C. for 5 minutes to solubilize cellular proteins.
[2] The solubilized cell protein concentration was adjusted to a final concentration of 1 ng/μL with a solution (3 mM SDC, 3 mM N-lauroyl sarcosinate sodium, 25 mM Tris-HCl [pH 9.0], and 37.5 mM weight Ammonium carbonate buffer) After diluting and adjusting with 100 μL, 1 μL of it was added to 50 μL of ethyl acetate solution added to a 1.5 mL Eppendorf tube, and the surface was covered with ethyl acetate solution. Drops were prepared. Such droplets contain 1 ng of cellular protein in a droplet (3 mM SDC, 3 mM sodium N-lauroylsarcosinate, 25 mM Tris-HCl [pH 9.0], and 37.5 mM ammonium bicarbonate buffer). is.
[3] In order to reduce and alkylate cysteine side chains in cellular proteins, a solution containing DTT (dithiothreitol) and iodoacetamide (IAA) was added so that the final concentrations of DTT and IAA were 10 mM and 50 mM, respectively. was mixed with droplets containing cellular proteins.
[4] A solution containing trypsin is mixed with droplets containing cell protein so that 1 ng of trypsin is added, and subjected to proteolytic treatment overnight at 37° C., and peptide fragments obtained by such treatment are separated. A droplet was prepared containing
[5] In order to transfer the SDC contained in the droplets to the organic phase (ethyl acetate solution), a TFA solution was mixed with the peptide fragment-containing droplets so that the final concentration of TFA was 10%. After shaking the Eppendorf tube up and down for 1 minute and incubating at room temperature for 5 minutes, the organic phase (ethyl acetate solution) was aspirated and removed using a Pipetman.
[6] The aqueous phase (liquid containing the peptide fragment) is concentrated and dried under reduced pressure for 3 minutes using a centrifugal concentrator (manufactured by TOMY), and then suspended in 30 μL of a liquid containing 5% acetonitrile and 0.5% TFA. muddy.
[7] Desalting and concentrating using SDB-XC StageTip (manufactured by GL Sciences) to purify the peptide fragment, and then resuspending in 0.1% TFA solution to prepare Comparative Example 3 sample, A liquid chromatography tandem mass spectrometer (manufactured by Sciex) was used to analyze the signal intensity of the entire mass spectrum (see “Comparative Example 3” in FIG. 1).

1-2 Results The signal intensities of the total mass spectrum in the samples of Comparative Example 2 and Comparative Example 3 are 3176.5 and 1617.3, respectively, and the intensity of the total mass spectrum of the sample of Comparative Example 1 (920.8) and were about 3.4 times and about 1.8 times higher, respectively (see FIG. 1).

This result is based on whether the solubilized cell protein is subjected to degradation treatment in a droplet whose surface is covered with an organic solvent solution, and the resulting peptide fragment is recovered (Comparative Example 3 sample preparation method), Alternatively, when the peptide fragment obtained by the degradation treatment is bound to a peptide-binding solid-phase carrier and the peptide-binding solid-phase carrier is recovered (Comparative Example 2 sample preparation method), the solubilized cell protein When the decomposition treatment is not performed in a droplet whose surface is covered with an organic solvent liquid, and the peptide fragment obtained by the decomposition treatment is not recovered using a peptide-binding solid-phase carrier ( Compared to Comparative Example 1 sample preparation method), the peptide fragment recovery rate is higher.

In addition, the signal intensity of the total mass spectrum of the example sample is 7880.2, which is about 2.5 compared to the intensity of the comparative example 2 sample and the comparative example 3 sample (3176.5 and 1617.3, respectively). times and about 4.9 times higher (see Figure 1).

This result shows that the solubilized cellular protein is subjected to degradation treatment in a droplet whose surface is covered with an organic solvent liquid (Condition 1), and the peptide fragment obtained by the degradation treatment is subjected to peptide bond (Condition 2) and (Preparation method of example sample), and only one of condition 1 and condition 2 is performed (comparative example Compared to the preparation method of Sample 2 and the preparation method of Comparative Sample 3), the recovery rate of the peptide fragment is further increased.

[Discussion]
The reason why the recovery rate of the peptide fragments in the sample of Comparative Example 2 was higher than that of the sample of Comparative Example 1 was that the peptide fragments generated by the decomposition treatment formed a complex with the FG beads, so that the peptide fragments were recovered and purified. It is thought that the proportion of peptide fragments bound to the surface of the container was suppressed during the process, and the proportion of finally recovered peptide fragments increased.

In addition, the reason why Comparative Example Sample 3 had a higher peptide fragment recovery rate than Comparative Example Sample 1 was that the solubilized cell protein was degraded in a droplet whose surface was covered with an organic solvent liquid. Therefore, the percentage of peptide fragments bound to the surface of the container during the decomposition treatment was suppressed, and the percentage of finally recovered peptide fragments was increased.

In addition, the reason why the recovery rate of the peptide fragment was higher in the example sample than in the comparative example 2 sample and the comparative example 3 sample is that the decomposition treatment of the solubilized cell protein was performed and the surface was covered with an organic solvent liquid. Since the peptide fragment generated during the decomposition treatment formed a complex with the FG beads, during the decomposition treatment of the solubilized cell protein and the recovery and purification process of the peptide fragment, It is conceivable that the percentage of peptide fragments bound to the surface of the container was further suppressed, and the percentage of finally recovered peptide fragments was further increased.

2. Confirmation of Detecting Peptide Fragments Derived from Cellular Proteins The above [Example sample preparation method] was performed without adding the obtained cell protein, and peptide fragments were identified (see FIG. 2). As comparative controls, the above [Preparation method of Example sample], the above [Preparation method of Comparative Example 2 sample], and the above [Preparation method of Comparative Example 2 sample] without adding solubilized cell protein were performed. , identified peptide fragments for each.

When the above [Example sample preparation method] was performed, the average number of identified peptide fragments was 43.3, and the above [Example sample preparation method] was performed without adding solubilized cell protein. It was about 7 times higher than the value (6.3) in the case of
This result shows that the majority (85% or more) of the peptide fragments identified by performing the above [Example sample preparation method] are peptide fragments that constitute cellular proteins, and other peptide fragments ( For example, peptide fragments derived from FG beads and peptide fragments derived from sample carryover) are almost absent.

3. Investigation of peptide-binding solid-phase carriers other than FG beads In order to investigate peptide-binding solid-phase carriers other than FG beads, magnetic beads coated with carboxyl groups (particle size: about 2.2 μm, Magnosphere MX200/Carboxyl , manufactured by JSR Life Sciences, hereinafter simply referred to as "Magnosphere"). Specifically, experiments were conducted according to the following procedures [1] to [2].

3-1 Method [1] HEK293 cells were trypsinized, detached from the culture vessel, collected, and added to 1 mL of surfactant solution (12 mM SDC, 3 mM N-lauroyl sarcosinate sodium, 50 mM Tris-HCl [pH 9.0]). 0]), sonication was performed for 20 minutes, followed by heating at 95° C. for 5 minutes to solubilize cellular proteins.
[2] The solubilized cell protein concentration was adjusted to a final concentration of 300 ng/μL with a solution containing 1120 μg/μL Magnospheres or 120 μg/μL FG beads (12 mM SDC, 12 mM N-lauroyl sarcosinate sodium, and 100 mM Tris-HCl [pH 9.0]) After diluting and adjusting with 100 μL, 20 μL of it was added to a 1.5 mL Eppendorf tube, and according to the instructions attached to the product, the cell proteins bound to each magnetic bead were collected and collected. After purification, Coomassie Brilliant Blue (CBB) staining was performed according to a standard method (see FIG. 3).

3-2 Results When the solubilized cellular proteins were bound to Magnospheres and purified, almost the same amount of cellular proteins could be purified as in the case of binding to FG beads and purification (see FIG. 3). This result indicates that a similar peptide recovery effect can be expected even when a peptide-binding solid-phase carrier such as Magnosphere is used in place of the FG beads in the above [Example sample preparation method]. there is

4. Examination of cell solubilizers other than surfactants Cell solubilizers other than surfactants such as SDC were examined. Specifically, in the above [Example sample preparation method], HEK293 cells were solubilized using 24 mM SDC or 1 M urea, and 0.1 ng of Example samples were prepared from cell proteins, and the recovery rate of peptide fragments was analyzed by mass spectrometry (“urea” in FIG. 4). In addition, the recovery rate of peptide fragments is defined as the amount (0.1 ng) at which all the solubilized cell proteins before recovery are fragmented and recovered, and all peptide fragments constituting the protein are detected (0.1 ng). Shown as relative values. Further, in the analysis systems of Examples 1 and 2, the sample was introduced into the analysis column having an inner diameter of 75 μm using an autosampler, whereas the analysis system of Example 4 and Example 5 described later In the analysis system of , the sample is directly introduced into an analysis column with an inner diameter of 50 μm, and the analysis systems of Examples 4 and 5 have theoretically higher sensitivity than the analysis systems of Examples 1 and 2. It is 1.5 times higher.

When the above [Example sample preparation method] was performed using cellular proteins solubilized with urea, the median recovery rate of peptide fragments was 0.19, and the peptide fragment was solubilized with a surfactant (SDC). Compared to the value (0.40) obtained when the above [Example sample preparation method] was performed using cellular protein, it was reduced to about 1/2 (see FIG. 4).

This result indicates that in order to efficiently recover peptide fragments in the above [Example sample preparation method], it is necessary to solubilize cellular proteins using a surfactant such as SDC. there is

5.1 Mass spectrometry at the cell level In order to examine whether the present preparation method can detect cellular proteins in one cell, 0.1 ng of cellular protein extract equivalent to one HeLa cell was used to perform the above [Preparation method of Example sample] and the above [Preparation method of Comparative Example 2 sample] as a comparative control. In addition, such cell protein extract was obtained by sonicating HeLa cells (obtained from ATCC) in 1 mL of surfactant solution (12 mM SDC, 3 mM N-lauroyl sodium sarcosinate, 50 mM Tris-HCl [pH 9.0]). Solubilized protein obtained by heating for 5 minutes at 95° C. after treatment for 20 minutes.

The number of identified peptide fragments in the example sample was 343, which was about 2.1 times greater than the number (160) in the comparative example 2 sample (see FIG. 5A). In addition, the number of cell proteins (types of cell proteins) identified in the example sample was 44, which was about 2.3 times greater than the value (19) in the comparative example 2 sample (see FIG. 5B).

This result shows that even when a peptide sample for mass spectrometry is prepared from an amount of cell protein equivalent to one cell using the preparation method of the present invention, the decomposition treatment of the cell protein is performed in a state in which it is in contact with the container surface. It shows that peptide fragments constituting cellular proteins can be recovered more efficiently than in the case of using the conventional method performed in liquid.

INDUSTRIAL APPLICABILITY The present invention contributes to the development of therapeutic agents that target cells with a limited number of cells, such as hematopoietic stem cells.

Claims (7)

A method for preparing a peptide sample for mass spectrometry, comprising the following steps (a) and (b).
(a) one or more proteins of mammalian cells solubilized with a surfactant are added to a peptide-binding solid-phase carrier and a proteolytic enzyme in a droplet whose surface is covered with an organic solvent liquid; Preparing a peptide-binding solid-phase carrier bound with a peptide fragment constituting a mammalian cell protein by proteolytic treatment in the presence;
(b) separating the peptide-binding solid-phase carrier to which the peptide fragment constituting the mammalian cell protein is bound, and preparing a peptide sample for mass spectrometry; A method for preparing a peptide sample for mass spectrometry, comprising the following steps (a-1), (a-2), and (b).
(a-1) proteolysis of one or more mammalian cell proteins solubilized with a surfactant in the presence of a proteolytic enzyme in a droplet whose surface is covered with an organic solvent liquid; processing;
(a-2) A peptide-binding solid-phase carrier is added to the droplets obtained in step (a-1) to prepare a peptide-binding solid-phase carrier to which a peptide fragment constituting a mammalian cell protein is bound. the step of
(b) separating the peptide-binding solid-phase carrier to which the peptide fragment constituting the mammalian cell protein is bound, and preparing a peptide sample for mass spectrometry; A method for preparing a peptide sample for mass spectrometry, comprising the following steps (A) and (b).
(A) Solubilization treatment of one or more mammalian cells in droplets whose surface is covered with an organic solvent liquid in the presence of a surfactant, a protease, and a peptide-binding solid-phase carrier and proteolytic treatment to prepare a peptide-binding solid-phase carrier to which a peptide fragment constituting a mammalian cell protein is bound;
(b) separating the peptide-binding solid-phase carrier to which the peptide fragment constituting the mammalian cell protein is bound, and preparing a peptide sample for mass spectrometry; 4. The preparation method according to any one of claims 1 to 3, wherein the peptide-binding solid-phase carrier is a magnetic bead whose surface is coated with carboxyl groups. The preparation method according to any one of claims 1 to 4, characterized in that the surfactant is a surfactant having properties of a phase transfer solubilizer. Before separating the peptide-binding solid-phase carrier to which the surfactant having the property of a phase transfer dissolution agent contains sodium deoxycholate and to which a peptide fragment constituting a mammalian cell protein is bound in step (b) 6. The preparation method according to claim 5, further comprising the step (x) of adding acid to the droplets to oil-solubilize the sodium deoxycholate in the organic solvent liquid. 7. The preparation method according to any one of claims 1 to 6, wherein the organic solvent liquid is a liquid containing ethyl acetate.

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